Molecular insights into the regulation of GNPTαβ by LYSET

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
In vertebrates, newly synthesized lysosomal enzymes traffick to lysosomes through the mannose-6-phosphate (M6P) pathway. The Golgi membrane protein LYSET was recently discovered to regulate lysosome biogenesis by controlling the level of GlcNAc-1-phosphotransferase (GNPT). However, its working mechanism remained unclear. In this study, we demonstrate that LYSET is a two-transmembrane protein essential for GNPT stability, cleavage by Site-1 Protease (S1P), and enzymatic activity. We reconcile conflicting models by showing that LYSET enhances GNPT cleavage and prevents its mislocalization to lysosomes for degradation. We further establish that LYSET achieves this by interacting with GOLPH3 and retromer complexes to anchor the LYSET-GNPT complex at the Golgi. Alanine mutagenesis identified an F4XXR7 motif in LYSET’s N-tail for GOLPH3 binding. The retromer further promotes Golgi retention by binding to the C-terminal of LYSET and recycling it from endolysosomes. Together, our findings reveal LYSET’s multifaceted role in stabilizing GNPT, retaining it at the Golgi, and ensuring the fidelity of the M6P pathway, thereby providing insights into its molecular function.

Introduction
In vertebrates, most lysosomal enzymes are delivered to the organelle lumen through the mannose-6-phosphate (M6P) pathway. At the cis-Golgi, GlcNAc-1-phosphotransferase (GNPT) transfers GlcNAc-1 phosphate from UDP-GlcNAc to specific mannose residues on the high mannose glycan chains of lysosomal enzymes, following which an uncovering enzyme (UCE) removes GlcNAc, forming an M6P monoester. At the trans-Golgi, M6P receptors (M6PRs) recognize the M6P modification and selectively bind to these enzymes. M6PRs traffic from the trans-Golgi network (TGN) to the endosomes, releasing the lysosomal enzymes due to increasing acidity. These enzymes further reach the lysosome through endomembrane trafficking, while the M6PRs are recycled back to the TGN through the retromer machinery1,2. Disruption of the M6P pathway results in the mistargeting of most lysosomal enzymes and a severe lysosome storage disease, Mucolipidosis type II (MLII)3.

TMEM251/LYSET/GCAF is a recently identified gene essential for the M6P pathway and the proper trafficking of lysosomal enzymes4-7. Patients with a loss-of-function mutation in this gene die in childhood or early adulthood. They present symptoms like those seen in MLII, such as severe skeletal dysplasia, coarsened facial features, short stature, and protruding abdomen8. Intriguingly, research shows that cells deficient in LYSET are refractory to several viral infections7. This gene is also critical for the propagation of certain tumors under nutrient-poor conditions6. Therefore, LYSET is crucial for human health and a potential drug target for treating cancers and viral infections.

LYSET's role within the M6P pathway is uncertain, specifically its effect on GNPT. In early September 2022, we and two other groups published simultaneously the discovery of LYSET and its connection to the M6P pathway. The consensus is that LYSET interacts with GNPT at the Golgi, which is essential for cellular GNPT activity. GNPT is an α2β2γ2 hexamer encoded by two genes, GNPTAB (encoding α,β subunits) and GNPTG (encoding γ subunit). Site-1-Protease (S1P) cleaves the α/β precursor upon its arrival at the Golgi, activating GNPT. We observed that the deletion of LYSET resulted in the loss of cleaved β subunit and the accumulation of some uncleaved precursor5. Therefore, we proposed that LYSET is critical for GNPT cleavage and activation of its enzymatic activity. However, our model did not explain the reduction of total GNPT protein levels following LYSET deletion. An alternative model proposed that the processing of GNPT is unaffected following LYSET knockout; instead, the LYSET-GNPT interaction is necessary for retaining cleaved GNPT at the Golgi. The transmembrane helices of GNPT contain multiple hydrophilic residues that could destabilize the enzyme in the membrane. Without LYSET, cleaved GNPT is trafficked to the lysosome and degraded by lysosomal proteases6,7,9,10. However, this model seems counterintuitive since most luminal enzymes fail to reach the lysosome in LYSET knockout cells, thereby abolishing their proteolytic activity. Both models account for the absence of cleaved GNPT observed following LYSET knockout yet occur through different mechanisms.

In this study, we aimed to resolve this uncertainty and elucidate the molecular details of LYSET function. First, we confirmed that LYSET is essential for the M6P pathway and lysosomal digestion. LYSET interacts with GNPT to regulate its cleavage efficiency, enzymatic activity, and protein stability. Deleting LYSET does result in the mislocalization of GNPT to the lysosome and its degradation. Through topology analysis, we determined that LYSET is a 2-transmembrane protein with both termini facing the cytosol. Importantly, in addition to the N-terminal cytosolic tail of GNPT interacting with COPI, the cytosolic tails of LYSET associate with the COPI adaptor GOLPH3 and retromer to maintain the LYSET-GNPT complex at the Golgi. Thus, we propose that LYSET actively contributes to maintaining GNPT Golgi localization by recruiting recycling machinery, including both coatomer and retromer.

Results
Reevaluating the relationship between LYSET and GNPTαβ in the M6P pathway
Because of the differences between the models, we felt it was necessary to reexamine the relationship between LYSET and GNPTαβ carefully. Using a GNPTαβ-3HA knock-in strain in HEK293T background5, we confirmed a loss of the cleaved β subunit upon LYSET knockout, accompanied by the accumulation of a higher molecular weight band in the knockout cells (Fig. 1A-B). The higher molecular weight band is attributed to the full-length GNPTαβ being further glycosylated (Fig. 1A, red arrow). There was a ~72% reduction in total GNPTαβ protein levels (cleaved + uncleaved). Importantly, this reduction was not due to decreased transcription, as q-PCR results indicated comparable levels of GNPTAB mRNA between WT and LYSET knockout cells (Fig. 1C). Additionally, using an antibody recognizing the α subunit, we observed a parallel abolishment of the cleaved α subunit and a reduction in total GNPTαβ levels post LYSET knockout (Fig. 1D-E). It's worth noting that the α subunit, after cleavage by S1P, develops more complex-type glycans and migrates similarly to uncleaved GNPTαβ11. Following deglycosylation by PNGase F treatment, it migrates faster than the uncleaved GNPTαβ (Fig. 1D, PNGase F-treated panel).

Extending our analysis beyond HEK293T cells, we generated LYSET and GNPTAB knockouts in the melanoma SKMEL30 cell line, known for its heightened expression of LYSET and GNPTαβ7. In line with HEK293T cell results, LYSET deletion significantly reduced GNPTαβ levels (~70% reduction) and nearly abolished the cleaved α subunit (Fig. 1F-G). Immunostaining using the α subunit antibody revealed that LYSET deletion led to a loss of most endogenous GNPTαβ Golgi signal (Fig. 1H). Instead, we observed a dot-like artifact within the Golgi cisternae of cells lacking GNPTαβ, most likely generated by the antibody utilized. We did not, however, observe GNPTαβ mislocalization to lysosomes.

Without activated GNPTαβ, one should expect no M6P modifications on lysosomal enzymes. Indeed, using a single-chain antibody against M6P (scFv M6P)12, we observed no differences between samples from LYSET deletion cells, GNPTAB deletion cells, or double deletion cells (Fig. S1A). The minor bands observed in knockout lanes likely represent non-specific binding by the antibody13. Without M6P modification, luminal enzymes, represented by CTSC and CTSD, cannot traffic to lysosomes and mature, resulting in a significant fraction being secreted into the media as unprocessed proenzymes (Fig. S1B). Enzyme assays of lysosomal hydrolases following CI-MPR-Sepharose pull-down supported these findings, as phosphorylated β-hexosaminidase (β-Hex), α-galactosidase (α-Gal), and β-galactosidase (β-Gal) activity were detected only in whole cell lysates from WT cells (Fig. S1C). Finally, the lack of functional hydrolases was also verified by the accumulation of undigested lysosomal substrates, represented by LAPTM4A and LC3B-II (Fig. S1D).

In summary, using two distinct cell lines (HEK293T and SKMEL30) and antibodies targeting both α and β subunits (after 3HA tagging), our findings consistently support that LYSET is essential for the processing and stability of endogenous GNPTαβ. In the absence of LYSET, the cleaved α and β subunits are undetectable, and M6P modifications on lysosomal enzymes are absent, leading to trafficking defects of luminal enzymes and the accumulation of undigested substrates.

The multifaceted role of LYSET in GNPTαβ stability, cleavage, and enzymatic activity
The absence of cleaved α and β subunits could be due to either a lack of S1P-dependent GNPTαβ cleavage (processing model)5 or rapid trafficking of the cleaved α and β subunits to the lysosome for degradation (trafficking model)6,7. However, the processing model cannot explain the ~70% loss of total GNPTαβ.

A key experiment to differentiate these models involves using a pulse-chase to track the fate of newly synthesized GNPTαβ in the absence of LYSET. To this end, we expressed GNPTαβ-V5 under a tetracycline-inducible (Tet-on) promoter and established stable cell lines in both WT and LYSET knockout backgrounds. Following a 2-hour doxycycline induction, we added cycloheximide to halt protein synthesis and monitored the fate of newly synthesized GNPTαβ-V5 at hourly intervals. In WT cells, the majority of GNPTαβ-V5 was cleaved into the β subunit. Total GNPTαβ-V5 (uncleaved + cleaved) remained relatively stable over a 5-hour chase (Fig. 2A-B). In contrast, in LYSET knockout cells, significantly less β subunit (Fig. 2A-B) and the accumulation of a higher molecular weight band (Fig. 2A, red arrow) were observed. Total GNPTαβ-V5 also decreased after 5 hours (Fig. 2A). These results indicate that 1) the processing is nearly abolished, and 2) even uncleaved GNPTαβ-V5 is degraded in the absence of LYSET. They are inconsistent with the hypothesis that LYSET has no impact on GNPTαβ cleavage, as suggested by the trafficking model, but they do support the notion that LSYSET serves as an anchor to maintain GNPTαβ at the Golgi, regardless of cleavage.

The processing model proposes that LYSET acts as a bridge between S1P and GNPTαβ to enhance the cleavage of the latter. So, we asked if increasing the amount of S1P would bypass the dependence on LYSET to cleave GNPTαβ. To this end, we expressed GNPTαβ at low levels (400 ng) in LYSET knockout cells with or without overexpression of S1P and measured GNPT activity. The presence of the additional S1P clearly increased GNPT activity, nearly doubling its value (from 296 to 544 pmol/hr/mg protein, Fig. 2C-D, compare 2 & 3). However, low-level co-expression of LYSET with endogenous S1P had an even greater impact on GNPT activity (3.4 fold increase), whereas the greatest stimulation (almost 5 fold) occurred when both LYSET and S1P were co-expressed (Fig. 2C-D). These data further support that, unlike the assumption that LYSET has no impact on GNPTαβ cleavage, LYSET works synergistically with S1P to enhance its cleavage/activation efficiency.

We next asked if co-expression of LYSET could stabilize the GNPTαβ protein and increase its PTase activity. Keeping the amount of GNPTAB cDNA constant (400 ng), while increasing the amount of LYSET cDNA, resulted in rising levels of PTase activity in a dose-dependent manner. Cellular levels of phosphorylated endogenous β-Hex also increased, but were quickly saturated (Fig. 2E). Further, the amount of the processed β subunit also increased in a LYSET dose-dependent manner (Fig. 2F). These results provide direct evidence that LYSET is critical for stabilizing GNPTαβ and enhancing its PTase activity within a cell.

Lastly, we asked if LYSET is required for GNPTαβ activity after its cleavage/activation. To answer this, we built a GNPTαβ construct that can bypass S1P-dependent processing by replacing its recognition sequence with that of Furin14. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at the Golgi15, allowing efficient GNPTαβ cleavage in the absence of LYSET (Fig. 2G, lanes 4 and 5). As an alternative approach to activating GNPTαβ, we directly deleted its auto-inhibition motif and S1P cleavage site (amino acids 820-928)14. As shown in Fig. 2G, when stably overexpressed, both constructs can rescue the mutant phenotypes of GNPTAB knockout cells but not those of LYSET knockout cells, indicating that LYSET is required for the GNPTαβ activity in vivo even after its cleavage.

In summary, we propose that LYSET is necessary to keep GNPTαβ in the Golgi, where it is essential for the stability of both cleaved and uncleaved GNPTαβ and consequently facilitates S1P-mediated cleavage efficiency and activated GNPTαβ enzyme activity.

Both cleaved and uncleaved GNPTαβ undergo lysosomal degradation without LYSET
What causes the reduction of GNPTαβ after LYSET knockout? In Fig. 1H, we cannot determine if endogenous GNPTαβ is mislocalized to the lysosome. To investigate further, we performed a longer exposure of the endogenous GNPTαβ-3HA immunoblot and detected two prominent bands between 15 and 20 kDa in LYSET KO cells (Fig. 3A), which likely represent degradation products of GNPTαβ-3HA (Red arrow, Fig. 3B). With the longer exposure, we also observed a minor band corresponding to the β subunit (Fig. 3A).

Since proteasomal degradation typically yields small peptides ranging from 6-10 residues16,17, the proteasome is unlikely to generate the observed degradation products. Therefore, we explored the possibility of lysosomal degradation. To investigate, we isolated lysosomes using magnetic beads after incubating cells with dextran-coated magnetite (DexoMAG®), which is endocytosed and delivered to the lysosome (Fig. 3C). In lysosomes from WT cells, the β subunit was clearly detected, while levels of the uncleaved GNPTαβ precursor were negligible (Fig. 3D, lanes 4 vs. 5). Conversely, lysosomes from LYSET KO cells contained dramatically increased amounts of both cleaved and uncleaved GNPTαβ-3HA, accompanied by several prominent degradation intermediates (Fig. 3D, lanes 7 vs. 8). To answer if S1P cleavage is required for the mislocalization of GNPTαβ-3HA, we treated cells with S1P inhibitor, PF-429242, for 19 hours before isolating lysosomes. As shown in Fig. 4E, even though the cleaved β subunit is much reduced after PF-429242 treatment, uncleaved GNPTαβ-3HA still was mislocalized to the lysosome, and the total level of lysosomal GNPTαβ-3HA was largely unchanged (lanes 8 vs. 10). These results support that endogenous GNPTαβ-3HA is mislocalized to the lysosome without LYSET, regardless of its cleavage state.

We also treated cells with BafA1 as an alternative method to confirm lysosomal degradation. As shown in Fig. 3F, 16 hours of BafA1 treatment significantly reduced the prominent degradation products between 15 and 20 kDa and partially stabilized the β subunit. We then investigated whether BafA1 treatment would reveal lysosomal mislocalization of GNPTαβ. Surprisingly, immunostaining with the α antibody showed that the stabilized GNPTαβ was localized to the Golgi rather than the lysosome (Fig. 3G), suggesting that BafA1 stabilizes GNPTαβ by preventing its exit from the Golgi rather than by inhibiting lysosomal degradation. This result is consistent with that found by Richards et al. and Pechincha et al.6,7.

In conclusion, our findings independently verified that LYSET knockout leads to the mislocalization and degradation of GNPTαβ in the lysosome, regardless of its cleavage state. Interestingly, as noted before, BafA1 treatment prevents GNPTαβ exit from the Golgi rather than solely blocking lysosomal degradation.

LYSET has two transmembrane helices with both termini facing the cytosol
To understand how LYSET retains GNPTαβ in the Golgi, we first need to establish LYSET's membrane topology. AlphaFold predicted LYSET to be a three-transmembrane (TM) helices-containing protein18, while other prediction tools, such as TOPCONS and OCTOPUS, suggested it had two-TM helices19,20. A recent publication proposed that LYSET has two transmembrane domains with both N- and C-termini facing the lumen8. However, no experimental evidence was presented.

Four reasonable topology models may exist for LYSET, as depicted in Fig. 4A. We first determined if the loop between TM helices 1 and 2 is located in the cytoplasm, as was previously proposed8. A native 51EGT53 sequence within the loop was mutated to create an NxS/T N-linked glycosylation site (E51N, Fig. 4B) with no detrimental effect on the function of LYSET in stimulating GNPTαβ activity (Fig. S2A). The E51N mutant migrated more slowly than WT LYSET, suggesting it was glycosylated. To confirm this, cell lysate was treated with either Endo H, which had minimal effect, or PNGase F, which collapsed the slower-migrating band to the size of WT, confirming that the E51N mutant had acquired complex N-linked glycans (Fig. 4C). This result demonstrates that the loop region located between residues 28 and 68 is in the lumen of the Golgi, ruling out topology models 1 and 3.

The C-terminal tail faces the cytosol in model 2, while in model 4, it faces the lumen (Fig. 4A). To distinguish between these possibilities, we employed a proteinase K sensitivity assay in which cells partially permeabilized with digitonin were treated with proteinase K, which can then only digest cytosolic proteins, but not those in organelle lumens (Fig. 4D).

For this assay, the C-terminus of LYSET was tagged with GFP, which does not disrupt the normal function of LYSET (Fig. S2B-C). As controls, we used Man1A1-GFP, a Golgi membrane protein with its C-terminal in the lumen (proteinase K resistant), and CTNS-GFP, a lysosome membrane protein with the C-terminus facing the cytosol (proteinase K sensitive). Over a 2-minute time period, Man1A1-GFP remained fluorescent, while both CTNS-GFP and LYSET-GFP were quenched (Fig. 4E-F). This result shows that the C-terminal tail of LYSET is located in the cytoplasm, eliminating model 4. Therefore, LYSET contains two transmembrane helices, with both its N and C termini facing the cytosol (i.e., topology model 2).

Alanine scan of LYSET reveals residues critical for its function and localization
Next, we performed a comprehensive alanine scanning analysis to identify critical residues for LYSET function. We systematically mutated each amino acid of LYSET to alanine, grouping around five residues at a time. Because the anti-LYSET antibody only recognizes the C-terminal tail, mutations closer to the C-terminus (residues 102 to 131) cannot be detected by the LYSET antibody. To address this, either an N-terminal 3xFLAG tag or a 3xHA tag was incorporated for immunoblotting and immunostaining.

In total, 31 mutants covering the entire more common, short-isoform of LYSET were expressed in LYSET KO HEK293T cells under the Tet-on promoter. This promoter, when uninduced, maintains a low level of protein expression (leaky expression), preventing potential overexpression artifacts. To assess the functionality of LYSET mutants, we utilized mature CTSD levels as a readout (Fig. 5A). The deletion of LYSET resulted in the abolishment of mCTSD (Fig. 5A, compare lanes 1 to 2). Reintroducing LYSET mutants restored CTSD maturation to varying degrees. A disease mutant, R7W, served as a positive control in this screen8. A 50% or less cutoff of WT mCTSD levels identified ten defective mutants with loss of LYSET function (highlighted in red, bottom panel, Fig. 5A). Additionally, two regions in the C-terminal of LYSET showed defective CTSD maturation (highlighted in orange, Fig. 5A,C), although mCTSD levels remained above the cutoff level.

The subcellular localization of these mutants were determined by stably overexpressing them in SKMEL30 LYSET knockout cells and immunostaining. Under these conditions, nine out of ten mutants localized to the Golgi and co-localized with GM130 (Fig. S3A-B). Only LYSET(93-96A) lost its Golgi localization and was trapped in the ER (Fig. S3A-C).

We also examined whether these mutations led to the loss of GNPTαβ. While all other mutations led to a significant reduction in GNPTαβ level, LYSET(67- 71A) rescued it to the same extent as WT (Fig. 5B). This suggests that mutations in this region do not disrupt LYSET's function of anchoring GNPTαβ in the Golgi, but potentially impair some other function of this protein in the M6P pathway.

In summarizing these results, we plotted the critical regions in the topology model (Fig. 5C). It indicates that the two TM helices (13-18, 24-28, 67-71, 77-81, 82-86) and the immediate cytosolic regions next to them (2-7, 87-91, 93-96) are crucial for LYSET function. Among them, residues 93-96 are particularly vital for the ER exit. Lastly, a small region within the lumenal loop (39-43) and the very C-terminus (127-131) also play a critical role in LYSET function.

The N-terminal tail of LYSET interacts with GOLPH3 and facilitates its Golgi localization.
We next sought to dissect the molecular mechanism that holds the LYSET-GNPTαβ complex in the Golgi. The N-terminal cytoplasmic tail (N-tail) of GNPTαβ is known to bind directly to the β/δ and γ/ζ subunits of COPI, and this interaction is critical for the Golgi localization of GNPTαβ21. Patient mutations disrupting this interaction result in the enzyme's mislocalization to the lysosome, leading to Mucolipidosis III21. However, the mislocalization of GNPTαβ observed upon LYSET depletion implies that the GNPTαβ-COPI interaction alone is insufficient to retain the enzyme at the cis-Golgi, underscoring the additional contribution of LYSET. Then, how does LYSET perform this role?

We first determined if the N-terminal cytoplasmic tail of LYSET was sufficient to redirect a GFP reporter from the plasma membrane to the Golgi. For this assay, we utilized a construct, SIN-SITM-GFP, encoding the N-tail and transmembrane segment of the plasma membrane protein, sucrase isomaltase (SI), fused to GFP. We then switched its N-tail with the first seven amino acids of the short isoform of LYSET to generate LYSETN-SITM-GFP (Fig. 6A). We and others have shown that while SIN-SITM-GFP localizes to the plasma membrane, replacement of its N-tail with the N-tails from several other Golgi glycosyltransferases, including GNPTαβ, restricts the chimera to the Golgi21,22. Immunofluorescence microscopy of WT HeLa cells transfected with LYSETN-SITM-GFP shows tight colocalization with the Golgi marker, GM130, whereas SIN-SITM-GFP localized predominantly on the plasma membrane (Fig. 6B-C). This result shows that the N-tail of LYSET has sufficient information to retain the chimera in the Golgi. Further investigation via an alanine mutagenesis scan revealed that residues F4 and R7 are critical for the Golgi localization (Fig. 6D-F). Importantly, the R7W mutation in humans is known to cause dysostosis multiplex, also known as Mucolipidosis type V5,8.

To further test the importance of the F4XXR7 motif, we tagged full-length LYSET mutants (F4A, R5A, or R7A) with a 10xGCN4 tag23 (Fig. S4A) and expressed them under the leaky expression of a Tet-on promoter. Both F4A and R7A mutations failed to rescue the LYSET KO phenotype in HEK293T cells, as indicated by the lack of CTSD maturation (Fig. S4B-C). Moreover, when moderately induced by a 100 ng/ml doxycycline, these two mutations resulted in reduced GM130-LYSET colocalization and mislocalization of some LYSET to the plasma membrane in SKMEL30 cells (Fig. S4D-E). In contrast, LYSET(R5A) retained the ability to rescue mCTSD (Fig. S4B-C) and was still localized to the Golgi, although an increased ER signal was observed (Fig. S4D-E).

The adapter protein GOLPH3 has multiple recognition motifs, one of which is an LXXR motif. The F4XXR7 motif at the N-terminal of LYSET is similar to the LXXR motif, it suggesting that its interaction with COPI might be mediated by GOLPH3 22 24-26. The Munro group has shown that a GST-GOLPH3 fusion protein efficiently bound GalNT2-SITM-GFP that carries the N-tail of the Golgi enzyme NA-acetylgalactosaminyltransferase-222. We prepared GST-GOLPH3 and performed the binding assays using GalNT2-SITM-GFP and SIN-SITM-GFP as positive and negative controls, respectively. As shown in Fig. 6G, LYSETN-SITM-GFP bound well to GST-GOLPH3, while there was no binding of GST-GOLPH3 to SIN-SITM-GFP as previously reported22.

We also tested whether the coatomer complex directly interacts with the N-terminus of LYSET. To this end, we synthesized a biotinylated peptide corresponding to the first 7 amino acids (N-tail) of LYSET, immobilized it on streptavidin beads, and performed pull-down assays with Sf9 insect cell lysates expressing the β/δ or γ/ζ subcomplexes of COPI21. While the GNPTαβ N-tail (positive control) robustly bound both β/δ and γ/ζ dimers, no binding was detected with the LYSET N-tail (Fig. 6H).

To confirm GOLPH3’s role in vivo, we deleted GOLPH3 in HeLa cells, which caused LYSETN-SITM-GFP to lose most of its Golgi localization (Fig. 6B-C). U2OS cells lacking GOLPH3/3L also had reduced colocalization of induced LYSET-10xGCN4 with GM130 (Fig. S5A-B). Furthermore, immunostaining of SKMEL30 cells confirmed diminished Golgi signals for GNPTαβ in GOLPH3-deficient cells (Fig. S5C-E), underscoring the importance of LYSET for maintaining GNPTαβ at the Golgi.

Finally, knocking down GOLPH3 disrupted lysosomal enzyme trafficking in HeLa cells, as evidenced by impaired CTSC and CTSD maturation (Fig. S5F-G).

In summary, our findings demonstrate that LYSET’s N-tail mediates Golgi localization through its GOLPH3 interaction.

The retromer complex recycles LYSET from the endo-lysosome to the Golgi.
Another critical molecular machinery for maintaining membrane proteins at the Golgi is the retromer complex27-30. Retromers actively retrieve cargo proteins, such as CI-MPR and sortilin, from endosomes and transport them back to the trans-Golgi network, preventing their degradation in the lysosome. To assess whether the retromer complex also contributes to the Golgi localization of LYSET, we deleted VPS35, a key component of the VPS26/29/35 retromer subcomplex, in HEK293T cells. Deletion of VPS35 resulted in a significant reduction in total endogenous GNPTαβ protein levels, while LYSET levels remained largely unchanged (Fig. 7A-B). Lysosome purification from two independent knockout colonies revealed that significant fractions of GNPTαβ and LYSET were mislocalized to the lysosome and partially degraded (Fig. 7C, lanes 6, 8 vs. 4). Furthermore, we observed a decrease in VPS26, another essential component of the retromer complex, indicating that the stability of retromer subunits is dependent on an intact retromer subcomplex (Fig. 7A). Similar observations have been reported before31.

We then utilized the SKMEL30 cell line to investigate the localization of endogenous LYSET and GNPTαβ through immunostaining. As shown in Fig. 7D-E, deletion of VPS35 led to the mislocalization of endogenous LYSET from the Golgi to punctate structures. Moreover, the Golgi localization of GNPTαβ was markedly reduced (Fig. 7D-E). These results underscore the critical role of the retromer complex in maintaining both proteins at the Golgi.

We then explored whether overexpression of LYSET could restore the Golgi localization of GNPTαβ in VPS35 deletion cells. Indeed, upon overexpression, a substantial amount of LYSET localized to the Golgi, leading to the restoration of GNPTαβ Golgi localization (Fig. 7D-E). These findings suggest that the loss of GNPTαβ Golgi localization following VPS35 deletion is attributable to the depletion of LYSET from the Golgi.

Since retromer is required to maintain LYSET at the Golgi, our next goal was to identify the retromer-binding motif within LYSET. We first asked whether the cytosolic C-terminal region of LYSET contains sufficient information for Golgi localization. To test this, we generated a chimera by replacing the cytosolic tail of the plasma membrane protein interleukin-2 receptor alpha chain (IL-2Rα) with the C-terminal tail of LYSET (amino acids 88–131) (Fig. 8A)32. In WT cells, this chimera displayed increased Golgi localization, indicating that the LYSET C-terminal tail contains a functional Golgi-targeting signal (Fig. 8B-C). However, in VPS35 knockout cells, this Golgi localization was lost, and the chimera redistributed into punctate structures (Fig. 8B-C). These results suggest that the C-terminal tail of LYSET confers Golgi localization in a retromer-dependent manner.

To further map the motif responsible, we performed an alanine scanning mutagenesis of the C-terminal region (residues 87–131) in the LYSET-10xGCN4 construct and analyzed subcellular localization in SKMEL30 cells. Of these, we observed decreased Golgi localization and increased punctate signal for mutations clustered in two regions: residues 97 and 99, and residues 108–110. Notably, these regions partially reduced mCTSD levels in earlier experiments, though not below our predefined threshold (Fig. 5A,C – orange).

To test whether these motifs function cooperatively, we combined the mutations. Constructs bearing either the 97A,99A or 108–110A mutations alone showed only a slight increase in LAMP2 colocalization, without a significant reduction in Golgi signal (Fig. 8D-E). However, the combined mutant, LYSET-10xGCN4(97,99,108–110A), showed significantly reduced colocalization with the Golgi marker and markedly increased colocalization with LAMP2 (Fig. 8D-E), suggesting additive disruption of Golgi retention. These findings imply that retromer binds cooperatively to both motifs for efficient LYSET recycling to the Golgi (Fig. 8F).

Bringing together our findings from alanine scanning, GOLPH3 interaction, and retromer dependence, we propose a model in which LYSET actively maintains GNPTαβ localization and function at the Golgi (Fig. 9). First, LYSET forms a stable complex with GNPTαβ. Second, the N-termini of both LYSET and GNPTαβ interact with COPI machinery, either directly(GNPTαβ) or via GOLPH3 (LYSET), promoting retrograde recycling from the trans-Golgi to the cis-Golgi. Third, under normal conditions, a fraction of the LYSET–GNPTαβ complex escapes the Golgi and traffics to endo-lysosomes, but this population is retrieved by the retromer complex, which recognizes the C-terminal motifs of LYSET. Thus, LYSET plays an active role in Golgi retention, bridging COPI-mediated retrograde trafficking and retromer-mediated retrieval, ensuring proper localization and activity of the GNPTαβ complex.

Methods
Mammalian cell culture
Cell lines used in this study are listed in Supplemental Table S1. HEK293T (CRL-3216) and HeLa (CCL-2) were purchased from ATCC, and SKMEL30 (SK1980-526) was purchased from Memorial Sloan Kettering Cancer Center. The U2OS cell line was a kind gift from Prof J. Tan. Cells were cultured in DMEM (Invitrogen) containing 10% Fetal Bovine Serum (Thermo Fisher, and 20% serum for SKMEL30 cells), 1% penicillin and streptomycin (Invitrogen), and 1 µg/ml plasmocin (Invivogen) at 37°C, 5% CO2. All cells were tested negative for mycoplasma using the Mycoalert™ mycoplasma detection kit (Lonza).

Plasmids
Plasmids used in this study are listed in Supplemental Table S2. The CDS of human LYSET was purchased from the DNASU plasmid Repository (Arizona State University).

Transient transfection
Cells were cultured in DMEM containing 10% (20% for SKMEL30 cells) serum media to 40-50% confluency before transfection. Cells were transfected with individual overexpression plasmids (2 µg DNA for a 3.5cm2 dish) using either Lipofectamine 2000 (Invitrogen) or jetOPTIMUS transfection reagent from Polyplus (Sartorius) according to the manufacturer's protocol. Cells in 6-well plates were harvested 48 h post-transfection and lysed in 150 µl of buffer A (25 mM Tris-Cl, pH 7.2, 150 mM NaCl, 1% Triton-X 100 and protease inhibitor cocktail).

Generation of lentiviral stable cell lines
HEK293T cells were transfected with transfer plasmid, psPAX2 (Addgene 12260), and pMD2.G (Addgene 12259) at a 3.5:3.5:1 ratio using Lipofectamine 2000 according to the manufacturer's instructions. 72 hours after transfection, the supernatant was collected and passed through a 0.45 µm filter. HEK293T, SKMEL30, or HeLa cells were seeded in 6cm2 dishes and infected with the collected supernatant. After 24 hours of infection, the cells were kept in selection (1 µg/ml for puromycin, and 10 µg/ml for blasticidin) for at least 10 days before subsequent analysis.

Generation of CRISPR-Cas9 KO and KI cell lines
LYSET and GNPTAB knockout cells were generated as described in Ran et al37. In brief, sgRNA guides were ligated into pspCas9 (BB)-2A-Puro (Addgene, 48139) or Lenti-multi-CRISPR (Addgene 85402) plasmids. For single colony isolation, cells were transfected with CRISPR-Cas9 knockout plasmids using Lipofectamine 2000 according to the manufacturer's instructions. 24 hours after transfection, cells were treated with 1 µg/ml puromycin (Invitrogen) for 2-3 days. Cells were then diluted into 96-well plates to a final concentration of 0.5 cell per well. The colonies were further screened and verified via western blot and/or sequencing analysis to confirm clean knockout colonies (Fig. S 6) .

The generation of GNPTAB knock-in (KI) cell line was described in Zhang et al 5. A 300 bp homology arms (upstream and downstream from the stop codon) were amplified from the genomic DNA. The 3HA coding sequence was inserted in between the homology arms by overlapping extension. The resulting DNA fragment was ligated into the pGEM-T Easy vector and transfected (4 µg) into HEK293T cells together with CRISPR-Cas9 plasmid (2 µg). 24 hours after transfection, cells were treated with 1 µg/ml puromycin (Invitrogen) for 2-3 days. Cells were then diluted into 96-well plates to a final concentration of 0.5 cell per well. The single colonies were screened by PCR using a 3HA internal forward primer and a reverse primer located 600 bp downstream of the stop codon. The candidate KI colonies were further verified by western blot and sequencing analysis.

The following reported sgRNAs and shRNAs were used in this study:
LYSET sgRNA1: 5' – TGTCCACACCCAAAAAGGCA – 3',
GNPTAB sgRNA1: 5' – ACTCATTGCGATCTATCGAG – 3',
GNPTAB sgRNA2 (KI): 5' – CTTCTATACTCTGATTCGAT – 3',
VPS35 sgRNA1: 5' –CTCAAGGGATGTTGCACACC– 3'
GOLPH3 sgRNA1: 5’ – AGGGCGACTCCAAGGAAACG – 3',
GOLPH3L sgRNA2: 5’ – GGATATCCGCCTTACTCTTA – 3',
GOLPH3 shRNA1: 5’ – GAGAAACAGAACTTCCTACTT – 3'

Sample preparation and western blotting
Cells were collected in ice-cold 1X PBS, pelleted at 2700 g for 1 min, and lysed in lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% Triton) containing protease inhibitor cocktail (Bimake) at 4°C for 20 min. Cell lysates were centrifuged at 18,000 g for 15 min at 4°C. The protein concentration of the supernatant was further measured by Bradford assay (Bio-rad) and normalized. After adding 2X urea sample buffer (150 mM Tris pH 6.8, 6 M Urea, 6% SDS, 40% glycerol, 100 mM DTT, 0.1% Bromophenol blue), samples were heated at 65°C for 8 min before further analysis.

Normally, 30 µg of each lysate was loaded and separated on SDS-PAGE gels. Note that for the LYSET blot in Fig. S2, only 1/20 of the samples were loaded in the LYSET overexpression lanes. Protein samples were transferred to a nitrocellulose membrane for western blot analysis. After incubation with primary and secondary antibodies, membranes were scanned using the Odyssey CLx imaging system (LI-COR) or developed with CL-XPosure film (Thermo Scientific).

The following primary antibodies were used for western blotting in this study: rabbit anti-GFP (1:3000, TP401, Torrey Pines Biolabs), mouse anti-actin (1:5000, 66009-1-lg, Proteintech), mouse anti-GAPDH (1:2000, 60004-1-1g, Proteintech), rabbit anti-CTSD (1:1000, 21327-1-AP, Proteintech), rabbit anti-LC3 (1:2000, 14600-1-AP, Proteintech), rabbit anti-HRD1 (1:4000, 13473-1-AP, Proteintech), rabbit anti-Golgin160 (1:1000, 21193-1-AP, Proteintech), mouse anti-HA (1:500, 16B12, BioLegend), mouse anti-CTSC (1:500, D-6, Santa Cruz Biotechnology), mouse anti-VPS35 (1:500, sc-374372, Santa Cruz Biotechnology), rabbit anti-LAPTM4A (1:1000, HPA068554-1, Millipore-Sigma), rabbit anti-LYSET (1:1000, HPA-48559, Millipore-Sigma), mouse anti-V5 (1:3000, 46-0705, Invitrogen), rabbit anti-δ-COP (PA5- 21484, Invitrogen), rabbit anti-VPS26 (1:1000, ab23892, Abcam), rabbit anti-GOLPH3 (1:2000, AB236296, Abcam), rabbit anti-β-COP (ab2899, Abcam), anti-GCN4 [IPI-C11L34] (1:1000, 218104-rAb, Addgene), mouse anti-LAMP2 (1:1000 H4B4-c, DSHB). Rabbit serum containing antibodies against the α-subunit of GNPTαβ was kindly provided by William Canfield, and the IgG fraction was purified using CaptivA Protein A affinity resin. The generation of single-chain antibodies against M6P was described in Zhang et al. 5,38.

The following secondary antibodies were used in this study: goat anti-mouse IRDye 680LT (926-68020), goat anti-mouse IRDye 800CW (926-32210), goat anti-rabbit IRDye 680LT (926-68021), goat anti-rabbit IRDye 800CW (926-32211). These secondary antibodies were purchased from LI-COR Biosciences and used at 1:10,000 dilution.

To detect LYSET, M6P (scFv M6P), GNPT αβ-3HA KI, or the uninduced 10xGCN4, the anti-protein A HRP (PA00-03, Rockland, for LYSET and M6P) or mouse HRP (115-035-046, Jackson labs, for GNPTαβ-3HA KI) secondary antibodies were used at 1:10,000 dilution. The signal was detected with the Pierce ECL kit (Thermo Scientific). All unprocessed blots are included in the Source Data file.

Membrane isolation
The membrane isolation protocol was adapted from Shao and Espenshade39, with some modifications. Essentially, cells with 70-80% confluency from a 10cm2 dish were collected in ice-cold 1X PBS, and pelleted at 2700 g for 1 min. The pelleted cells were resuspended in 1 ml ice-cold membrane isolation buffer (1 mM EDTA and 1 mM EGTA in 1X PBS, with protease inhibitor) and homogenized. The homogenate was centrifuged at 900 g for 5 min at 4°C, and the supernatant was transferred to a new tube and centrifuged at 20,000 g for 20 min at 4°C to collect membranes. After centrifugation, the membrane pellet was further dissolved in lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 1% Triton) containing 1X protease inhibitor cocktail (Biomake) at 4°C for 20 min. The undissolved membranes were removed by another round of centrifugation at 20,000 g for 15 min at 4°C, and the protein concentration from the supernatant was measured by Bradford assay and normalized. Samples were incubated with 2X urea sample buffer samples at 65°C for 8 min before western blot analysis.

Phosphotransferase Activity Assays
The protocol for measuring the GlcNAc-phosphotransferase activity was adapted from van Meel et al.13, with some modifications. Essentially, the cells were transfected at 50–60% confluency in six-well plates with a total of 2 μg plasmid DNA (pcDNA3.1 vector, GNPTAB cDNA, and LYSET cDNA in the indicated combinations) and 3 μL jetOPTIMUS transfection reagent according to the manufacturer's protocol. Two days post-transfection, the cells were harvested and lysed in 150 µl of buffer A. Whole cell lysate (50 µg) was assayed in a final volume of 50 µl in buffer containing 50 mM Tris (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, 2 mg/ml BSA, 2 mM ATP, 75 μM UDP-GlcNAc, and 1 μCi UDP-[3H] GlcNAc, with 100 mM α-methylmannoside (α-MM) as acceptor. The reactions were performed at 37°C for 1 h, quenched with 950 µl of 5 mM EDTA (pH 8.0), and the samples applied to a QAE-Sephadex column (1 ml of packed beads equilibrated with 2 mM Tris, pH 8.0). The columns were washed twice, first with 3 ml of 2 mM Tris (pH 8.0), then with 2 ml of the same buffer. Elution was performed twice, first with 3 ml of 2 mM Tris (pH 8.0) containing 30 mM NaCl, then once again with 2 ml of the same buffer. 8 ml of EcoLite scintillation fluid (MP Biomedicals) was then added to the vials, and the radioactivity in the collected fractions was measured using a Beckman LS6500 scintillation counter. All CPM values that were obtained after subtracting buffer-only background were converted to pmol of GlcNAc-phosphate transferred to α-MM per hour per mg total protein lysate (pmol/hr/mg).

Lysosomal enzyme assay
Soluble bovine cation-independent mannose 6-phosphate (CI-MPR),purified from fetal bovine serum40was conjugated to CNBr-activated Sepharose 4B (Millipore-Sigma) as per the manufacturer's instructions. For lysosomal enzyme assays, 600 µg of whole cell lysates prepared from either WT HEK293T cells, or from untransfected or 48 h transfected LYSET knockout, GNPTαβ knockout, or double knockout cells were incubated with equivalent amounts of the CI-MPR affinity beads for 2 h at 4C, washed twice with cold Tris-buffered saline containing 1% Tx-100 (pH 7.4) (TBST), then assayed for bound lysosomal enzymes. Briefly, β-hexosaminidase (β-Hex), α-galactosidase (α-Gal) and β-galactosidase (β-Gal) were assayed in a final volume of 50 µl with 5 mM 4-methylumbelliferyl(MU)-N-acetyl-β-d-glucosaminide (Sigma-Aldrich), 5 mM 4-MU-α-d-galactopyranoside and 5 mM 4-MU-β-d-galactopyranoside (Calbiochem, San Diego, CA), respectively, in 50 mM citrate buffer containing 0.5% Triton X-100 (pH 4.5) at 37°C. Reactions were stopped after 1 h by addition of 950 µl of 0.4 M glycine-NaOH (pH 8.0), samples were vortexed and spun down briefly, and the supernatant transferred to a round-bottomed glass tube. The fluorescence was then measured using a TURNER Model 450 Fluorometer (Barnstead Thermolyne Corporation, Dubuque, Iowa), with excitation and emission wave lengths of 360 and 450 nm, respectively.

GST pull-down assays
In order to assess the binding of the LYSET N-tail to GOLPH3, GST-GOLPH3 was first expressed and purified from E. coli BL21 Codon-Plus (RIL) cells (Agilent). For binding assays, 100 µg of the purified GST-GOLPH3 fusion protein was first immobilized on 50 µl of packed glutathione-agarose beads at room temperature for 1 h in TBST. The beads were washed once with cold TBST, then 300 µl of whole cell lysates prepared from cells transfected with the various GFP chimeras were added, and the beads tumbled for 2 h at 4C. The beads were washed 3 times with cold TBST, and the pellets were resuspended in sample buffer and heated at 100C for 10 min before SDS-gel loading.

Secretion analysis
Cells were cultured to reach 70-80% confluency to collect secreted proteins from the media. They were washed with serum-free DMEM three times and incubated with serum-free DMEM for 16 hours. The conditioned media were collected and centrifuged at 500 g for 5 min to remove cell debris, filtered with a 0.45 µm filter, and concentrated to ~100 µl using 10 kDa cutoff Amicon Centrifugal filters (Millipore-Sigma). The protein concentration from the concentrated media was measured by Bradford assay and normalized to its cell lysate. Samples were then mixed with 2X Urea buffer and heated at 65°C for 8 min before further analysis. For the induced secretion assay in Fig. S1B, the cells were incubated with serum-free DMEM containing 10 mM NH4Cl for 16 hours before collection and further processing.

In-vitro deglycosylation
The deglycosylation assay was performed as described in Venkatarangan et al. 41, with some modifications. For the whole cell lysate, cells were rinsed once with 1X ice-cold PBS and then harvested in 1 ml of PBS, split into three equal parts, and centrifuged at 1000 g, 1 min, 4°C. For deglycosylation assay from the membrane fraction, the pelleted cells were resuspended in 1 ml ice-cold membrane isolation buffer (1 mM EDTA and 1 mM EGTA in 1X PBS, with protease inhibitor) and homogenized. The homogenate was centrifuged at 900 g for 5 min at 4°C, and the supernatant was split into three equal parts, transferred to a new tube, and centrifuged at 20,000 g for 20 min at 4°C to collect membranes. Two of the three pellets were resuspended in 40 μl of lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% Triton, 1x PIC), while the third pellet was resuspended in 1X glyco-buffer 2 (50 mM sodium phosphate pH 7.5, 1% NP-40, 1X PIC). All pellets were allowed to lyse for 20 min at 4°C. Lysates were subsequently centrifuged at 18,000 g, 15 min, 4°C, and the protein concentration of the resulting supernatants was measured using Bradford assay. All supernatants were normalized to equal protein concentration. One lysate in the lysis buffer was used as an untreated control. To the second lysate in lysis buffer, 3.9 μl of 10X Denaturation buffer (5% SDS, 400 mM DTT) was added to 35 μl of lysate and boiled at 98°C for 10 min. The lysate was then allowed to cool to room temperature for 5 min, and a further 4.7 μl of 10X glyco-buffer 3 (500 mM sodium acetate pH 6) and 2 μl of Endo H enzyme were added and incubated at 37°C overnight. To the lysate in glyco-buffer 2, 10 μl of glyco-buffer 2 was added to 35 μl of the lysate, and 2 μl of PNGase F was added further added to it and incubated at 37°C overnight. After incubation, all lysates were mixed with an equal volume of 2X urea sample buffer, heated at 65°C for 10 min before further analysis.

Fluorescence Protease Protection (FPP) assay
HeLa cells were transfected with GFP tagged construct for 48 hours and grown to a density of about 1-5 × 106 cells/ml (~80% confluency for a 6 cm dish). The cells were resuspended, and 10 μL were loaded onto a cell microfluidic plate (Millipore-Sigma, CellASIC ONIX M04S-03) through the cell loading sequence for adherence overnight in 37°C, 5% CO2 incubator42. Cells were first washed with KHM buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl2) for 2 min at 6 psi (41.4 kPa), then treated with 20 μ M of digitonin for 3 min at 6 psi, and with 50 μg/ml proteinase K at 6 psi. Images were captured on the DeltaVision imaging system (GE Healthcare Life Sciences) at intervals of 30 seconds.

Immunostaining, microscopy, and image processing
Immunostaining was performed as described in Zhang et al.38, with some modifications. Cells grown on 1.5 circular glass coverslips were washed with ice-cold 1X PBS and fixed in 4% paraformaldehyde for 10 min at room temperature38. Cells were permeabilized with 0.3% Triton in PBS for 15 min. The samples were blocked in 3% BSA for 30 min at room temperature, followed by incubating with primary and secondary antibodies. The cell nucleus was stained using Hoechst (1:8000, Invitrogen). Coverslips were mounted in Fluoromount-G (SouthernBiotech) and cured for 24 hours before imaging.

The following primary antibodies were used for immunostaining in this study: rabbit anti-GNPT α subunit (1:100, homemade), rabbit anti-LYSET (1:100, HPA-48559, Millipore-Sigma), mouse anti-GM130 (1:100, 610822, BD Biosciences), and anti-GCN4 [IPI-C11L34] (1:100, 218104-rAb, Addgene), mouse anti-IL-2Ra (1:100, 05-170, Millipore-Sigma).

The following secondary antibodies were used at 1:100: FITC goat anti-rabbit (111-095-003, Jackson ImmunoReseach) and TRITC goat anti-mouse (115-025-003, Jackson ImmunoReseach), sheep anti-mouse (NA931, Cytiva) and donkey anti-rabbit (NA934, Cytiva).

Microscopy was performed with a DeltaVision system (GE Healthcare Life Sciences) or a Dragonfly Confocal Microscope System (Andor Technology). The DeltaVision microscope was equipped with a scientific CMOS camera and an Olympus UPLXAP0100X objective. The following filter sets were used: FITC (excitation, 475/28; emission, 525/48), TRITC (excitation 542/27; emission 594/45), and DAPI (excitation 390/18; emission 435/48). Image acquisition and deconvolution were performed with the SOFTWORX program. For spinning disk confocal microscopy, the following filter sets were used: FITC (excitation, 475/28; emission, 525/48), TRITC (excitation 542/27; emission 594/45), and DAPI (excitation 390/18; emission 435/48). Image acquisition and deconvolution were performed with the Fusion program. Images were further cropped or adjusted using ImageJ (National Institutes of Health). The Pearson coefficient analysis was performed using the FIJI ImageJ JACOP BIOP program.

Magnetic Isolation of Lysosomes
HEK293T cells were seeded in 150 mm dishes containing DMEM with 10% FBS. Once they reached 40% confluency, cells were incubated in DMEM containing 10% FBS, 10 mM HEPES, 10% DexoMAG40 for 24 hours. Following this, cells were incubated in DMEM with 10% FBS for another 24 hours to allow the endocytosis of the magnetic nanoparticles. Following the incubation, cells were rinsed twice and then harvested in 1X ice-cold PBS, using a cell scraper, and spun down at 60 g, 10 min, 4°C. The supernatant was discarded, and the pellet was resuspended in 3 ml of homogenization buffer (HB: 5 mM Tris, 250 mM Sucrose, 1 mM EGTA in mass-spectrometry grade water (ThermoFisher), pH 7.4. HB was supplemented with protease inhibitor cocktail (Roche) immediately before use and degassed. The cell suspension was homogenized in a Dounce homogenizer for 30 strokes and then passed through a 22G needle eight times. The homogenate was then centrifuged at 800 g, 10 min, 4°C. The supernatant was collected, and 80 µl was taken as the whole cell lysate (WCL), mixed with equal volume of 2X urea buffer (150 mM Tris pH 6.8, 6 M Urea, 6% SDS, 40% glycerol, 100 mM DTT, 0.1% Bromophenol blue) and heated at 65°C for 10 min. The remaining supernatant was allowed to flow freely through the magnetic LS column (130-042-401, Miltenyi Biotech). The column was pre-equilibrated with HB and attached to a platform that provided the magnetic field (130-091-051, Miltenyi Biotech). The column was then washed with 20 mL of HB using gravity. The columns were then detached from the magnetic field, and the lysosomes were eluted by flushing 1 ml of HB through the column using a syringe. The eluted lysosomes were further concentrated by spinning the eluate at 14,000 rpm, 1 hour, 4°C. The pelleted lysosome fraction was resuspended in 80 µl lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% Triton, 1X PIC) and heated at 65°C for 10 min with an equal volume of 2X urea buffer.

Quantification and statistical analysis
For the quantification of western, the band intensity was quantified using Image Studio software version 6.0 (LI-COR). For film, band intensity was calculated using ImageJ. Graphs were generated using Prism version 10.6.1 (GraphPad). Statistical analysis was performed with the two-tailed unpaired t-test with Welch’s correction or one-way ANOVA with Tukey’s or Dunnett’s multiple comparison test. Error bars represent the standard deviation. Representative images and blots were independently repeated at least two times with similar results.

Data availability
Data supporting this study is provided within the paper and supplementary files. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Yang, et al. Kornfeld and Li describe some details on how the recently identified TMEM251- also termed\nLYSET controls the GlyNAc-1-phosphotransferase (GNPT) which is required for mannose 6 phosphorylation of many\nlysosomal hydrolases. LYSET/TMEM251 was discovered and characterized in two recent publications in Science (PMID:\n36074821, 36074822) and NCOMM (PMID: 36096887). In the current study some confirmatory experiments are presented\nillustrating the essential role of TMEM251/LYSET for the M6PR-dependent delivery of acid hydrolases to the lysosomal\ncompartment. It is confirmed (as outlined in detail below) that a lack of TMEM251/LYSET causes GNPT to be localized to\nlysosomes where is gets degraded most likely by residual active lysosomal proteases. Topology studies of\nTMEM251/LYSET suggest that both polypeptide termini are facing the cytosol. Two binding partners of TMEM251/LYSET\nwere characterized, Golph3 and retromer, that may be contributing to Golgi localization of TMEM251/LYSET.\nAs such the study appears too preliminary and is in large parts not presenting novel information. Especially in the first part\n(Fig 1-4; results sections (lines 107 to 264) and statements in the introduction (lines 97-99)) of the study there is a general\nlack of conceptual advance. In its current version – with the focus in topology determination, alanine scan and binding\npartner description- I would see the manuscript much better suited for a specialized biochemical journal. The analysis of the\nTMEM251/LYSET topology, the reported alanine scan, potential interaction with GOLPH3 and retromer are new. However,\nit remains open what are the exact and specific roles of the newly described binding partners of TMEM251/LYSET.\nExamples for a lack of novelty:\no Loss of GNPTAB (Figs 2 and S2) was reported before (both Science papers)\no Interaction of TMEM251 and GNPT was demonstrated before (both Science papers and NCOMM paper)\no Mislocalization and lysosomal degradation of GNPT in TMEM251 KO cells was reported before\no Impaired M6P tagging (Fig. 1E) in TMEM251 or GNPTAB KO was demonstrated in both Science papers before\no Hypersecretion of proforms of cathepsin and lack of mature forms intracellularly was reported before in TMEM251 KO cells\n(Fig. S1B, C), accumulation of LC3 was previously reported (in the other two studies but also already by this group)\nConcerns:\nFigure 1 represents in part already knowledge presented earlier and relies on over-expression in HEK cells since apparently\nGNPT ptase activity was too low to be measured. Why were no experiments designed in a more suitable cell line allowing to\nendogenously monitor the consequences of a loss of TMEM251/LYSET or/and GNPT? The conclusion from the presented\nexperiment that both TMEM251/LYSET and GNPT are crucial for the M6P pathway are not new. There is a strong claim that\nTMEM251 and GNPTAB steady state Golgi localization is achieved by vesicular recycling, but evidence is merely indirect\n(COP, Golph3, Retromer interactions).\nPtase activity assays (Fig 1B): Immunoblot panels for these transfected cells need to be included to (i) confirm TMEM251 KO\nand (ii) be able to judge whether GNPT expression levels correlate with detected activity; this similarly applies to Fig. 1C; in\nthis case, however, data in Fig. 1 D appear to be from a similar experimental setup; it would be good to have panels 1C and\n1D from one experiment, but they should then be included in one joint subpanel; this similarly applies to panels 3B and 3C;\ncontrol immunoblots are also missing for Fig. S3 and S4 - this actually appears to be a more general issue; Ptase activity\nassays should be supplemented with immunoblot panels showing KO and overexpression controls.\nThe authors observed that high-level ectopic expression of GNPTAB leads to substantial Ptase activity also in TMEM251\nKO cells (Fig. 1). What could be the underlying mechanism? Is the retention mechanism the authors described before (direct\ninteraction of GNPTAB with COPI) sufficient at such high GNPTAB levels? Or is overexpressed GNPTAB still lost from the\nGolgi due to absence of TMEM251 and simply constantly replenished due to high overexpression reaching sufficient steady\nstate Golgi levels? This should be addressed experimentally. Also, can overexpression of GNPTAB rescue the TMEM251\nphenotype (e.g. cathepsin maturation defects)?\nThe lack of novelty holds - in large part – also true for the reported interdependent protein stability of TMEM251 and GNPT\nand the observation that TMEM251 is required for the processing efficiency and therefore enzymatic activity of GNPTab.\nThe conclusions and interpretations of the data in Fig.2/3 have also been reported before. In Fig.2B it is reported that after HA-\ndetection there should be an accumulation of a higher molecular weight fragment on the TMEM251KO cells. However, such\na fragment cannot be seen.\nLysosomal degradation of GNPTab (and the explanation why GNPTab is largely invisible in TMEM251 KO cells) has also\nbeen previously demonstrated, i.e. the experiments here do not add much to the previous knowledge.\nAs an actual first new part of this study - in terms of a gain in knowledge - the determination of the discussed topology of\nTMEM251/LYSET is presented. With state-of-the-art technology and artificial introduction of a new glycosylation site\n(Fig.5C) and proteinase K protection assays (Fig.5E) it could be revealed that TMEM251 contains two transmembrane\nhelices with both termini facing the cytosol. Studying 31 alanine mutants of TMEM251 and rescue of cathepsin D maturation\nin TMEM251 KO cells revealed ten mutants with an apparent loss of TMEM251 function. The two transmembrane regions\nand amino acids close to them appear especially critical for TMEM251 function. How the identified regions modulate GNPT\nbinding and TMEM251 functions remain -however- unsolved.\nThis alanine scan is a comprehensive approach to identify sequences critical to TMEM251 function, but requires further\ncontrols. In Figure 6A abundance of mature cathepsin D is used as only (indirect!) readout for assessing the capacity of\nTMEM251 variants to rescue the TMEM251 KO phenotype in 293T cells. TMEM251 mutant expression levels need to be\nshown (and possibly be included in the quantification) here along with cathepsin D and actin levels, as it obviously matters\na) whether TMEM251 mutants are expressed at all (an unstable TMEM251 mutant could be informative re: critical structural\nmotifs) and b) whether their potency to rescue the KO phenotype is really different and not merely due to different expression\nlevels. A selection of mutants found to display impaired TMEM251 function was subsequently tested in SKMEL30 cells (with\nnotable differences in expression among the mutants!), but using a different readout (ability to rescue GNPTAB levels).\nReadouts in both cell lines should be the same (i.e. also GNPTAB levels need to be shown in 293T cells and cathepsin D\nlevels in SKMEL30 cells) and SKMEL30 data should be similarly quantified to make any robust and cell line-independent\nconclusions. This is in particular required as the authors observe marked differences for e.g. 67-71A in both systems. In this\nparticular case, it would also be interesting if localization differs between 293T and SKMEL30 cells.\nIn their study, the authors exclusively use TMEM251 isoform 2 (i.e. a short isoform with only 7 cytoplasmic amino acids\npreceding TMD1 (according to topology model 2)). The authors only briefly comment on this – “the more common short\nisoform of TMEM251” (line 301). It is indeed true that most cell lines appear to predominantly express this isoform at the\nendogenous level – but the SKMEL30 cells used quite extensively in this study are a notable exception that have a\nrobust/dominant endogenous expression of isoform 1 (see Fig. 8A in this manuscript, but also data in Richards et al. and\nPechincha et al.). Also, both the long and the short isoform were previously shown to be fully capable of rescuing a\nTMEM251 deficiency phenotype, i.e. are fully functional. That in mind, the authors should also check whether the critical\nmotifs identified in their alanine scan of the short isoform also impair function of the long isoform and in particular their\nexperiments with the Golgi localization reporter (Fig. 7) should be repeated with the longer N-terminus of the long TMEM251\nisoform.\nThe N-terminal region of TMEM251 contained sufficient information to retain a GFP-plasma membrane reporter in the Golgi.\nSince the N-terminal sequence met the criteria for binding of a COP1 adaptor, Golph3 its interaction with the N-terminus of\nTMEM251 was demonstrated in a GST pull down approach. However, direct binding of Golph3 in the streptavidin-binding\nassay to the N-terminus of TMEM251 could not be shown. Importantly, no experiments are presented to show the\nimportance of Golph3 (e.g. in KO cells) for lysosomal sorting and GNPT stability and function.\nGFP reporter constructs like the one used in Figure 7 are well established tools to study the determinants of Golgi\nlocalization of type II membrane proteins. Indeed, the observations made here suggest that the first seven amino acids of\nTMEM251 could dictate a Golgi localization and potentially also an interaction with GOLPH3. However, this system is highly\nartificial for many reasons and the findings from these experiments should be taken with a grain of salt. TMEM251 is not a\ntype II membrane protein and thus cannot be simply reduced to only its first 7 amino acids - in particular when the alanine\nscan data show that the 2-7A mutant still exhibits Golgi localization and instead mutation of other motifs impair Golgi\nlocalization. If this region is important for Golgi localization of TMEM251, shouldn’t this mutant be mislocalized/lost from the\nGolgi apparatus? N-termini of type II Golgi membrane proteins are frequently rather short and Arg-rich – the capacity of\nTMEM251 aa 2-7 to direct the reporter to the Golgi may thus simply a coincidence. Also, these experiments need to be\nfurther substantiated by constructs with TMEM251’s longer N-terminus and additional mutants as well as full-length\nTMEM251.\nVPS35 deletion (retromer member) did not affect TMEM251 expression levels but slightly reduced the level of GNPT as has\nbeen reported earlier. TMEM251 localization was apparently changed although the punctate Golgi-like structures remain\nunexplained. A fraction of TMEM251 could be found stabilized in lysosomes suggesting a retromer involvement in recycling\nof missorted TMEM251.\nThe imaging data depicting the mislocalization of TMEM251 and GNPTAB in retromer-deficient cells needs to be assessed\nmore quantitatively. There still appears to be a substantial portion of TMEM251 in the Golgi and also the single-dot Golgi\nstaining of GNPTAB is not as evident as in Fig. 4. The authors also write that “the TMEM251-GNPT complex may exit the\nGolgi and mislocalize to endo-lysosomes” (line 412/413), but co-staining of TMEM251 and GNPTAB in retromer-deficient\ncells would be required to substantiate that indeed the TMEM251-GNPT complex is mislocalized (and not its two\ncomponents independently). Also, the authors do see a destabilization/loss of GNPTAB under these conditions, but not of\nTMEM251. Is GNPTAB more prone to lysosomal degradation than TMEM251?\nQuantification and statistical evaluation of multiple independent experiments and biological replicates are missing for most\nof the (immunoblot and microscopy-based) experiments.\nMinor comments:\nDifferent amounts of plasmid DNA (encoding in particular GNPTAB or TMEM251) were transfected transiently for numerous\ndata panels to emphasize dose-dependent effects (e.g. of TMEM251 on GNPTAB stability). The figure legends should state\nthat these absolute DNA amounts given in the figure panels apply to six-well culture dishes.\nFigure legends are rather cryptic in general and lack important information here and there (e.g. in Fig. 4E and 4G inhibitor\nconcentrations should be provided in the figure panels or the figure legends)\nThe authors write that all genome editing cell clones/lines (KO and KI) were validated/confirmed by Sanger sequencing.\nThese data should be made available in the supplementary figures\nFig 2B: From the context it can be assumed that GNPTαβ and GNPTβ were detected with an anti-HA antibody in these KI\ncells, but that needs to be stated in the legend\nSuppl. Fig. 2B – TMEM251 panel, between lane 3 and 4: Cutting/Splicing? Or why does TMEM251 in GNPTAB KOs exhibit\na different running behavior compared to parental cells (cf to Fig.1E)\nContradictory statements: line 158 (section heading; “Tmem251 is required for stability of GNPTαβ) vs. line 189 to 191 (“In\nthe absence of TMEM251, […] the uncleaved GNPTαβ level remains relatively unchanged”); in particular the latter statement\ndoes not appear to adequately reflect observations in e.g. Fig. 2C and Fig. S2B)\nLine 244: WT cells should be TMEM251 WT cells (lanes 4 and 5 are GNPT KI cells, so no parental cells)\nLines 260 to 262 – reference to Fig. 4G missing in main text\nFig. 2E: Dot-like Golgi (-proximal) localization of GNPTAB upon TMEM251 deletion in SKMEL30 cells: This observation\n(in SKMEL30 cells) stands in contrast to observations by Richards et al. showing dot-like, but not really Golgi-proximal\nGNPTAB staining in TMEM251 KO 293FT cells. What is the nature of this unique dot-like structures? What happens if cells\nare treated with protease inhibitors, does GNPTAB then also accumulate in lysosomal structures (in light of the lysosomal\nenrichment data presented)?\nCould GNPTAB mutants (K4Q, S14Y) shown previously to impair binding of GNPTAB to COPI also impair binding to\nTMEM251? (previously looked at?)\nLine 384: “Deletion of VPS35 resulted in a significant reduction in endogenous GNPTab protein levels in both cell lines” –\nthis statement is not overtly supported by the data presented. In SKMEL30 there is only a modest (at best) reduction and this\nwould need to be properly quantified in light of this statement; the 293T data appear to be supporting this statement better,\nbut quantification would be required here as well to exclude transfer/ECL issues\nFig. 5C – The E51N mutant generated to test the topology model appears to be not glycosylated quantitatively. Is this an\nissue with heavy overexpression or could this indicate the simultaneous presence of topologically different variants of\nTMEM251?\nWhen transiently transfected, low-level overexpression of GNPTAB is insufficient to compensate for the lack of TMEM251\nand a drastic difference in Ptase activity is observed between WT and TMEM251 KO cells (Fig. 1B, lane 5 vs 6). When\nstably expressing low-level GNPTAB-V5 variants (Fig. 3D), no notable difference in GNPTAB-V5 levels is evident between\nTMEM51 WT and TMEM251 KO cells (despite the latter clearly having a phenotype). How can that be explained?", "reviewer_2": "In this research, the authors conducted a thorough analysis of the role of TMEM251/LYSET protein in the processing,\nstability, and localization of GlcNAc-1-phosphotransferase 35 (GNPT), a key enzyme in the M6P pathway. TMEM251 is\nessential for human health and is a potential drug target for treating cancers and viral infections. While two studies in 2022\nlinked TMEM251 to the M6P pathway, its specific role remained unclear. The authors used gene editing, microscopy,\nmutagenesis analysis, and in vitro methods to show that TMEM251 is crucial for the processing, activity, and maintenance of\nGNPT in the Golgi apparatus. They also conducted biochemical analysis and alanine mutagenesis scanning to determine\nthe membrane topology of TMEM251 and identified critical residues in its cytosolic region that affect protein function and\nlocalization. The findings confirm the importance of TMEM251 and GNPT in the M6P pathway at the physiological level and\nsupport the hypothesis that GNPT’s steady localization in the Golgi is maintained through vesicular recycling mediated by\nTMEM251, COPI, Golph3, and retromer. This work is suitable for publication in this journal, although several critical\nquestions remain unanswered. Addressing these questions could further elevate the significance of the findings: What is the\nstoichiometry of the GNPT-TMEM251 complex? Is this a stable complex, or is it regulated temporally and spatially? Are\nother Golgi enzymes regulated by TMEM251? What is the molecular mechanism by which TMEM251 influences GNPT\nprocessing?\nSpecific comments and suggestions for improvement:\nThe study predominantly utilized knockout cells and overexpression conditions. This might lead to secondary defects,\ncompensatory responses, and other artifacts. Implementing acute protein downregulation techniques and focusing on\nendogenous proteins—perhaps tagged using gene-editing methods—would significantly enhance the robustness of this\nstudy.\nIn Figure 1E, it is crucial to display a complete GNPTAB blot to confirm the effect of TMEM251 knockout on the processing of\nthe endogenous enzyme in HEK cells. Additionally, clarification on the role and relevance of HRD1 in this context would be\nhelpful.\nTMEM251 KO cells showed mislocalization of GNPT, forming a \"single-dot-like structure\" at the Golgi rather than\nmislocalizing to lysosomes. It's crucial to investigate the nature of this structure. Immuno-electron microscopy (immune-EM)\ncould be a suitable further exploration method.\nThe observation that in TMEM251 KO cells treated with\nBafA1, stabilized GNPT remains in the Golgi instead of moving to the lysosome is significant. The authors suggest that\n\"BafA1 stabilizes GNPT by impeding its Golgi exit rather than inhibiting lysosomal degradation.\" This novel finding warrants\nfurther investigation to understand the underlying mechanisms.\nThe effects of GOLPH3 depletion (in the context of GOLPH3/GOLPH3L double KO) on the M6P pathway and the\nlocalization of TMEM251 and GNPT should be explored. This could provide insights into the regulatory mechanisms\ninvolving GOLPH3 in this pathway.", "reviewer_3": "The study by Yang, Doray and colleagues analyzes the role of TMEM251 in the activity of the GNPT enzyme which\nconstitutes the fulcrum of the M6P pathway, and which is essential for the function of lysosomal enzymes. The most\ninnovative part of the work concerns the role of TMEM251 in the GNPT traffic mechanisms. This is a work of considerable\ninterest which, however, in its current form presents some important gaps of both an interpretative and experimental nature.\nThe work is very complex and, as such, intrinsically difficult to analyze, but it is also written in a poorly organized way so that\nthe logical connections between the parts are even more difficult to follow\nBelow we will try to analyze these weaknesses and where possible indicate how to overcome them.\nMajor points:\nThe authors describe the effects of TMEM251 on the stability of the GNPT enzyme and give evidence that TMEM251 is\nessential for the activating GNPT cleavage and is also essential for the stability of the cleaved portion of GNPT, in the sense\nthat the levels of this protein decrease in the absence of TMEM251. In this regard the authors note that GNPT is intrinsically\nunstable due to the composition of its transmembrane domains, but in the following experiments the authors demonstrate\nthat in the absence of TMEM251 the uncleaved GNPT is degraded in lysosomes. This is probably the reason for the\ninstability of GNTP. The flow here is difficult to understand and the authors should logically connect the two data sets.\nThe authors show that TMEM251 is necessary for GNPT activity even after cleavage has occurred ie, after the activation\nstep has been carried out. This is attributed to the physical interaction of TMEM251 with GNPT, which would form a stable\ncomplex which would have the effect of stabilizing the active conformation of the enzyme.\nIn the next part of the work, however, the authors demonstrate that TMEM251 is essential to prevent lysosomal degradation\nof both the cleaved and uncleaved enzyme. From what we understand, it is therefore probable that the property of TMEM251\nto maintain the activity of the cleaved enzyme is simply due to the fact that it localize GNPT at the Golgi preventing its\nlysosomal degradation. This possibility does not seem to be grasped by the authors. It is an ambiguity that the authors must\nresolve either with textual changes and/or with new experiments.\nThe authors perform an elegant series of experiments that leads them to the conclusion that TMEM251 interacts with Golph3\nin the trans Golgi to maintain the localization of the TMEM251/GNPT complex in the cis Golgi and for the prevention of\nlysosomal degradation of GNPT.\nA perplexing point here is the observation that mutants in the cytosolic tail of TMEM251 that abolish the binding between the\ntail and Golph3 do not change the localization of TMEM251 in the Golgi, in other words, that the interaction between\nTMEM251and Golph3 is not essential for the retention of TMEM251 and GNPT in the Golgi. The authors suggest that there\nmay be additional regions beyond the cytosolic tail that maintain TMEM251 in the Golgi. This is possible, but the finding\nraises concerns that should be addressed with textual changes or new experiments.\nThe authors show evidence that the retention of GNPT in the Golgi requires both the binding of GNPT itself to COP1 via the\nmotif previously identified by Doray and Kornfeld, and the binding between TMEM251 and Golph3, as shown in this study.\nThen, in the last paragraph, the authors propose the existence of a further mechanism for the retention of GNPT in the Golgi\nwhich would be based on the activity of the retromer, the complex that transports proteins from the endo-lysomal system to\nthe Golgi, which thus be responsible for maintaining the localization and functionality of GNPT/ TMEM251. This last\nconclusion is consistent with the data, which however are simply based on depletion and overexpression experiments of a\nprotein crucial for the function of the retromer, VPS35.\nBased on these collective evidence, the authors suggest that a triple recycling/retention mechanism is necessary for the\nmaintenance of GNPT/ TMEM251 in the Golgi and present a model in figure 9.\nThe model shows a single retrotransport step from the trans to the cis Golgi. This is difficult to understand based on current\nknowledge and also inconsistent with the data shown in the manuscript and with the localization of the adaptor GOLPH3\nwithin Golgi. The overall model could be modified by proposing a mechanisms of Golgi retention in three steps: first step of\nretrotransport from Golgi trans to medial cisternae due to the binding between TMEM251 and Golph3 which would then be\nfollowed by a second retrograde step dependent on the binding between the cytosolic tail of GNPT and COP1. The third\nstep, mediated by the retromer, could recycle the fraction of GNPT / TMEM251 complex that escaped from the first two\nrecycling mechanisms, from the endo-lysosomes to the trans Golgi. Here, GOLPH3 first and COPI then could bring the\ncomplex back to the cis Golgi. Organized in this way the model could be coherent with the majority of the data and highlight\nthe novelty of the paper.\nOverall, we believe that the information brought by this study potentially represents a notable progress in an area of research\nwhich is currently under strong development and is of great importance, namely, the mechanisms of selective retrotransport\nof Golgi enzymes by specialized adaptors, with regulatory effects on glycosylation. However, the authors need to address\nthe points we have raised with textual changes and new experiments.\nMinor points:\n1) One hypothesis put forward by the authors suggests that TMEM251 may serve as a bridging factor, linking GNPT with the\nS1P protease necessary for GNPT activation. It would be important to investigate the localization and levels of S1P in\nTMEM251 knockout cells.\n2) While GOLPH3 is suggested as a mechanism for retaining the TMEM251-GNPT complex at the Golgi, its involvement\nremains unproven in GOLPH3 knockdown (KD) or knockout (KO) cells. The authors establish that the N-terminal tail of\nTMEM251 contains sufficient information for GOLPH3-dependent Golgi localization. They should define the motif.\nHowever, this is the sole evidence implicating GOLPH3 in TMEM251 retention at the Golgi, and using flow cytometry could\nquantitatively clarify this aspect.\n3) Despite an interaction between the N-terminal tail and GOLPH3, the TMEM251 2-7A mutant, expected to lack GOLPH3\ninteraction, still localizes to the Golgi. The authors propose that additional regions beyond the N-terminal tail are responsible\nfor retaining TMEM251 in the Golgi. However, the TMEM251 2-7A mutant fails to restore GNPT levels in TMEM251 KO cells\ndespite its Golgi localization. The authors should discuss this discrepancy or address it with new experiments; perhaps the\nTMEM251 2-7A mutant is present in the Golgi but not in its precise subcellular location.\n4) The authors suggest that there is a fraction of GNPT /TMEM251 which undergoes recycling back to the Golgi through the\nretromer machinery. They support this with findings that VPS35 KO results in TMEM251 and GNPT appearing in lysosomal\npuncta, where they degrade due to defective retromer function. However, a discrepancy arises in VPS35 KO, where\nTMEM251 remains at lysosomes but is not degraded, while GNPT is degraded. The authors should discuss this\ndiscrepancy or address it with new experiments. This might be due to the loss of proteases that degrade TMEM251 but not\nthose that target GNPT.\n5) The authors hypothesize that the TMEM251 cytosolic C-terminal tail, especially residues 93-96, might interact with the\nretromer for recycling back from endo-lysosomes to the Golgi, although co-immunoprecipitation failed to detect such\ninteraction. Investigating the localization of the 93-96A mutant in lysosomes could further corroborate this hypothesis.\n6) A topological issue arises from proposing the TMEM251-GNPT complex being recycled by GOLPH3 from trans Golgi to\ncis in Fig9. GOLPH3 is indeed localized at the trans-Golgi network (TGN). The authors could introduce the 3 steps model in\nthe figure 9, which is more coherent with data they are showing.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "The manuscript by Yang, et al. Kornfeld and Li describe some details on how the recently\nidentified TMEM251- also termed TMEM251 controls the GlyNAc-1-phosphotransferase\n(GNPT) which is required for mannose 6 phosphorylation of many lysosomal hydrolases.\nTMEM251/TMEM251 was discovered and characterized in two recent publications in\nScience (PMID: 36074821, 36074822) and NCOMM (PMID: 36096887). In the current\nstudy some confirmatory experiments are presented illustrating the essential role of\nTMEM251/TMEM251 for the M6PR-dependent delivery of acid hydrolases to the\nlysosomal compartment. It is confirmed (as outlined in detail below) that a lack of\nTMEM251/TMEM251 causes GNPT to be localized to lysosomes where is gets degraded\nmost likely by residual active lysosomal proteases. Topology studies of\nTMEM251/TMEM251 suggest that both polypeptide termini are facing the cytosol. Two\nbinding partners of TMEM251/TMEM251 were characterized, Golph3 and retromer, that\nmay be contributing to Golgi localization of TMEM251/TMEM251.\nAs such the study appears too preliminary and is in large parts not presenting novel\ninformation. Especially in the first part (Fig 1-4; results sections (lines 107 to 264) and\nstatements in the introduction (lines 97-99)) of the study there is a general lack of\nconceptual advance. In its current version – with the focus in topology determination,\nalanine scan and binding partner description- I would see the manuscript much better\nsuited for a specialized biochemical journal. The analysis of the TMEM251/TMEM251\ntopology, the reported alanine scan, potential interaction with GOLPH3 and retromer are\nnew. However, it remains open what are the exact and specific roles of the newly\ndescribed binding partners of TMEM251/TMEM251.\nWe must respectfully disagree with this reviewer on the novelty and conceptual advance.\nLYSET/TMEM251 was reported in 2022, by three independent groups (Pechincha et al.,\n2022a; Richards et al., 2022; Zhang et al., 2022). All three agreed that TMEM251 plays a\ncritical role in the M6P pathway by regulating GNPT, but the underlying mechanism is\nunder debate. In addition, there were inconsistencies in the data between the two Science\npapers (Pechincha et al., 2022a; Richards et al., 2022). It is imperative that this\ncontroversy be resolved before the field can move forward, which is what the first part of\nour study has accomplished. More importantly, our paper reveals that both GOLPH3 and\nretromer are crucial to the TMEM251 Golgi localization, serving as the underlying\nmechanism for maintaining GNPT at the Golgi, which is a novel and mechanistic finding.\nExamples for a lack of novelty:\nin order to strengthen the manuscript and contribute good science to the field, Reviewer\n1’s comments make it difficult to do so. The reviewer has overlooked the careful work we\nhave invested in resolving the current controversy and elucidating the detailed\nmechanism of TMEM251-GNPTαβ regulation (Fig. 1-4). Additionally, the amount of work\nsuggested by Reviewer 1 for the subsequent figures (Fig. 5-8) is unfeasible and would\nnot provide additional information to this study. Nevertheless, we have made a huge effort,\nspending more than a year, to address their concerns and provide valid reasons refuting\ntheir claims as outlined below.\no Loss of GNPTAB (Figs 2 and S2) was reported before (both Science papers)\nIndeed, both Science papers showed lysosomal degradation of GNPTαβ in the absence\nof TMEM251, but their model for how GNPTαβ degrades is not entirely correct.\nSpecifically, they claimed that GNPTαβ processing at the Golgi is unaffected by pulse-\nchase experiments. After processing, only the mature subunits (both α and β) will\nmislocalize to the lysosome and degrade.\nIn contrast, by pulse-chase experiment, we showed that the processing efficiency of\nGNPTαβ is low in TMEM251 KO cells (Fig. 3A, now Fig. 2A), and both processed and\nunprocessed GNPTαβ are mislocalized to the lysosome for degradation (Fig. 4D-E, now\nFig. 3D-E). Therefore, the current manuscripts highlight that both the processing and\nlocalization of GNPTαβ are affected in TMEM251 KO cells.\no Interaction of TMEM251 and GNPT was demonstrated before (both Science papers\nand NCOMM paper)\nIndeed, all three groups showed this interaction before; thus, we did not show the\ninteraction again in the previous submission. Therefore, we believe Reviewer 1’s\ncomment is unfounded.\no Mislocalization and lysosomal degradation of GNPT in TMEM251 KO cells was reported\nbefore\nThis is a repetitive comment and has been addressed above.\no Impaired M6P tagging (Fig. 1E) in TMEM251 or GNPTAB KO was demonstrated in both\nScience papers before.\nWe have moved it to Supplementary Figure 1A. Please also see the following response\n2\no Hypersecretion of proforms of cathepsin and lack of mature forms intracellularly was\nreported before in TMEM251 KO cells (Fig. S1B, C), accumulation of LC3 was previously\nreported (in the other two studies but also already by this group)\nIndeed, Fig. 1E and S1B-C have been shown before. Since the purpose of Fig. 1 was to\nresolve the controversy related to TMEM251, it was necessary to first verify some of the\nkey results from previous studies. However, we must emphasize that Fig. 1 was not a\nsimple repeat of published results. Using biochemistry, we measured the enzymatic\nactivities of PTase as well as the direct M6P phosphorylation of several representative\nlysosomal enzymes (Previous Fig. 1B-D, and S1A), which are new and independently\nverify the importance of TMEM251. In addition, we included the phenotype of TMEM251\nGNPTAB double KO, which was also new data. The purpose of Fig. 1 was to familiarize\nthe reader with GNPTαβ and TMEM251’s role in the M6P pathway and show that, at\nphysiologically relevant levels, both proteins are required. In summary, the majority of Fig.\n1 presents unpublished data, and we disagree with Reviewer 1’s conclusion that Fig. 1\nlacks novelty. However, to satisfy the reviewer, we have removed most of Fig. 1 and\nmoved Fig. 1E to the supplementary (now Fig. S1A).\nConcerns:\nFigure 1 represents in part already knowledge presented earlier and relies on over-\nexpression in HEK cells since apparently GNPT ptase activity was too low to be\nmeasured. Why were no experiments designed in a more suitable cell line allowing to\nendogenously monitor the consequences of a loss of TMEM251/TMEM251 or/and\nGNPT? The conclusion from the presented experiment that both TMEM251/TMEM251\nand GNPT are crucial for the M6P pathway are not new.\nWe have addressed Reviewer 1’s concerns about the apparent lack of novelty in Fig. 1\nabove.\nReviewer 1 misconstrued the findings of Fig. 1. We highlight that TMEM251 is required\nfor GNPT activity at closer to endogenous levels (~0.4 µg cDNA), but that overexpression\n(0.8-2 µg cDNA) can largely bypass this requirement. By using overexpression, we could\ncontrol the amount of GNPT/TMEM251 expressed in the cell and thus show the dose-\ndependent increase in activity that occurs. No other labs, including ours, have shown this\nbefore.\nWe have a paper in press in STAR Protocols (Doray et al., 2025) on how to measure the\nendogenous activity of GNPT in SKMEL30 cells. There, we show that GNPT activity is\nreduced in both GNPTAB KO and TMEM251 KO cell lines. Additionally, we demonstrate\nthat the endogenous activity of GNPT in SKMEL30 cells is approximately eight times\nhigher compared to HEK293T cells.\nachieved by vesicular recycling, but evidence is merely indirect (COP, Golph3, Retromer\ninteractions).\nWe completely disagree with this statement. Our conclusion regarding vesicular recycling\nis supported by multiple experiments, and the evidence we provided is strong and\ncompelling.\n- We showed that the N-terminal tail of TMEM251 contains sufficient information to\nmaintain this construct in the Golgi (Fig. 7A-B, now Fig. 6A-C) using a well-\nestablished SITM-GFP fusion strategy in the field, as acknowledged by this reviewer\nin a later comment (Liu et al., 2018; Welch et al., 2021).\n- We showed the N-terminal tail of TMEM251 is maintained in the Golgi in a\nGOLPH3-dependent manner using immunofluorescent imaging (Fig. 7E, now Fig.\nbinding assay (Fig. 7D, now Fig. 6G)\n- We demonstrated that the Retromer complex is important for maintaining\nTMEM251 and GNPTαβ protein levels in the Golgi using western blot (Fig. 7A-B),\nlysosome purification (Fig. 7C), and immunofluorescent imaging (Fig. 8C, now Fig.\n7D-E).\nHowever, to satisfy the reviewer, we have since provided additional evidence to further\nsupport the vesicular recycling of TMEM251.\n- We determined the critical residues (F4XXR7) in the TMEM251 N-terminal for\nGOLPH3-mediated Golgi retention (Fig. 6D-F, Fig. S4A-E).\n- We provided further evidence that TMEM251 is maintained in the Golgi via\nGOLPH3 by investigating the colocalization between the Golgi and TMEM251/\nGNPTαβ in U2OS and SKMEL30 cell lines lacking GOLPH3 (Fig. S5A-D).\n- We showed that lysosome enzyme maturation is affected in HeLa GOLPH3 KD\ncells (Fig. S5E-F)\n- Using another well-established IL-2Rα chimera strategy, we showed that the C-\nterminal of TMEM251 contains enough information to confer Golgi localization and\nthat this localization is dependent on VPS35 (Fig. 8A-C).\n- Finally, we narrowed down the likely binding sequence of the retromer in the C-\nterminal of TMEM251 (V97XY99---V108IC110) (Fig. 8D-F)\nIn summary, our vesicular recycling model provides a well-thought-out explanation for\nwhy TMEM251 is necessary to maintain GNPT in the Golgi. We demonstrate that, unlike\nwhat was claimed by Pechincha et al., TMEM251 does more than passively shield the\ncharged residue in the transmembrane region of the GNPT enzyme.\nincluded to (i) confirm TMEM251 KO and (ii) be able to judge whether GNPT expression\nlevels correlate with detected activity; this similarly applies to Fig. 1C; in this case,\nhowever, data in Fig. 1 D appear to be from a similar experimental setup; it would be good\nto have panels 1C and 1D from one experiment, but they should then be included in one\njoint subpanel; this similarly applies to panels 3B and 3C; control immunoblots are also\nmissing for Fig. S3 and S4 - this actually appears to be a more general issue; Ptase\nactivity\nassays should be supplemented with immunoblot panels showing KO and overexpression controls.\nTo improve the flow of the paper we have removed or redistributed most of the data from\nFig. 1B and Fig. S3.\nBoth Fig. 1C-D (Previous Figures 1C, D, see below), as well as Fig. 3B-C (now Fig. 2C-\nD in the revised manuscript) were from the same experiment. We have included a\nsentence in the figure legends of Fig. 3B-C (now Fig. 2C-D) to make this more obvious.\nThank you!\nThe activity data from Fig. 2B is from three independent transfections (i.e., biological\nreplicates), while the data from Fig. S2A-B is from two independent transfections.\nPrevious Figure 1C, D – TMEM 251 is essential for the Ptase activity of GNPTαβ at\nphysiological levels, but a high-level expression of GNPTαβ can overcome the\nrequirement. (C) In TMEM251 KO cells, the enzyme activities of GNPTαβ and β\nHexosaminidase can be restored by either gradually increasing the transfected GNPTαβ cDNA\nor co-transfecting TMEM251. Values shown are the average of two assays from two\nindependent transfections. (D) Western blot showing the expression levels of GNPTαβ and its\nprocessing efficiency under the same experimental setup as (C).\nThe authors observed that high-level ectopic expression of GNPTAB leads to substantial\nPtase activity also in TMEM251 KO cells (Fig. 1). What could be the underlying\n5\nmechanism? Is the retention mechanism the authors described before (direct\ninteraction of GNPTAB with COPI) sufficient at such high GNPTAB levels? Or is overexpressed\nGNPTAB still lost from the Golgi due to absence of TMEM251 and simply constantly\nreplenished due to high overexpression reaching sufficient steady state Golgi levels?\nThis should be addressed experimentally.\nThis is an excellent question, but it was already answered in Fig. 7F (See Previous Figure\n7F below) and lines 360-373 of our initial submission. In that figure, we showed that\noverexpressed WT GNPTαβ has a high PTase activity (1997 pmol/hr/mg protein) in\nTMEM251 KO cells. In contrast, the overexpressed K4Q mutant, which nearly abolishes\nthe interaction between GNPT and COPI, had very little PTase activity (164 pmol/hr/mg\nprotein, a 92% reduction). This result indicated that overexpressed GNPTαβ still depends\non its interaction with COPI to be maintained in the Golgi. Importantly, when TMEM251 is\npresent, we observed stimulation of PTase activity even for K4Q mutant (716 pmol/hr/mg\nprotein, 4.4-fold, Previous Fig. 7F). Our data indicated that COPI and TMEM251 work\nindependently and synergistically to maintain GNPT at the Golgi. For better cohesion,\nwe have since removed Fig. 7F, as well as most of Fig. 1.\nPrevious Figure 7F –Comparing PTase activities between WT GNPTαβ and K4Q/ S15Y\nmutants, with or without TMEM251. Values shown are the average of two assays from two\nindependent transfections.\nAlso, can overexpression of GNPTAB rescue the TMEM251 phenotype (e.g. cathepsin\nmaturation defects)?\nYes, we showed in Fig. 1C (See Previous Figure 1C above) that overexpression of\nGNPTαβ restores β-Hex activity in a dose-dependent manner. In addition, the PTase\n6\nactivity is also increasing, bypassing the requirement of TMEM251. As mentioned before,\nwe have removed the majority of the original Fig. 1.\nThe lack of novelty holds - in large part – also true for the reported interdependent protein\nstability of TMEM251 and GNPT and the observation that TMEM251 is required for the\nprocessing efficiency and therefore enzymatic activity of GNPTab.\nThe conclusions and interpretations of the data in Fig.2/3 have also been reported before.\nAs we emphasized above, one important aim of this paper was to resolve the controversy.\nThe major argument was whether the GNPTαβ cleavage (or processing) was affected by\nTMEM251 deletion. In the previous NC paper, we proposed that GNPTαβ processing was\ndefective without TMEM251. However, the other two Science papers stated that the\nprocessing was normal, and after processing, the cleaved subunits would mislocalize to\nthe lysosome and degrade. Using a pulse chase experiment, they observed no difference\nin GNPTαβ processing after knocking out TMEM251 (Pechincha et al., 2022a).\nTo resolve the controversy, it is necessary to re-examine the processing and localization\nof endogenous GNPTαβ again, which is why we generated Fig. 2 (now Fig. 1). As one\ncan tell, the processed β- (Fig. 2B, now Fig. 1A) and α-subunits (Fig. 2C-D, now Fig. 1F)\nwere largely abolished, supporting that the processing might be defective. Interestingly,\nwe did not observe the mislocalization of GNPTαβ by immunostaining. Instead, we\nobserved only a reduction in endogenous GNPTαβ at the Golgi (Fig. 2E, now Fig. 1H).\nThis is very different from what was reported by Richards et al. (Fig. 4A and S14G of\ntheir paper) and similar to the results obtained by Pechincha et al. (Fig. 6F of their paper).\nAgain, when there is a controversy in the literature, it is necessary to carefully repeat and\nreport the findings, instead of stating “it is not novel.” In an era when more than 50% of\nbiomedical research has reproducibility issues (Baker, 2016; Errington et al., 2021), we\nsincerely hope this reviewer and the editor do not punish careful reevaluation of\ncontroversial literature.\nWe also performed a pulse-chase experiment to directly examine the processing of newly\nsynthesized GNPTαβ. As shown in Fig. 3A (now Fig. 2A), the processing of GNPTαβ is\nmarkedly reduced after knocking out TMEM251. This is inconsistent with Fig. S10 in the\nScience paper published by Pechincha et al. The field deserves to know the\ninconsistency.\nFig. 3B-C (now Fig. 2C-D) is novel information. In Fig. 3B-3C (now Fig. 2C-D), we\ndemonstrated that overexpression of TMEM251 can stimulate the processing of GNPTαβ\nand enhance its PTase activity in a dose-dependent manner. Fig. 3D (now Fig. 2E)\ndemonstrated that even after the processing, TMEM251 is still required for PTase activity.\nWe are not aware that any group has done these sets of experiments before.\nmolecular weight fragment on the TMEM251KO cells. However, such a fragment cannot\nbe seen.\nThis statement is incorrect. A clear upper band is visible in Fig. 2B (now Fig. 1A) just\nabove the FL GNPT. Similarly, an upper band is also visible in Fig. 3A (now Fig. 2A) in\nthe TMEM251 KO cell line. For clarity, we have added an arrow indicating the upper band\nin Fig. 1A and Fig. 2A and mentioned it in the figure legends.\nLysosomal degradation of GNPTab (and the explanation why GNPTab is largely invisible\nin TMEM251 KO cells) has also been previously demonstrated, i.e. the experiments here\ndo not add much to the previous knowledge.\nAlthough both Science papers concluded that GNPTαβ mislocalizes to the lysosome for\ndegradation after knocking out TMEM251, we missed this important conclusion in our\ninitial NC paper. It was therefore necessary to verify this result independently (Fig. 4A,\nnow Fig. 3A).\nTherefore, because we never observed the mislocalization of GNPTαβ to the lysosome\nby immunostaining (Fig. 2E, now Fig. 1H vs. Fig. 4A of Richards et al.), we relied on the\nbiochemical purification of lysosomes to test the mislocalization (Fig. 3D). In fact, we\nrepeated the GNPTαβ immunostaining in HAP1 cells, as reported by Richards, but were\nunable to detect any GNPTαβ signal.\nAdditionally, both Science papers showed that BafA1, but not MG132, can stabilize\nGNPTαβ. However, it is essential to point out that, even though the two science papers\nare consistent in their conclusion, their BafA1 treatment results were contradictory and\nconfusing. Richards et al. showed that the BafA1-stabilized GNPTαβ is localized to the\nlysosome (Fig. S14F-G). In striking contrast, Pechincha et al. showed that BafA1-\nstabilized GNPTαβ is actually localized to the Golgi (Fig. S10E).\nIn Fig. 4D (now Fig. 3D) we observed a striking accumulation of both unprocessed and\nmature GNPTαβ subunits and their degradation products in the lysosome. It was\nassumed that only processed mature subunits would mislocalize. Our data in Fig. 4D-E\n(now Fig. 3D-E) clarifies that this is NOT the case because, in the presence of a Site-1-\nprotease inhibitor, unprocessed GNPTαβ still mislocalizes to the lysosome.\nLastly, our immunostaining of BafA1-treated cells in Fig. 4G (now Fig. 3G) demonstrated\nthat GNPTαβ is stabilized at the Golgi, supporting the observation by Pechincha et al.\nour previous Nature Comm. and the two Science papers but also clarifies the discrepancy\nbetween the two Science papers.\nAs an actual first new part of this study - in terms of a gain in knowledge - the\ndetermination of the discussed topology of TMEM251/TMEM251 is presented. With state-\nof-the-art technology and artificial introduction of a new glycosylation site (Fig.5C) and\nproteinase K protection assays (Fig.5E) it could be revealed that TMEM251 contains two\ntransmembrane helices with both termini facing the cytosol. Studying 31 alanine mutants\nof TMEM251 and rescue of cathepsin D maturation in TMEM251 KO cells revealed ten\nmutants with an apparent loss of TMEM251 function. The two transmembrane regions\nand amino acids close to them appear especially critical for TMEM251 function.\nWe thank Reviewer #1 for the positive comments.\nHow the identified regions modulate GNPT binding and TMEM251 functions remain -\nhowever- unsolved.\nThis is true; however, determining the membrane topology and critical regions within\nTMEM251 are important first steps. Figuring out the molecular basis for all 10 critical\nregions will take years to complete and is beyond the scope of this paper. In our current\nmanuscript, we focus on the molecular basis for maintaining GNPTαβ in the Golgi.\nThis alanine scan is a comprehensive approach to identify sequences critical to\nTMEM251 function, but requires further controls. In Figure 6A abundance of mature\ncathepsin D is used as only (indirect!) readout for assessing the capacity of TMEM251\nvariants to rescue the TMEM251 KO phenotype in 293T cells. TMEM251 mutant\nexpression levels need to be shown (and possibly be included in the quantification) here\nalong with cathepsin D and actin levels, as it obviously matters a) whether TMEM251\nmutants are expressed at all (an unstable TMEM251 mutant could be informative re:\ncritical structural motifs) and b) whether their potency to rescue the KO phenotype is really\ndifferent and not merely due to different expression levels.\nWe thank Reviewer #1 for these thoughtful suggestions. The goal of the alanine scanning\nexperiment was to identify functionally important regions of TMEM251, using CTSD\nmaturation as an indirect readout of TMEM251 activity. Unfortunately, due to the lack of\na reliable TMEM251 antibody, we are currently unable to determine TMEM251 protein\nexpression levels as suggested. Unfortunately, the new batch of Sigma TMEM251\nantibody (HPA048559) stopped working.\nA selection of mutants found to display impaired TMEM251 function was subsequently\ntested in SKMEL30 cells (with notable differences in expression among the mutants!),\nbut using a different readout (ability to rescue GNPTAB levels). Readouts in both cell lines\n9\nshould be the same (i.e. also GNPTAB levels need to be shown in 293T cells and cathepsin D\nlevels in SKMEL30 cells) and SKMEL30 data should be similarly quantified to make any robust and cell line-independent\nconclusions. This is in particular required as the authors observe marked differences for e.g. 67-71A in both systems. In this\nparticular case, it would also be interesting if localization differs between 293T and SKMEL30 cells.\nWe agree that, in theory, measuring GNPTαβ levels in HEK293T cells would provide a\nmore direct readout. However, this approach is technically challenging given the number\nof mutants analyzed. Each sample requires at least one 10-cm dish for total membrane\nisolation, and performing this for all 31 cell lines with a minimum of three biological\nreplicates would require at least 93 membrane preparations, which is not feasible.\nInstead, we utilized the SKMEL30 cell line, which expresses higher endogenous levels of\nboth TMEM251 and GNPTαβ, to focus on a subset of critical mutants for further validation.\nImportantly, the results from Fig. 6A (now Fig. 5A) and Fig. 6C (now Fig. 5B) are largely\nconsistent: mutants that showed impaired CTSD maturation in Fig. 5A also exhibited\nreduced GNPTαβ levels in Fig. 5B, with the exception of 67–71A.\nIn conclusion, we respectfully argue that performing the full experimental set proposed by\nReviewer 1 (i.e., two independent readouts across two cell lines) would not yield\nadditional mechanistic insights beyond what is already presented. We believe our efforts\nare better directed toward strengthening other aspects of the study.\nIn their study, the authors exclusively use TMEM251 isoform 2 (i.e. a short isoform with\nonly 7 cytoplasmic amino acids preceding TMD1 (according to topology model 2)). The\nauthors only briefly comment on this – “the more common short isoform of TMEM251”\n(line 301). It is indeed true that most cell lines appear to predominantly express this\nisoform at the endogenous level – but the SKMEL30 cells used quite extensively in this\nstudy are a notable exception that have a robust/dominant endogenous expression of\nisoform 1 (see Fig. 8A in this manuscript, but also data in Richards et al. and\nPechincha et al.). Also, both the long and the short isoform were previously shown to be fully capable\nof rescuing a TMEM251 deficiency phenotype, i.e. are fully functional. That in mind, the\nauthors should also check whether the critical motifs identified in their alanine scan of the\nshort isoform also impair function of the long isoform and in particular their experiments\nwith the Golgi localization reporter (Fig. 7) should be repeated with the longer N-terminus\nof the long TMEM251 isoform.\nAs acknowledged by Reviewer 1, we previously demonstrated in our previous Nature\nCommunications paper that the short isoform of TMEM251 is functionally equivalent to\nthe long isoform. Specifically, we showed that the short isoform alone is sufficient to\nrescue the TMEM251 knockout phenotype. Repeating all experiments with the long\n10\nisoform would not only require substantial time and resources, but more importantly,\nwould not yield additional scientific insight.\nWe would also like to respectfully point out a contradiction in Reviewer 1’s attitude. While\nthe reviewer criticizes Figures 1–4 as lacking novelty, despite our careful efforts to resolve\nambiguities in the literature, they also recommend repeating numerous experiments using\nthe long isoform, which we believe would not enhance the mechanistic understanding of\nTMEM251 function. Given the demonstrated functional redundancy between the\nisoforms, we feel that repeating the entire dataset with the long isoform is unnecessary.\nThe N-terminal region of TMEM251 contained sufficient information to retain a GFP-\nplasma membrane reporter in the Golgi. Since the N-terminal sequence met the criteria\nfor binding of a COP1 adaptor, Golph3 its interaction with the N-terminus of\nTMEM251 was demonstrated in a GST pull down approach. However, direct binding of\nGolph3 in the streptavidin-binding assay to the N-terminus of TMEM251 could not be shown.\nImportantly, no experiments are presented to show the importance of Golph3 (e.g. in KO\ncells) for lysosomal sorting and GNPT stability and function.\nGFP reporter constructs like the one used in Figure 7 are well established tools to study\nthe determinants of Golgi localization of type II membrane proteins. Indeed, the\nobservations made here suggest that the first seven amino acids of TMEM251 could\ndictate a Golgi localization and potentially also an interaction with GOLPH3. However,\nthis system is highly artificial for many reasons and the findings from these experiments\nshould be taken with a grain of salt. TMEM251 is not a type II membrane protein and thus\ncannot be simply reduced to only its first 7 amino acids - in particular when the alanine\nscan data show that the 2-7A mutant still exhibits Golgi localization and instead mutation\nof other motifs impair Golgi localization. If this region is important for Golgi localization of\nTMEM251, shouldn’t this mutant be mislocalized/lost from the Golgi apparatus? N-termini\nof type II Golgi membrane proteins are frequently rather short and Arg-rich – the capacity\nof TMEM251 aa 2-7 to direct the reporter to the Golgi may thus simply a coincidence.\nAlso, these experiments need to be further substantiated by constructs with TMEM251’s\nlonger N-terminus and additional mutants as well as full-length TMEM251.\nThank you for the suggestions.\nAs requested by the reviewer, we examined the effect of GOLPH3 depletion in HeLa\ncells. Knockdown of GOLPH3 initially led to a reduction in lysosomal enzyme maturation\n(Fig. S5E–F). However, we observed a strong compensatory response over time, with\nGNPTαβ protein levels increasing after 14 days (see Supporting Figure 1 below).\nboth TMEM251-10xGCN4 and endogenous GNPTαβ (Fig. S5A–D). Notably, GOLPH3\nappears to be essential for SKMEL30 cell viability, as its knockdown led to a marked\ndecrease in cell survival.\nSupporting Figure 1. Western blots show that GNPTαβ levels initially decrease but subsequently\nincrease in GOLPH3 knockdown HeLa cells.\nRegarding Reviewer 1’s additional concerns:\nWe focused on the N-terminal region of TMEM251 because of its critical functional\nimportance, as highlighted by the R7W mutation (R45W in the long isoform), which is a\nknown disease-causing mutation in humans. Our findings indicate that the N-terminal tail\nmediates interaction with GOLPH3, which is essential for TMEM251 retention in the Golgi.\nTo further support the significance of the TMEM251 N-terminus, we now provide\nadditional evidence:\n The R7W mutation abolishes the interaction between TMEM251 and GOLPH3 in\nvitro (Supporting Fig. 2A).\n The R7W mutation disrupts Golgi localization of the 251N-SITM-GFP chimera\n(Supporting Fig. 2B).\n The R7W mutant undergoes faster degradation than the wild-type protein\n(Supporting Fig. 2C).\nThese findings reinforce the functional relevance of the TMEM251 N-terminal tail in Golgi\nlocalization and stability.\nthe interaction between the 251N(WT)-SITM-GFP and 251N(R7W)-SITM-GFP chimeras and GST-\nGOLPH3. (B) Immunostaining images and quantification showing the localization of SIN-SITM-\nGFP, 251N(WT)-SITM-GFP, and 251N(R7W)-SITM-GFP in WT HeLa cells. (C) A cycloheximide chase\nassay showing the degradation of WT and R7W TMEM251 over 2 hours. Scale bar: 10 µm.\nRegarding the observation that 2–7A and R7W mutants still localize to the Golgi in Fig.\n6B (now Fig. S3A), two factors may contribute to this result:\n1) Overexpression of TMEM251 mutants was required for localization experiments\ndue to the low sensitivity of the available TMEM251 antibody (i.e., HPA048559,\nwhich is now discontinued). As shown in Fig. 8C (now Fig. 7E–F), overexpressed\nTMEM251 can bypass the requirement for retromer to restore Golgi localization.\nAdditionally, despite being a disease-associated allele in humans, overexpressed\nR7W mutant can even complement TMEM251 knockout phenotypes (see\nSupporting Fig. 3 below), further indicating that overexpression may compensate\nfor functional defects.\n13\n2) The C-terminal region of TMEM251 also contributes to its Golgi localization via\nretromer-dependent recycling (Fig. 8A-C), which may partially compensate for\ndefects in the N-terminal domain.\nSupporting Figure 3. Western blots showing that overexpression of either TMEM251WT or\nTMEM251R7W in TMEM251 knockout cells can rescue CTSD maturation and restore degradation\nof lysosomal cargoes, including LC3-II and LAPTM4A.\nTo investigate the motif within the N-terminal tail of TMEM251 responsible for Golgi\nlocalization, we generated individual alanine substitutions at residues 2–7 in the short N-\nterminal tail of the 251N-SITM-GFP construct. This analysis identified F4XXR7 as a critical\nmotif required for GOLPH3-mediated Golgi localization (Fig. 6D–F).\nTo assess the functional significance of these residues, we performed rescue experiments\nin HEK293T TMEM251 knockout cells using the TMEM251-10xGCN construct, either\nwild-type or carrying F4A, R5A, or R7A mutations. Under leaky expression from a Tet-on\npromoter to approximate endogenous levels, F4A and R7A mutants failed to rescue\nTMEM251 knockout phenotypes, whereas R5A retained partial function (Fig. S5A–C).\nLastly, we evaluated the Golgi localization of these mutants in SKMEL30 cells. Upon\nmoderate induction with 100 ng/µl doxycycline for 18 hours, both F4A and R7A mutants\nexhibited reduced Golgi localization compared to WT and R5A constructs (Fig. S5D–E).\nVPS35 deletion (retromer member) did not affect TMEM251 expression levels but slightly\nreduced the level of GNPT as been reported earlier.\nThis statement is incorrect. Several members of our team have studied GNPT for\ndecades, and to our knowledge, there are no published reports demonstrating that GNPT\nlevels are reduced following VPS35 knockout.\nstructures remain unexplained. A fraction of TMEM251 could be found stabilized in\nlysosomes suggesting a retromer involvement in recycling of missorted TMEM251.\nThe imaging data depicting the mislocalization of TMEM251 and GNPTAB in retromer-\ndeficient cells needs to be assessed more quantitatively.\nAs suggested by the reviewer, we quantified the mislocalization of TMEM251 and\nGNPTαβ by calculating the Pearson correlation coefficient (Fig. 8C, now Fig. 7D–E).\nAdditionally, we found that the punctate signal observed near the Golgi in GNPTαβ-\ndeficient cells is a staining artifact resulting from non-specific binding by the antibodies\nused in this study (Fig. 1H).\nThere still appears to be a substantial portion of TMEM251 in the Golgi and also the\nsingle-dot Golgi staining of GNPTAB is not as evident as in Fig. 4.\nWe did not claim that VPS35 deletion phenocopies TMEM251 knockout. However, we did\nobserve a partial but significant reduction of TMEM251 from the Golgi, along with a\ncorresponding decrease in GNPTαβ signals in the Golgi (Fig. 7D-E).\nIn our proposed model (Fig. 9), COPI, GOLPH3, and the retromer complex all contribute\nto the proper Golgi localization of TMEM251 and GNPTαβ. Consistent with this model,\ndeletion of VPS35 would be expected to result in a partial mislocalization of both proteins.\nThe authors also write that “the TMEM251-GNPT complex may exit the Golgi and\nmislocalize to endo-lysosomes” (line 412/413), but co-staining of TMEM251 and GNPTAB\nin retromer-deficient cells would be required to substantiate that indeed the TMEM251-\nGNPT complex is mislocalized (and not its two components independently).\nBoth antibodies were raised in rabbits, which prevents us from performing any co-\nimmunostaining experiments. However, in purified lysosomes from WT cells, we observed\na small but significant portion of both GNPTαβ and TMEM251 (Fig. 3D-E, 7C), supporting\nour statement.\nAlso, the authors do see a destabilization/loss of GNPTAB under these conditions, but\nnot of TMEM251. Is GNPTAB more prone to lysosomal degradation than TMEM251?\nYes, we agree with this interpretation. Uncleaved GNPTαβ is a 143 kDa protein with the\nmajority of its mass located in the lumen, making it susceptible to degradation by\nlysosomal proteases when mislocalized. In contrast, TMEM251 has only a short luminal\n15\ndomain of approximately 40 amino acids, which likely limits its exposure to luminal\nproteolysis.\nQuantification and statistical evaluation of multiple independent experiments and\nbiological replicates are missing for most of the (immunoblot and microscopy-based)\nexperiments.\nWe have quantified and performed statistical analysis as requested (see Figs. 1, 2, 5, 6,\n7, 8, S3, S4, and S5).\nMinor comments:\nDifferent amounts of plasmid DNA (encoding in particular GNPTAB or TMEM251) were\ntransfected transiently for numerous data panels to emphasize dose-dependent effects\n(e.g. of TMEM251 on GNPTAB stability). The figure legends should state that these\nabsolute DNA amounts given in the figure panels apply to six-well culture dishes.\nThis information was stated in the Materials and Methods section of the manuscript.\nFigure legends are rather cryptic in general and lack important information here and there\n(e.g. in Fig. 4E and 4G inhibitor concentrations should be provided in the figure panels or\nthe figure legends)\nWe have added additional information to the figure legends, including concentrations and\ntreatment time for the inhibitors used.\nThe authors write that all genome editing cell clones/lines (KO and KI) were\nvalidated/confirmed by Sanger sequencing. These data should be made available in the\nsupplementary figures\nWe verified our cells using either Western blot or sequencing (Fig. S6). We also added\nthe cell line information in the Materials and Methods section.\nFig 2B: From the context it can be assumed that GNPTαβ and GNPTβ were detected with\nan anti-HA antibody in these KI cells, but that needs to be stated in the legend\nThis was already indicated in the figure panel, but we also updated the legend to make it\nmore straightforward.\nSuppl. Fig. S2B – TMEM251 panel, between lane 3 and 4: Cutting/Splicing? Or why does\ncells (cf to Fig.1E)\nThe TMEM251 overexpression lysates were diluted to maintain them within the sensitivity\nrange of the TMEM251 antibody. We have added an explanation in the figure legend (now\nFig. 1D).\nContradictory statements: line 158 (section heading; “Tmem251 is required for stability of\nGNPTαβ) vs. line 189 to 191 (“In the absence of TMEM251, […] the uncleaved GNPTαβ\nlevel remains relatively unchanged”); in particular the latter statement does not appear to\nadequately reflect observations in e.g. Fig. 2C and Fig. S2B)\nThank you for pointing this out. We have revised the wording and quantified both the total\nGNPTαβ and α subunit levels to provide a more numerical description (now Fig. 1D-G).\nLine 244: WT cells should be TMEM251 WT cells (lanes 4 and 5 are GNPT KI cells, so\nno parental cells)\nTMEM251 WT cells are a confusing nomenclature. We believe that leaving it as WT cells\nis sufficient to make the point. For further clarity, lane numbers are indicated in the text.\nLines 260 to 262 – reference to Fig. 4G missing in main text\nThank you! We included it in the text of this submission (now Fig. 3G).\nFig. 2E: Dot-like Golgi (-proximal) localization of GNPTAB upon TMEM251 deletion in\nSKMEL30 cells: This observation (in SKMEL30 cells) stands in contrast to observations\nby Richards et al. showing dot-like, but not really Golgi-proximal GNPTAB staining in\nTMEM251 KO 293FT cells. What is the nature of this unique dot-like structures? What\nhappens if cells are treated with protease inhibitors, does GNPTAB then also\naccumulate in lysosomal structures (in light of the lysosomal enrichment data\npresented)?\nWe acknowledge the differences between our imaging data and that reported by\nRichards et al. These discrepancies may be due, in part, to differences in the epitopes\nrecognized by the GNPTα antibodies used in the two studies. In our hands, we were\nunable to detect a lysosomal pool of GNPTαβ in TMEM251 knockout cells by\nimmunostaining (Fig. 1H). To confirm GNPTαβ mislocalization to the lysosome, we\nfound it necessary to perform lysosome purification, which allowed us to detect this\npopulation biochemically (Fig. 3D-E).\nlysosomal degradation, but by inhibiting its exit from the Golgi (Fig. 3G). This\nmechanism was also independently observed by Pechincha et al. We are unsure how\nRichards et al. observed stabilization of GNPTαβ at the lysosome following BafA1\ntreatment, as it is inconsistent with both our data and that of others.\nRegarding the punctate \"dot\" signal, we concluded that it is an artifact generated by the\nGNPTαβ antibody, as it persists even in GNPTAB knockout cells (Fig. 1H).\nCould GNPTAB mutants (K4Q, S14Y) shown previously to impair binding of GNPTAB to\nCOPI also impair binding to TMEM251? (previously looked at?)\nThis question was originally addressed in Fig. 7F (now removed for improved flow, see\nPrevious Fig. 7F above). The K4Q and S14Y mutants clearly retain partial binding to\nTMEM251. This is supported by the observation that co-overexpression of TMEM251 with\nthese mutants enhances phosphotransferase activity, suggesting a residual physical\ninteraction. Although we have removed Fig. 7F to streamline the manuscript, the\nunderlying data support the conclusion that these mutants are still capable of interacting\nwith TMEM251 to some extent.\nLine 384: “Deletion of VPS35 resulted in a significant reduction in endogenous GNPTab\nprotein levels in both cell lines” – this statement is not overtly supported by the data\npresented. In SKMEL30 there is only a modest (at best) reduction and this would need to\nbe properly quantified in light of this statement; the 293T data appear to be supporting\nthis statement better, but quantification would be required here as well to exclude\ntransfer/ECL issues\nWe have quantified the reduction of GNPTαβ in the VPS35 KO cell lines and made\ncorresponding changes in the text (see Fig. 7A-B).\nFig. 5C – The E51N mutant generated to test the topology model appears to be not\nglycosylated quantitatively. Is this an issue with heavy overexpression or could this\nindicate the simultaneous presence of topologically different variants of TMEM251?\nThis may be due to overexpression of the E51N mutant. Excessive production of the\nprotein could saturate the glycosylation machinery, resulting in a subset of the protein\nremaining unglycosylated.\nWhen transiently transfected, low-level overexpression of GNPTAB is insufficient to\ncompensate for the lack of TMEM251 and a drastic difference in Ptase activity is observed\n18\nbetween WT and TMEM251 KO cells (Fig. 1B, lane 5 vs 6). When stably expressing low-\nlevel GNPTAB-V5 variants (Fig. 3D), no notable difference in GNPTAB-V5 levels is\nevident between TMEM51 WT and TMEM251 KO cells (despite the latter clearly having\na phenotype). How can that be explained?\nTMEM251 regulates GNPTαβ at two levels: cleavage and enzymatic activity. In Fig. 3D\n(now Fig. 2E), we use a modified GNPTαβ construct (GNPTαβ-Furin-3V5) engineered to\nbe cleaved by Furin instead of S1P, as well as a second construct (GNPTαβ-Δ820–928-\n3V5) lacking the autoinhibitory domain. Both constructs bypass the requirement for S1P-\nmediated cleavage and, as expected, their expression levels are largely unaffected in\nTMEM251 knockout cells. However, despite successful cleavage, these GNPTαβ variants\nremain non-functional specifically in the absence of TMEM251. This suggests that\nTMEM251 exerts an additional layer of regulation by directly modulating GNPTαβ’s\nphosphorylation enzyme activity, independent of its role in cleavage.\nReviewer 2\nIn this research, the authors conducted a thorough analysis of the role of\nTMEM251/TMEM251 protein in the processing, stability, and localization of GlcNAc-1-\nphosphotransferase 35 (GNPT), a key enzyme in the M6P pathway. TMEM251 is\nessential for human health and is a potential drug target for treating cancers and viral\ninfections. While two studies in 2022 linked TMEM251 to the M6P pathway, its specific\nrole remained unclear. The authors used gene editing, microscopy, mutagenesis analysis,\nand in vitro methods to show that TMEM251 is crucial for the processing, activity, and\nmaintenance of GNPT in the Golgi apparatus. They also conducted biochemical analysis\nand alanine mutagenesis scanning to determine the membrane topology of TMEM251\nand identified critical residues in its cytosolic region that affect protein function and\nlocalization. The findings confirm the importance of TMEM251 and GNPT in the M6P\npathway at the physiological level and support the hypothesis that GNPT’s steady\nlocalization in the Golgi is maintained through vesicular recycling mediated by TMEM251,\nCOPI, Golph3, and retromer. This work is suitable for publication in this journal, although\nseveral critical questions remain unanswered. Addressing these questions could further\nelevate the significance of the findings: What is the stoichiometry of the GNPT-TMEM251\ncomplex? Is this a stable complex, or is it regulated temporally and spatially? Are\nother Golgi enzymes regulated by TMEM251? What is the molecular mechanism by which\nTMEM251 influences GNPT processing?\nSpecific comments and suggestions for improvement:\nmight lead to secondary defects, compensatory responses, and other artifacts.\nImplementing acute protein downregulation techniques and focusing on endogenous\nproteins—perhaps tagged using gene-editing methods—would significantly enhance the\nrobustness of this study.\nThank you for these thoughtful suggestions. In our experience, CRISPR-mediated gene\nknockout generally yields cleaner and more consistent results than protein knockdown\napproaches. To ensure reproducibility, we routinely analyze two or more single-cell\nclones and test across multiple cell lines. Additionally, rescue experiments are typically\nperformed to confirm the specificity of the observed phenotypes.\nIn the original Fig. 1 (now removed), we used GNPTαβ overexpression to demonstrate\nthat the requirement for TMEM251 could be bypassed, but only when GNPTαβ is\nexpressed at levels significantly above physiological conditions. This supported the idea\nthat TMEM251 is essential for GNPTαβ function under endogenous expression levels.\nWhile we have since removed and redistributed the data from Fig. 1 to improve\nmanuscript flow, the conclusions remain supported.\nTo focus on endogenous protein behavior, we used SKMEL30 cells, which express\nhigher natural levels of both GNPTαβ and TMEM251. In HEK293T cells, we employed a\nGNPTαβ-3xHA knock-in line that allows us to monitor endogenous levels of both full-\nlength GNPTαβ and its β subunit via HA antibody.\nA key limitation of this study is the lack of a reliable antibody for detecting endogenous\nTMEM251. Unfortunately, during the course of manuscript revision, the new batch of the\nTMEM251 antibody (HPA048559 from Sigma) lost sensitivity and could no longer detect\nthe protein. To address this, we developed a Tet-on TMEM251-10xGCN expression\nsystem, and used its leaky expression to approximate endogenous TMEM251 levels for\nour experiments.\nIn Figure 1E, it is crucial to display a complete GNPTAB blot to confirm the effect of\nTMEM251 knockout on the processing of the endogenous enzyme in HEK cells.\nAdditionally, clarification on the role and relevance of HRD1 in this context would be\nhelpful.\nFig. 1E is now presented as Fig. S1A. In this experiment, HRD1 was used as a loading\ncontrol for membrane isolation, and GNPTαβ protein levels were normalized to HRD1,\nas indicated in the figure legend.\nDue to the low endogenous expression of GNPTαβ in HEK293T cells, detection by\nimmunoblotting is challenging. To improve signal specificity, we cut the membrane at\n20\n~100 kDa to concentrate antibody binding on the appropriate region of the blot (now\nFig. S1A).\nTo directly address the reviewer’s concern, we used a GNPTαβ-3xHA knock-in HEK293T\ncell line (Fig. 2B, now Fig. 1A), which allowed us to more clearly monitor GNPTαβ\nprocessing in the context of TMEM251 knockout.\nTMEM251 KO cells showed mislocalization of GNPT, forming a \"single-dot-like structure\"\nat the Golgi rather than mislocalizing to lysosomes. It's crucial to investigate the nature of\nthis structure. Immuno-electron microscopy (immune-EM) could be a suitable further\nexploration method.\nWe now have evidence that the dot-like structure observed near the Golgi is likely due to\nnon-specific antibody binding, as similar puncta are still present in GNPTAB knockout\ncells (Fig. 1H).\nThe observation that in TMEM251 KO cells treated with\nBafA1, stabilized GNPT remains in the Golgi instead of moving to the lysosome is\nsignificant. The authors suggest that \"BafA1 stabilizes GNPT by impeding its Golgi exit\nrather than inhibiting lysosomal degradation.\" This novel finding warrants further\ninvestigation to understand the underlying mechanisms.\nThis is indeed an interesting observation that warrants further investigation. Importantly,\nPechincha et al. also reported that BafA1 can stabilize GNPTαβ at the Golgi (Pechincha\net al., 2022b). However, a detailed exploration of this mechanism is beyond the scope of\nthe current study.\nThe effects of GOLPH3 depletion (in the context of GOLPH3/GOLPH3L double KO) on\nthe M6P pathway and the localization of TMEM251 and GNPT should be explored. This\ncould provide insights into the regulatory mechanisms involving GOLPH3 in this pathway.\nThank you for this excellent suggestion. We have now included data examining the\neffects of GOLPH3 deletion or downregulation on the M6P pathway:\nGOLPH3 knockdown in HeLa cells: We knocked down GOLPH3 in HeLa cells and\nobserved a decrease in lysosomal enzyme maturation (Fig. S4E–F). However, over\ntime, we noted a compensatory response in which GNPTαβ expression was elevated\ncompared to wild-type (See supporting Figure 1 above). A similar compensatory effect\nwas also observed in U2OS cells (data not shown). These findings suggest that such\ncompensatory effects are not limited to CRISPR-mediated knockouts but can also occur\nin knockdown experiments.\nSKMEL30 cell line to assess endogenous GNPTαβ localization. GOLPH3 depletion\nresulted in reduced GNPTαβ localization to the Golgi (Fig. S5C–D). Notably, GOLPH3\nappears to be essential in SKMEL30 cells (i.e., knockdown severely impaired cell\nviability, and complete knockout was lethal).\nAs there is currently no reliable antibody for immunostaining endogenous TMEM251,\nwe tagged TMEM251 with a 10xGCN4 epitope and expressed it under the leaky\nexpression of a Tet-on promoter. In U2OS cells, we observed a clear reduction in Golgi\nlocalization of TMEM251-10xGCN following GOLPH3/3L knockout (Fig. S5A–B), further\nsupporting the role of GOLPH3 in TMEM251 retention.\nReviewer 3\nThe study by Yang, Doray and colleagues analyzes the role of TMEM251 in the activity\nof the GNPT enzyme which constitutes the fulcrum of the M6P pathway, and which is\nessential for the function of lysosomal enzymes. The most innovative part of the work\nconcerns the role of TMEM251 in the GNPT traffic mechanisms. This is a work of\nconsiderable interest which, however, in its current form presents some important gaps\nof both an interpretative and experimental nature. The work is very complex and, as\nsuch, intrinsically difficult to analyze, but it is also written in a poorly organized way so\nthat the logical connections between the parts are even more difficult to follow\nBelow we will try to analyze these weaknesses and where possible indicate how to\novercome them.\nMajor points:\nThe authors describe the effects of TMEM251 on the stability of the GNPT enzyme and\ngive evidence that TMEM251 is essential for the activating GNPT cleavage and is also\nessential for the stability of the cleaved portion of GNPT, in the sense that the levels of\nthis protein decrease in the absence of TMEM251. In this regard the authors note that\nGNPT is intrinsically unstable due to the composition of its transmembrane domains, but\nin the following experiments the authors demonstrate that in the absence of TMEM251\nthe uncleaved GNPT is degraded in lysosomes. This is probably the reason for the\ninstability of GNTP. The flow here is difficult to understand and the authors should logically\nconnect the two data sets.\nThank you for your comments. We have rewritten a large portion of our manuscript and\nremoved some of the figures to increase the logic flow. In the absence of TMEM251,\nlack of GNPTαβ stability in cells lacking TMEM251, GNPTαβ cleavage and subsequent\nactivity is reduced leading to the observed lysosomal defects.\nThe authors show that TMEM251 is necessary for GNPT activity even after cleavage has\noccurred ie, after the activation step has been carried out. This is attributed to the physical\ninteraction of TMEM251 with GNPT, which would form a stable complex which would\nhave the effect of stabilizing the active conformation of the enzyme.\nIn the next part of the work, however, the authors demonstrate that TMEM251 is essential\nto prevent lysosomal degradation of both the cleaved and uncleaved enzyme. From what\nwe understand, it is therefore probable that the property of TMEM251 to maintain the\nactivity of the cleaved enzyme is simply due to the fact that it localize GNPT at the Golgi\npreventing its lysosomal degradation. This possibility does not seem to be grasped by the\nauthors. It is an ambiguity that the authors must resolve either with textual changes and/or\nwith new experiments.\nThank you for pointing this out. As mentioned above, we have revised the text to improve\nclarity. We propose that TMEM251 functions to retain both the uncleaved and cleaved\nforms of GNPTαβ in the Golgi, thereby supporting proper S1P-mediated cleavage and\nensuring the enzymatic activity of the cleaved form. In the absence of TMEM251,\nuncleaved GNPTαβ may prematurely exit the Golgi before it can be processed by S1P,\nwhile the cleaved form is similarly mislocalized before it can perform its function.\nThe authors perform an elegant series of experiments that leads them to the conclusion\nthat TMEM251 interacts with Golph3 in the trans Golgi to maintain the localization of the\nTMEM251/GNPT complex in the cis Golgi and for the prevention of lysosomal\ndegradation of GNPT.\nA perplexing point here is the observation that mutants in the cytosolic tail of TMEM251\nthat abolish the binding between the tail and Golph3 do not change the localization of\nTMEM251 in the Golgi, in other words, that the interaction between TMEM251and Golph3\nis not essential for the retention of TMEM251 and GNPT in the Golgi. The authors suggest\nthat there may be additional regions beyond the cytosolic tail that maintain TMEM251 in\nthe Golgi. This is possible, but the finding raises concerns that should be addressed with\ntextual changes or new experiments.\nTo address this concern, we have revised the text for greater clarity and conducted\nadditional experiments (see below).\non TMEM251-10xGCN construct in U2OS cells. Under moderate induction, we observed\nthat Golgi localization of TMEM251-10xGCN was reduced in cells lacking GOLPH3/3L\ncompared to wild-type controls (Fig. S5A–B). Furthermore, we provide compelling\nevidence that GOLPH3 mediates this retention through the N-terminal F4XXR7 motif of\nTMEM251: mutation of these residues leads to partial loss of TMEM251 from the Golgi\n(Fig. 6 and Fig. S4).\nIn addition, depleting GOLPH3 in SKMEL30 cells resulted in a loss of endogenous\nGNPTαβ from the Golgi (Fig. S5C–D), further supporting GOLPH3’s role in stabilizing the\nGNPTαβ–TMEM251 complex at the Golgi.\nBased on our proposed model (Fig. 9), TMEM251 and GNPTαβ are retained at the Golgi\nthrough the combined action of COPI, GOLPH3, and the retromer complex. Therefore, it\nis not unexpected that only a portion of TMEM251 is lost from the Golgi upon GOLPH3\ndepletion, as other retention mechanisms remain partially active.\nThe authors show evidence that the retention of GNPT in the Golgi requires both the\nbinding of GNPT itself to COP1 via the motif previously identified by Doray and Kornfeld,\nand the binding between TMEM251 and Golph3, as shown in this study. Then, in the last\nparagraph, the authors propose the existence of a further mechanism for the retention of\nGNPT in the Golgi which would be based on the activity of the retromer, the complex that\ntransports proteins from the endo-lysomal system to the Golgi, which thus be responsible\nfor maintaining the localization and functionality of GNPT/ TMEM251. This last\nconclusion is consistent with the data, which however are simply based on depletion and\noverexpression experiments of a protein crucial for the function of the retromer, VPS35.\nBased on these collective evidence, the authors suggest that a triple recycling/retention\nmechanism is necessary for the maintenance of GNPT/ TMEM251 in the Golgi and\npresent a model in figure 9.\nThe model shows a single retrotransport step from the trans to the cis Golgi. This is\ndifficult to understand based on current knowledge and also inconsistent with the data\nshown in the manuscript and with the localization of the adaptor GOLPH3 within Golgi.\nThe overall model could be modified by proposing a mechanisms of Golgi retention in\nthree steps: first step of retrotransport from Golgi trans to medial cisternae due to the\nbinding between TMEM251 and Golph3 which would then be followed by a second\nretrograde step dependent on the binding between the cytosolic tail of GNPT and COP1.\nThe third step, mediated by the retromer, could recycle the fraction of GNPT / TMEM251\ncomplex that escaped from the first two recycling mechanisms, from the endo-lysosomes\nto the trans Golgi. Here, GOLPH3 first and COPI then could bring the complex back to\n24\nthe cis Golgi. Organized in this way the model could be coherent with the majority of the\ndata and highlight the novelty of the paper.\nThank you for the suggestion. We have revised the model to better reflect the current\ndata.\nOverall, we believe that the information brought by this study potentially represents a\nnotable progress in an area of research which is currently under strong development and\nis of great importance, namely, the mechanisms of selective retrotransport of Golgi\nenzymes by specialized adaptors, with regulatory effects on glycosylation. However, the\nauthors need to address the points we have raised with textual changes and new\nexperiments.\nThank you for your positive comments. We have addressed the minor concerns as\ndescribed below.\nMinor points:\n1) One hypothesis put forward by the authors suggests that TMEM251 may serve as a\nbridging factor, linking GNPT with the S1P protease necessary for GNPT activation. It\nwould be important to investigate the localization and levels of S1P in TMEM251\nknockout cells.\nS1P cleaves a range of substrates in the Golgi, including SREBP1, SREBP2, ATF6, and\nGNPTαβ. Therefore, its localization and stability in the Golgi are likely independent of\nTMEM251. To confirm this, we performed immunostaining using an S1P-3xHA construct\nto assess S1P localization in TMEM251 knockout cells (see Supporting Fig. 5 below).\nAs expected, S1P remained localized to the Golgi in the absence of TMEM251.\nGRASP55 in WT and TMEM251 KO cells. Scale bar: 10 µm.\n2) While GOLPH3 is suggested as a mechanism for retaining the TMEM251-GNPT\ncomplex at the Golgi, its involvement remains unproven in GOLPH3 knockdown (KD) or\nknockout (KO) cells. The authors establish that the N-terminal tail of TMEM251 contains\nsufficient information for GOLPH3-dependent Golgi localization. They should define the\nmotif. However, this is the sole evidence implicating GOLPH3 in TMEM251 retention at\nthe Golgi, and using flow cytometry could quantitatively clarify this aspect.\nWe thank the reviewer for these thoughtful suggestions.\nAs requested, we identified the N-terminal motif required for GOLPH3-dependent Golgi\nlocalization of TMEM251, specifically the F4XXR7 sequence. Using the TMEM251N-SI-\nGFP chimera, we introduced point mutations in the N-terminal tail to determine which\nresidues are essential for Golgi retention (Fig. 6D–F). Since the imaging results clearly\nindicated the critical residues, we did not pursue flow cytometry for further analysis.\nTo assess the functional importance of these residues, we introduced F4A, R5A, and R7A\nmutations into our Tet-on TMEM251-10xGCN system and evaluated their ability to rescue\nmature cathepsin D (mCTSD) levels (Fig. S4A–C) and restore Golgi localization (Fig.\nS4D–E).\nFurthermore, we provided additional evidence for GOLPH3-dependent recycling of\nTMEM251 by analyzing TMEM251-10xGCN localization in U2OS GOLPH3/3L double\nknockout (DKO) cells and endogenous GNPTαβ localization in SKMEL30 GOLPH3\nknockdown cells (Fig. S5A-D). In both cases, Golgi localization of TMEM251 and\ncells led to reduced lysosomal enzyme maturation (Fig. S5E–F). However, over time, we\nobserved a compensatory response, with GNPTαβ protein levels increasing (see\nSupporting Fig. 1).\n3) Despite an interaction between the N-terminal tail and GOLPH3, the TMEM251 2-7A\nmutant, expected to lack GOLPH3 interaction, still localizes to the Golgi. The authors\npropose that additional regions beyond the N-terminal tail are responsible for retaining\nTMEM251 in the Golgi. However, the TMEM251 2-7A mutant fails to restore GNPT levels\nin TMEM251 KO cells despite its Golgi localization. The authors should discuss this\ndiscrepancy or address it with new experiments; perhaps the TMEM251 2-7A mutant is\npresent in the Golgi but not in its precise subcellular location.\nIn Fig. 5A, GNPTαβ levels rescued by the various TMEM251 mutants were assessed\nunder Tet-on leaky expression conditions, which approximate endogenous TMEM251\nlevels. In contrast, for imaging experiments (Fig. S3A), the TMEM251 mutants were\noverexpressed, which may explain the observed Golgi localization of the 2–7A mutant\ndespite its impaired function.\nTo further investigate this, we generated individual point mutations—F4A, R5A, and\nR7A—in TMEM251 within the Tet-on expression system (Fig. S4A). Under leaky\nexpression, both the F4A and R7A mutants failed to rescue lysosomal enzyme processing\n(Fig. S4B–C). Additionally, when moderately induced with doxycycline, these mutants\ndisplayed reduced Golgi colocalization compared to wild-type and R5A (Fig. S4D–E).\nThese results demonstrate that disrupting key residues involved in GOLPH3 interaction\ncompromises both the localization and functional activity of TMEM251.\n4) The authors suggest that there is a fraction of GNPT /TMEM251 which undergoes\nrecycling back to the Golgi through the retromer machinery. They support this with\nfindings that VPS35 KO results in TMEM251 and GNPT appearing in lysosomal puncta,\nwhere they degrade due to defective retromer function. However, a discrepancy arises in\nVPS35 KO, where TMEM251 remains at lysosomes but is not degraded, while GNPT is\ndegraded. The authors should discuss this discrepancy or address it with new\nexperiments. This might be due to the loss of proteases that degrade TMEM251 but not\nthose that target GNPT.\nThank you. We have addressed this point in the revised text. Full-length GNPTαβ is a\n143 kDa protein with the majority of its mass located in the lumen, rendering it highly\nsusceptible to degradation by lysosomal proteases if mislocalized. In contrast,\nTMEM251 contains only a short luminal segment of approximately 40 amino acids,\nmaking it more resistant to lysosomal degradation.\nfraction of VPS35 knockout cells, with a degradation product detected between 10 and\n15 kDa (Fig. 7C).\n5) The authors hypothesize that the TMEM251 cytosolic C-terminal tail, especially\nresidues 93-96, might interact with the retromer for recycling back from endo-lysosomes\nto the Golgi, although co-immunoprecipitation failed to detect such interaction.\nInvestigating the localization of the 93-96A mutant in lysosomes could further corroborate\nthis hypothesis.\nAs suggested, we examined the localization of the TMEM251 93–96A mutant and found\nthat it is retained in the ER, suggesting a potential protein folding defect (Fig. S3A–C).\nTo further support our hypothesis that the retromer interacts with the C-terminus of\nTMEM251, we utilized a C-terminal chimera expressed in both wild-type and VPS35\nknockout SKMEL30 cells. Fusion of the TMEM251 C-terminal tail to the N-terminal portion\nof IL-2Rα was sufficient to confer Golgi localization in wild-type cells (Fig. 8A-C). This\nlocalization was significantly reduced in VPS35 knockout cells, indicating that the\nretromer is required to retain the chimera at the Golgi.\nTo narrow down the putative retromer binding site, we performed an extensive alanine\nscan of individual residues in the C-terminal tail of TMEM251-10xGCN. Mutations at\nV97A, Y99A, V108A, I109A, C110A, and especially their combinations resulted in\ndecreased Golgi signal and increased punctate structures (Fig. 8D-E), consistent with\nimpaired Golgi retention and suggesting these residues are important for retromer-\nmediated recycling.\nAlthough we have not been able to demonstrate a direct interaction between VPS35 and\nTMEM251—potentially due to the transient nature of the interaction or the requirement of\nan adaptor protein—our findings strongly support a functional role for the retromer in\nrecognizing this C-terminal motif.\n6) A topological issue arises from proposing the TMEM251-GNPT complex being recycled\nby GOLPH3 from trans Golgi to cis in Fig9. GOLPH3 is indeed localized at the trans-Golgi\nnetwork (TGN). The authors could introduce the 3 steps model in the figure 9, which is\nmore coherent with data they are showing.\nThank you. We adjusted the model accordingly.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nThank you for taking the time to provide thoughtful and valuable suggestions to improve\nour manuscript.\nReferences:\nBaker, M. 2016. 1,500 scientists lift the lid on reproducibility. Nature. 533:452-454.\nDoray, B., D. Henn, X. Yang, M. Li, and P. Dickson. 2025. Protocol to measure\nendogenous GlcNAC-1-phosphotransfera activity in SK-MEL-30 cells. STAR\nProtocols.\nErrington, T.M., A. Denis, N. Perfito, E. Iorns, and B.A. Nosek. 2021. Challenges for\nassessing replicability in preclinical cancer biology. Elife. 10.\nLiu, L., B. Doray, and S. Kornfeld. 2018. Recycling of Golgi glycosyltransferases requires\ndirect binding to coatomer. Proceedings of the National Academy of Sciences.\n115:8984-8989.\nPechincha, C., S. Groessl, R. Kalis, M. de Almeida, A. Zanotti, M. Wittmann, M.\nSchneider, R.P. de Campos, S. Rieser, and M. Brandstetter. 2022a. Lysosomal\nenzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.\nScience. 378:eabn5637.\nPechincha, C., S. Groessl, R. Kalis, M. de Almeida, A. Zanotti, M. Wittmann, M.\nSchneider, R.P. de Campos, S. Rieser, M. Brandstetter, A. Schleiffer, K. Muller-\nDecker, D. Helm, S. Jabs, D. Haselbach, M.K. Lemberg, J. Zuber, and W. Palm.\n2022b. Lysosomal enzyme trafficking factor LYSET enables nutritional usage of\nextracellular proteins. Science. 378:eabn5637.\nRichards, C.M., S. Jabs, W. Qiao, L.D. Varanese, M. Schweizer, P.R. Mosen, N.M. Riley,\nM. Klüssendorf, J.R. Zengel, and R.A. Flynn. 2022. The human disease gene\nLYSET is essential for lysosomal enzyme transport and viral infection. Science.\n378:eabn5648.\nZhang, W., X. Yang, Y. Li, L. Yu, B. Zhang, J. Zhang, W.J. Cho, V. Venkatarangan, L.\nChen, and B.B. Burugula. 2022. GCAF (TMEM251) regulates lysosome\nbiogenesis by activating the mannose-6-phosphate pathway. Nature\nCommunications. 13:5351.\nWelch, L.G., S.-Y. Peak-Chew, F. Begum, T.J. Stevens, and S. Munro. 2021. GOLPH3\nand GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi\ntransport vesicles. Journal of Cell Biology. 220:e202106115.\nReviewer #1 (Remarks to the Author):\nReviewer Comments (Reviewer#1):\nThanks for revising the Yang et al. manuscript and trying to address my and the\ncomments of the other reviewers. As outlined in more detail below and despite the\nattempts to argue that there is an apparent controversy in literature which has to be\nsolved and that findings need to be repeated, I still remain reserved related to the\nconceptual advance and novelty presented in this manuscript. A large part of the\nprevious manuscript, yet still a substantial part of the revised manuscript (Figs. 1 to 3) is\ndedicated to resolving the finer details of the LYSET-GNPT interplay. I have previously\nvoiced that this particular part (on its own) does not represent a significant conceptual\nadvance and do have to maintain this view. As suggested in my previous review I find\nthe manuscript much more suited for publication in a specialized biochemical journal\nadding some extra information to the field. However, in a journal like NCOMM I would\nhave expected a significant body of novel and unexplored findings which is -according\nto my opinion- not the case in this study.\nRegarding the “novelty” of our manuscript, we would like to point out that our\nmanuscript not only covers the finer details of LYSET-GNPT interplay, as\nacknowledged by the reviewer, but also:\n- Confirm the topology of LYSET\n- Identify the critical residues of LYSET\n- Confirm GOLPH3’s role in maintaining LYSET in the Golgi,\n- Identified the recognition motif of GOLPH3 for LYSET,\n- Determined that the retromer plays a role in maintaining LYSET in the Golgi\n- Show evidence that the retromer binds to the C-terminal of LYSET.\nWe believe that all these discoveries contribute to the novelty of the paper.\nThe authors have now, though, included a paragraph (lines 148ff) that clarifies the\nsituation a bit better and differentiates between their own previously reported\ndata/model (“processing model”) and the data presented by two other independent\npapers (“trafficking model”). Indeed, resolving this controversy is of interest to the field\nof researchers interested in LYSET/GNPT biology, but in this particular case it also\nneeds to be noted that the newly presented data rather argue in favour of the trafficking\nmodel proposed by others previously and do not strongly support the initial model\nproposed by the authors (Zhang et al., 2022, Nat Commun.). Indeed, the authors should\nmore strongly voice to what extent their own data now support the model reported\nbefore by the other groups, e.g. in lines 193-195, line 217 and line 227/228. While the\nauthors raise a point that some discrepancies exist among the three studies on the\nfunction of LYSET, it would be helpful to clarify that the primary point of contention lies\nwith their own publication (Zhang et al., 2022, Nat Commun.), in which the role of\nLYSET was interpreted as the sole activator of MBTPS1-mediated cleavage of\nGNPTAB—an interpretation that remains under debate. The perceived “controversy”\nbetween the two back-to-back Science papers is relatively minor and may largely stem\nfrom methodological differences, such as the use of different cell lines, endogenous\nexpression versus overexpression systems, and antibodies that detect either the alpha\nor beta subunits of GNPTAB. Other variables, such as subunit stability and processing\nof the precursor protein, may also contribute. Notably, the immunofluorescence data in\nRichards et al. (Fig. S14) are based on endogenous protein detection using an antibody\ntargeting the alpha subunit. Following 24-hour Bafilomycin A1 treatment, a modest\nincrease in Golgi-localized signal was observed. The lysosomal staining might reflect a\npool of slowly degraded alpha subunit that had accumulated before Golgi exit was\ninhibited. Similarly, in Figure 3E, the 19-hour S1P inhibitor treatment showed only minor\neffects, likely due to the high stability of the endogenous protein. Since Pechincha et al.\nstudied only newly synthesized, overexpressed protein, the perceived discrepancies\nmay be less significant than suggested. As the authors themselves acknowledge, the\nprecursor protein may exhibit different stability in the presence of lysosomal proteases.\nTo conclusively demonstrate that the precursor is primarily degraded in lysosomes,\nexperiments using lysosomal protease inhibitors would be necessary.\nWe are unsure how to answer this criticism because we did not acknowledge that\n“the precursor protein may exhibit different stability in the presence of lysosomal\nproteases.” Please indicate the lines where we made such a statement. Instead,\nwe found that LYSET is resistant to lysosomal degradation.\nFinally, as far as I can judge the claim that Pechincha et al. and Richards et al.\nexclusively showed lysosomal degradation of the cleaved GNPTAB subunits is not\naccurate. While Richards et al. predominantly detect the endogenous alpha subunit at\nsteady state, they do not assert that cleavage is a prerequisite for degradation. The\nslightly higher molecular weight species observed in LYSET knockout cells may also\ninclude the precursor.\nWe are unsure about this criticism as we did not claim that “Pechincha et al. and\nRichards et al. exclusively showed lysosomal degradation of the cleaved GNPTAB\nsubunits”. We only stated “An alternative model proposed that the processing of\nGNPT is unaffected following LYSET knockout; instead, the LYSET-GNPT\ninteraction is necessary for retaining cleaved GNPT at the Golgi.” (Lines 86-88)\nIn Pechincha et al., it was stated, “Radioactive pulse-chase experiments\ndemonstrated that LYSET was dispensable for synthesis and proteolytic\nprocessing of ectopically expressed GNPTAB α/β precursor (fig. S10A).\nMoreover, Pechincha et al. provide clear evidence of an increase in cleavage fragments\nupon treatment with lysosomal protease inhibitors, strongly indicating that the precursor\nitself undergoes lysosomal degradation.\nWe thank the reviewer for pointing this out. This result strongly supports our\nconclusion that LYSET facilities S1P-mediated GNPTAB cleavage. All three papers\nconfirmed that the mRNA level of GNPTAB is not affected in LYSET KO cells. With\nthe degradation of the precursor, one should expect to see reduced processing.\n(Introduction): I agree that LYSET seems to have multiple functions in regulating\nGNPTAB localization and therefore function. However, the authors neglect here that a\nstabilizing role of LYSET for the TMD1 of GNPTAB has been clearly shown. The authors\nthemselves have (re-) demonstrated this in their recent MBoC publication (Doray et al.\n2025). Showing the interaction of LYSET with the Golgi-adaptor GOLPH3, however,\nagain lacks novelty, since this was shown by Brauer et al. before (EMBO J 2024) which\nthe authors only briefly mention in their discussion.\nIn our initial submission, we already included results showing the interaction\nbetween LYSET and GOLPH3. We have confirmed with the editorial office that we\nwere under the scoop protection policy from NC. Moreover, we identified the\nrecognition site as the FXXR motif using two different methods.\nFigure 1:\nThe new Figure 1 includes very few novel information but is primarily a reconfirmation of\ndata published by Richards et al, Pechincha et al. and Zhang et al. The whole figure\nincluding panel H serving as a control for their newly generated antibody would be\nbetter suited for the supplement. In Figure S1, again, the authors present no novel data\nbut replicate published results that never presented any controversy and could be\nexplained both by LYSET-mediated activation, retention or stabilization of GNPTAB.\nWe have addressed this comment in the first round of rebuttal. The reviewer has\nvery detailed knowledge of the field; however, most readers do not have that\nadvantage. It is necessary to familiarize the readers with the importance of LYSET\nin the M6P pathway. Moreover, the paper consists of a total of nine main figures\nand six supplementary figures, the majority of which are novel and contribute to\nthe field's understanding of the M6P pathway.\nFigure 2:\nPanel A: Quantification is missing. Since the overall expression of GNPTAB is reduced\nin LYSET KO, it is hard to judge whether there is a slight delay in MBTPS1- mediated\nprocessing.\nWe thank the reviewer for this suggestion. We included the quantification for Fig.\n2A (See Fig. 2B) to show that the overall processing rate of GNPTAB is reduced in\nLYSET KO cells.\nFig2B: A western blot should be added, otherwise it is impossible to distinguish between\naltered expression and altered activity.\nWe included the Western Blot for Fig. 2B (now Fig. 2C) as the reviewer suggested,\nthank you.\nIf the activity data is from three independent biological replicates, why is the standard\ndeviation not shown? Single data points including error bars should be presented.\nWe are unsure about this criticism because Figure 2B (Now Fig 2C) already\nshows the standard deviation with the individual data points. In addition, we are\ndeeply saddened to notify this reviewer that our beloved Stuart suddenly passed\naway on August 17. It will be impossible to ask his group to perform more\nexperiments at this point.\nFig. 2E The authors have to show the absence of LYSET and the purportedly mild\noverexpression. The conclusion of the panel is potentially important since it would be\nthe only piece of evidence that the phosphotransferase activity itself (at least of the\nmutants) might be affected by LYSET expression, so all the controls have to be shown.\nIf LYSET is overexpressed anyway, there is no reason not to use a tagged version.\nThe reviewer misunderstood Figure 2E (Now Fig. 2F). We did not overexpress\nLYSET. These cell lines were either WT or LYSET KO cell lines. Instead, we mildly\nOE GNPTAB-820-928deletion-3V5 or GNPTAB-Furin-3V5, the expression levels of\nwhich are shown. This experiment is to investigate if LYSET is required for\nGNPTAB activity after its cleavage.\nAt the same time, LYSET detection would be critical to e.g. take expression levels of\nLYSET into consideration for evaluating the various mutants generated (Fig. 5). This\ncontrol is essentially missing. Considering the newly included findings, it would also be\nimportant to check levels of endogenous LYSET in GOLPH3/GOLPH3L-deficient cells\nAs stated above, we are evaluating the function of different GNPTAB mutants\nmostly in LYSET KO cells background, and no overexpressed LYSET was used in\nthis assay. So, the expression of endogenous LYSET is not critical for drawing\nthe conclusions. We agree that having a LYSET blot will serve as a nice control\nand give a complete picture of the whole assay. Unfortunately, as we stated in the\nlast round of revision, the new batch of TMEM251 antibody (HPA048550 from\nSigma) lost sensitivity, and we can no longer access the LYSET western blot.\nFigure 3:\nFig. 3D Is the whole cell lysate shown or a WCL depleted from lysosomes? How do the\nauthors explain the low LAMP2 signal and the many (unspecific?) bands?\nWCL stands for whole cell lysate, which is the starting material used for\nlysosome purification. There might be two reasons for the many bands in LAMP2\nblots: 1) LAMP2 is a heavily glycosylated protein. It always migrates as a smear\nor several bands. 2) It is not uncommon for antibodies to recognize additional,\nnon-specific bands. In the “LYS” fraction, however, the two non-specific bands,\nwere removed by lysosome purification, confirming lysosome purification can\nremove non-specific bands from the WCL.\nFigure 4:\nFig. 4E The labelling of the panels is misleading, please clearly indicate that all the\nproteinase K-treated conditions are also treated with Digitonin for 3 minutes. Scale bars\nare missing.\nWe relabeled the panels of Figure 4E so that it is easier to understand and added\nthe scale bar.\nIf 4F is really the quantification of the images shown in E, the timepoints of addition.\nof digitonin and proteinase K are incorrectly labelled.\nWe carefully re-checked the time points in panel F. Digitonin was added at minute\n2 and incubated for three minutes (2–5 min), followed by adding proteinase K at\nminute 5. This timing is consistent with that shown in panel E. The main\ndifference between Figure 4E and 4F, is that the images with just the KHM buffer\nwashing were not included in Figure 4E. However, to make it easier to\nunderstand, we slightly changed the labeling on Figure 4F. We also revised the\nfigure legends to provide more detailed information.\nFigure 5:\nFig.5A/B How do the authors explain that some mutations in LYSET have completely\ndifferent functions in 293T cells than in SK-MEL30 cells (e.g. 67-71A, which shows\nabnormal CTSD processing/localization, but WT GNPTAB levels)? For 87-91A or 93-\n96A this is less pronounced, but there is way more GNPTAB than in 82-86A but similar\nCTSD signals.\nWe have addressed this point in our previously revised manuscript (Line 283,\nnow lines 287-289). These results suggest that mutations in this region do not\ndisrupt LYSET’s role in anchoring GNPTAB, but may instead impair other\nfunctions, such as GNPTAB enzymatic activity.\nFig. 5C: other topology predictors show a slightly different topology with R7 directly\nadjacent to the membrane (as in Fig. 8F), please indicate which tool was used and\nharmonize the different depictions.\nThank you for pointing this out. We corrected the topology models in Fig 5C and\n8F to be consistent with what was predicted by Topcon. We also mentioned this\nin the Figure legend.\nR7W does not decrease LYSET levels (confirming the data in Richards et al. 2022), but\ndecreases GNPTAB expression (as also shown in Richards et al.) The data in Fig.6F\nsuggests that R7 is important for GOLPH3 binding so the mutant would be expected to\ndestabilize LYSET levels. How do the authors explain this discrepancy?\nAlthough in SKMEL30 cells (Fig. 5B), the overexpressed R7W mutant does not\nseem to decrease the protein level, we did observe that R7W has a short half-life\ncompared to WT LYSET, which we have previously shown in Supporting Figure\n2C of our rebuttal letter. However, we will include the data again. Please see\nSupporting Figure 1C.\nSupporting Figure 1, R7W affects TMEM251 Golgi retention. (A) Pull-down assay showing\nthe interaction between the 251N(WT)-SITM-GFP and 251N(R7W)-SITM-GFP chimeras and GST-\nGOLPH3. (B) Immunostaining images and quantification showing the localization of SIN-SITM-\nGFP, 251N(WT)-SITM-GFP, and 251N(R7W)-SITM-GFP in WT HeLa cells. (C) A cycloheximide chase\nassay showing the degradation of WT and R7W TMEM251 over 2 hours. Scale bar: 10 µm.\nAdditionally, we show in Figure S4B, using Tet-on leaky expression to mimic\nendogenous levels, that mutating R7 not only affects the stability of LYSET, but\nalso subsequent mature CTSD levels. Finally, in Figure 6 A and B of Richards et\nal. (2022) and Figure 4E of Pechincha et al., (2022), there was clearly less LYSET\nin the R45W mutant compared to WT, indicating that this mutation is indeed less\nstable compared to WT. In fact, Pechincha et al stated that “LYSETR45W and\nLYSETY72X were barely detectable at the protein level, conceivably because they\nabrogated LYSET function by destabilization and premature truncation of the\nprotein, respectively (Fig. 4E and Fig. S8, B and C).”\nIn the initial version of the manuscript, the authors presented data demonstrating that\nthe N-terminal cytosolic amino acids of the short LYSET isoform (i) can mediate binding\nto GST-GOLPH3 in vitro and (ii) enforce GOLPH3-dependent Golgi retention when\nadded to N-terminally to an artificial type II membrane protein reporter. Overcoming the\nlimitations of the reporter data which I pointed out earlier, the authors have now\nincluded experiments arguing in favour of a GOLPH3-dependent Golgi localization of\neven full-length LYSET, showing mislocalization of an ectopically expressed full-length\nLYSET in GOLPH3/GOLPH3L KO U2OS cells and mislocalized endogenous GNPT in\nGOLPH3 knockdown cells (Fig. S5). I suggest that these data are all included in one of\nthe manuscript’s main figures, although, as pointed out earlier, this finding is not novel\nsince Brauer et al. (2024) have shown this before.\nAs stated above, we have confirmed with the editorial office that we are under\nscoop protection. We are pleased that an independent study has largely\nconfirmed our findings about Golph3, which accounts for only one-third of our\npapers. In addition, we identified the recognition motif to be FxxR.\nThank you for suggesting merging the two figures. We tried; however, we failed\nto merge them in a logical way. As you can tell, we performed a very extensive\nanalysis here using both Chimera and full-length LYSET. To maintain the flow of\nthe paper, we believe it is better to keep the images as they are.\nAn additional concern is that the authors do not show mislocalization of endogenous\nLYSET. Also, as a consequence of LYSET mislocalization, impaired cellular M6P\ntagging and maturation of lysosomal enzymes can be expected (see also ref #36) and\nexperiments in GOLPH3 KO HeLa and U2OS cells available to the laboratory (see\nFigure 6B, ref #21 and Fig. S5A) should be used to more clearly demonstrate this\n(instead of Fig. S5E/F).\nFirst, as we have repeatedly stated, there is no good LYSET antibody available to\nshow the mislocalization of endogenous LYSET. However, we did show\nmislocalization using both chimera (figure 5B-C) and mildly overexpressed\nLYSET-GCN4 (figure S5A-B). Second, we have shown that endogenous cathepsin\nD has maturation defects in Golph3 Knockdown (Figure S5E-F). We are unsure\nwhat new information could be drawn by showing more lysosomal enzymes,\nespecially in light of the fact that we have been repeatedly criticized as “not\nnovel” and Brauer et al. have already shown this in their publication. The main\nconclusion we want to present here is that Golph3 is critical for maintaining\nLYSET Golgi-localization, and its recognition motif is FXXR. We believe we have\nprovided sufficient evidence to justify them.\nIn addition, through comprehensive mutagenesis, the authors determined that F4 and\nR7 present in the LYSET N-terminus are critical to Golgi localization of the type II\nmembrane protein reporter, but – strongly supporting that these residues are indeed\nrelevant here – also of full-length LYSET ectopically expressed in 293T and SKMEL30\ncells (Fig. S4). Again, I feel that this dataset should be included in a main figure. In\naddition, it would be interesting if there is an additive effect of the two mutants and\nwhether any of the mutants shows impaired binding to GST-GOLPH3 in vitro. I also still\nfeel that it needs to be explored whether the F4 and R7 mutants exhibit mislocalization\nwhen in the context of the long LYSET isoform or whether the substantially longer N-\nterminus can somehow overrule those mutations (see Brauer et al. 2024).\nWe have shown before that long and short isoforms are functionally redundant in\nour previous Nature Communications publication. Although interesting, repeating\nall the experiments with the long isoform is not justified, as it does not add new\ninformation. In addition, as acknowledged by this reviewer, Brauer et al. have\ntested the long isoform and reached a similar conclusion to ours.\nRegarding combining the figures, please see our response above.\nRegarding combining F4A with R7A mutants, we would like to point out that F4A\nor R7A alone is sufficient to abolish the Golgi localization. It is reasonable to\nassume that combining the two mutations would NOT do otherwise.\nFinally, we showed reduced GST-GOLPH3 binding to the 251(R7W) peptide in our\nsupporting figure 2 of our previous rebuttal letter, and we are unsure why the\nreviewer would request this data again. However, as stated earlier, we have\nincluded the data (please see supporting Figure 1A,B) again for the reviewer.\nFigure 7:\nWhat’s the difference between the endogenously tagged GNPTAB expression in Fig. 7A\nand B? Why is there so much background signal in the WCL of C? Why is there a\nprecursor band detected in the 3HA-KI (-) cells? Shouldn’t the LYSET WCL levels be\ndecreased if there’s more LYSET mislocalized to lysosomes?\nFigure 7B is the quantification of Figure 7A. Thank you for pointing out the\nvariation in background signals of the Anti-HA antibody (16B12, BioLegend). We\nare unsure about the variation; it could be due to the different lot numbers of the\nantibody, as we did the two sets of experiments at different times. However, we\ndesigned both experiments carefully by including the non-tagged WT controls.\nWith this control, we are confident about our conclusion.\nWe disagree that “there is a precursor band detected in the 3HA-KI (-) cells”; the\nright interpretation should be that those bands are background bands.\nThe observation that LYSET does not reduce after knocking out VPS35 indicates\nthat LYSET is resistant to lumenal proteases, even after it mislocalizes to the\nlysosome. This is different from GNPTAB. The possible reason for the resistance\nis that LYSET is a small transmembrane protein with only 37 residues exposed to\nthe lumen (Figure 5C), whereas GNPTAB has 1256 amino acids, and the majority\nof the protein is in the lumen.\nFigure 8:\nFig.8B the IF data are of rather bad quality and the GRASP55 signal seems to be -in\npart- extremely overexposed. Please select better quality images.\nThank you for pointing this out. We have fixed this issue.\nFig. 8D, IF of poor quality, especially the WT LAMP2 staining. Please include higher\nquality (resolution) images. LYSET-GCN stainings seem to be overexposed which is\nprecluding proper colocalization analysis (especially the lower panel of LAMP2 co-\nstainings).\nWe have fixed this issue. Thank you!\nThe authors postulate a mechanism of LYSET binding both GOLPH3 and VPS35.\nPlease show the expression of a LYSET mutant both deficient in LYSET and VPS35\nbinding.\nWe are unsure about the request to show LYSET interacting with LYSET.\nRegarding LYSET interaction with VPS35, we have tried very hard to get this to\nwork. But this proves to be technically challenging. However, the mislocalization\nof endogenous LYSET (and GNPTAB) in VPS35 KO melanoma cells, as well as\nour extensive analysis using IL-2Ra-LYSETC chimera, together with our\nmutagenesis scan, provided compelling evidence to demonstrate the importance\nof retromer in maintaining LYSET at the Golgi.\nBrauer et al. (see above) show that LYSET interacts with GOLPH3 and GOLPH3L.\nLYSET is mislocalized and is destabilized in double KO cells. The authors rather\nsuggest VPS35 as being important for LYSET Golgi-localization. Are these cell-type-\nspecific differences?\nHere, we have shown that both GOLPH3 and VPS35 contribute to keeping LYSET\nin the Golgi, not just VPS35 alone. The GOLPH3(and likely GOLPH3L) interacts\nwith the N-terminus FXXR motif, whereas retromer likely interacts with the C-\nterminal V97xY99—V108I109C110 motif.\nRegarding GOLPH3-LYSET interaction, our conclusions are largely consistent.\nThe minor discrepancy may be due to differences in cell lines and protein\nexpression levels.\nFigure 9:\nThe authors did not present data that convincingly shows two independent steps of\ncoatamer-dependent recycling, although it’s conceivable that there may be redundant\nmechanisms. Neither the figure legend nor the main text do explain the two distinct\n“coatomer-dependent recycling” boxes included in this illustration. This needs to be\nexplained. Do the authors propose different modes of retrieving in the early and the late\nGolgi, with the latter being COPI-independent but GOLPH3-depdent? (with a label\n“coatomer component” still in the box?)\nThis was suggested by Reviewer #3 from the previous round. The two separate\nboxes are now merged into one.\nAgain, the authors postulate a role of LYSET in GNPTAB cleavage, which is not\nunambiguously shown. The slightly increased processing might well be explained by a\nstabilisation of GNPTAB in the cis-Golgi apparatus and therefore longer dwell-time in\nthe proximity of MBTPS1. In addition, the authors have demonstrated themselves in\nDoray et al. that the stabilising mutant of GNPTAB is efficiently cleaved by MBTPS1 in\nLYSET KO cells and stabilized upon overexpression of LYSET, so it is unclear to the\nreviewer why they deliberately omit this function in their summary figure.\nThe reviewer misconstrued the summary figure. We did not deliberately omit this\nin the summary figure. We believe we have clearly explained both in text and in\nthe figures that LYSET is necessary to keep GNPTAB in the Golgi. Without\nLYSET, GNPTAB is sent to the lysosome to be degraded, and without LYSET, to\nkeep GNPTAB in the Golgi S1P S1P-mediated cleavage can no longer occur.\nAdditionally, LYSET is kept in the Golgi via both Golph3 and the retromer\ncomplex. In fact, the main idea of the figure is to show that GNPTAB is necessary\nfor M6P modification, LYSET keeps GNPTAB in the Golgi, and that LYSET is kept\nin the Golgi via multiple mechanisms. Lastly, we have now added a sentence in\nFigure 9 to explain that LYSET keeps GNPTAB in the Golgi to facilitate its\ncleavage.\nAdditional issues:\nAs a side note I could not get access (despite asking twice at the editorial board) to a\nversion of the revised manuscript containing the marked changes which would be\nrequired to fully appreciate the authors changes and responses to my review.\nWe apologize. The editorial office requested a marked version on July 25th, and\nwe sent it to them on the same day. However, there might have been some\nmiscommunication between the editorial office and the reviewer, as it did not\nreach the reviewer.\nPage 15 of the rebuttal letter: the authors claim that they cannot do costainings due to\nantibody issues, but they have endogenously tagged GNPTAB-HA cells which they\ncould use.\nThe endogenous GNPTAB-3HA KI cell line is in a HEK293t background. Although\nwe can detect GNPTAB-3HA via western blot, we were unable to do so using\nimmunofluorescence. This is most likely due to the low expression levels of\nGNPTAB in Hek293t cells.\nLine 323ff. re: GOLPH3 recognition motif: The authors directly compare the FXXR motif\nfound to be important experimentally with a previously suggested LXXR GOLPH3\nrecognition motif. However, as evident from the data presented by Welch et al. this motif\nis not necessarily present in all GOLPH3/3L clients and a polybasic stretch directly\npreceding the transmembrane domain of a Golgi type II protein may be a more accurate\ndistinctive feature of a Golgi client. In fact, immediately after making this statement, the\nauthors introduce GALNT2 as a prototypic GOLPH3 client which is used as their\npositive control – which completely lacks the LxxR motif (MRRRSR). This should be\nrephrased accordingly.\nWe have rephrased this part in the manuscript (lines 327-330). Thank you for the\nsuggestion.\nThe authors now refer to TMEM251 as LYSET in the revised manuscript and it is good\nto further establish a uniform naming of the gene/protein in the literature. Yet, this\nmakes it hard for a reader to understand labelling in Fig. 6H, in which “251N peptide”\ncertainly refers to an N-terminal TMEM251 peptide. The authors should consider\nrephrasing the labelling of this panel to make this more obvious. Also, the exact\nsequence of the biotinylated peptide used should be provided.\nThank you for pointing this out. We have changed the labelling to “LYSET-N\npeptide” to make it more consistent with the rest of the manuscript. The exact\npeptide sequence is MMNFRQRM, which was described in the manuscript (line\n337).\nLine 458: Brauer et al showed that GOLPH3 and GOLPH3L are redundant in\nmaintaining LYSET and GNPTAB in the cis-Golgi. Were both deleted by the authors?\nDoes the siRNA the authors used target both isoforms? Is the antibody used\nspecific for GOLPH3 or does it equally detect GOLPH3L? Could this explain the\ncompensatory signal in the KD cells?\nAs shown in Figure S6 F,G we cleanly knocked out GOLPH3 in the U2OS cell line,\nwhile two of the three GOLPH3L chromosomes contained mutations. According\nto Ng MM, et al (2013), HeLa cells primarily express GOLPH3 and not GOLPH3L.\nThus, we used a shRNA specific to GOLPH3 to knock down GOLPH3 only. We\ndid, however, as a control, use shRNA targeting both GOLPH3 and GOLPH3L and\nhad similar observations. Thus, we believe it is unlikely that the slight\ncompensatory effect observed is due to GOLPH3L.\nHandwritten labelling can be found on some immunoblot panels. In particular in case of\nFig. 2D it would be good to mask this (as lane numbering is provided below the panels\nanyway).\nWe cropped the images to remove the handwritten labelling.\nIn panel 3A it would be good to highlight the degradation products mentioned with\narrows.\nWe used a red arrow to highlight the degradation products.\nPanel S3C appears to be very pixelated. Could a higher resolution panel be included in\nthe figure?\nThank you for pointing this out. This was most likely a conversion issue. We\nchanged the images so that they are no longer pixilated.\nLike in Fig. S4A, the box labelled EF1a in Fig. 8A should be additionally garnished with\nan arrow to clearly demonstrate it is the promoter driving the expression of the fusion\nconstruct.\nWe added the arrow to clarify the promoter region. Thank you for the suggestion.\nInconsistent naming of the detection tag used: The tag is mostly referred to as “10x\nGCN” but as 10x GNC4” (Fig. S4D) (with the latter naming corresponding to the one\nused by Tanenbaum et al. in the original manuscript reporting the tag)\nWe changed all the labeling to 10xGCN4 to keep it consistent throughout the text.\nHow strong a reduction in GOLPH3 was achieved following knockdown in SKMEL30\ncells (Fig. S5C) – such a control staining (GOLPH3 co-staining or immunoblot) is\nmissing.\nWe added a western blot to show the reduction of GOLPH3 in SKMEL30 cells\n(now Fig. S5C)\nThe disease mucolipidosis type 5 does not exist. LYSET deficiency is officially called\ndysostosis multiplex, Ain-Naz type. Ain et al. 2021 is incorrectly cited by the authors.\nWe previously proposed to call the disease Mucolipidosis Type V due to its\nsimilarities to Mucolipidosis Type II and III. However, as the reviewer\nrecommended, we have added the name dysostosis multiplex (see lines 316-317).\nReviewer #2 (Remarks to the Author):\nThe revised manuscript is significantly improved, both in terms of new experimental\ndata and clarity of writing. The authors have adequately addressed all of my previous\ncomments.\nWe thank the reviewer for all the constructive comments.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank the reviewer for all the constructive comments.\nRebuttal letter\nReviewer #1 (Remarks to the Author):\nAs clearly and in detail stated in my previous reviews my enthusiasm about this\nmanuscript remains dampened. It seems -in contrast to this reviewer´s view- that the\neditor is in favor of promoting this manuscript despite a number of experimental\nsuggestions were not addressed and only briefly discussed.\nWe apologize that we could not address all of the Reviewers comments to a satisfactory\ndegree. We tried our best to address all the comments we felt would improve the science\nof our paper and we thank the reviewer for all the thoughtful critique. We still feel that our\nmanuscript not only confirms previous work, but more importantly provides novel\nadvances to the field including:\n- Resolve the topology of LYSET\n- Identify the critical residues of LYSET\n- Confirm GOLPH3’s role in maintaining LYSET in the Golgi,\n- Identified the recognition motif of GOLPH3 for LYSET,\n- Determined that the retromer plays a role in maintaining LYSET in the Golgi\n- Show evidence that the retromer binds to the C-terminal of LYSET.\nSome additional remarks:\n“We are unsure how to answer this criticism because we did not acknowledge that\n“the precursor protein may exhibit different stability in the presence of lysosomal proteases.”\nPlease indicate the lines where we made such a statement. Instead, we found that LYSET\nis resistant to lysosomal degradation.“\nI refered in a long paragraph to the stability of GNPTAB and the authors´s comment\nrelated to the stability of LYSET is misplaced and does not fit to my remark which should\nbe thoroughly read and responded to.\nWe re-read the reviewers’ comments and hope that the following response is satisfactory.\nAs discussed in our previous rebuttal letter, we did not mention anything related to the\nstability of the GNPTAB precursor protein in the presence of lysosomal proteases in our\nmanuscript and was thus unsure what the reviewer was referring to. We did, however,\nmention that LYSET was resistant to lysosomal degradation in our manuscript and\ntherefore included it in our previous reply to the Reviewer.\nPlease see below a more in-depth response to the reviewers’ additional concerns.\nIn regards to the request by the Reviewer to “More strongly voice to what extent their own\ndata now support the model reported before by the other groups” and to “clarify that the\nprimary point of contention lies with their own publication (Zhang et al., 2022, Nat\nCommun.), in which the role of LYSET was interpreted as the sole activator of MBTPS1-\nmediated cleavage of GNPTAB—an interpretation that remains under debate.”\nWe believe we have comprehensively discussed the two models and acknowledged that\nLYSET is necessary to keep GNPT in the Golgi, as proposed in the Trafficking model.\nBelow we have outlined the discussion of the two models and our conclusions:\n- In Lines 82-94 we give an introduction to the two proposed models and\nacknowledge that the processing model does not account for the reduction of total\nGNPTAB in LYSET KO cells.\n- In line 98-99 we summarize that without LYSET, GNPT is mislocalized and\ndegraded by the lysosome.\n- In line 108-121 we confirmed that GNPTAB protein levels are reduced in LYSET\nKO Hek293t cells (Figure 1A-E).\n- In line 122-129 we used the SKMEL30 cells to again confirm that GNPTAB levels\nare reduced in cells lacking LYSET (Figure 1F-H).\n- In lines 142-147 we summarized our findings that LYSET is necessary for\nGNPTAB processing and stability.\n- In lines 149-152, we again discussed the two models and acknowledged that the\nprocessing model cannot explain the large loss of total GNPTAB.\n- The entire section between lines 148 and 197 is dedicated to show that LYSET is\nnecessary to keep GNPTAB in the Golgi and thus facilitates cleavage (Figure 2A-\nG).\n- We finalized the section by summarizing our findings and acknowledging that\nLYSET is necessary to keep both full length and uncleaved GNPTAB in the Golgi,\nand thus consequently facilitates S1P-mediated cleavage (lines 195-197).\n- Next, we again dedicated a section (lines 198-233) to validate that both full-length\nand cleaved GNPTAB gets degraded in the lysosome (Figure 3A-G).\n- Finally, we discussed the two models again (lines 421-431) clearly stating that\nwithout LYSET full length GNPT may rapidly exit Golgi and therefore reduce S1P\ncleavage efficiency by reducing the precursor concentration.\n- We acknowledged that as stated by Richards et al., and Pechincha et al., LYSET\nmaintains GNPT in the Golgi (lines 433-434)\nWe hope the reviewer takes note of our careful discussion of the literature, our attempts\nto resolve the controversy and our acknowledgement that LYSET maintains GNPT in the\nGolgi as proposed previously by Richards et al. and Pechincha et al.\nThe reviewer also raises the concern that “The perceived “controversy” between the two\nback-to-back Science papers is relatively minor and may largely stem from\nmethodological differences, such as the use of different cell lines, endogenous\nexpression versus overexpression systems, and antibodies that detect either the alpha\nor beta subunits of GNPTAB.”\nRegarding this, as mentioned in the previous rebuttal letter, we have since changed the\nwording to more accurately discuss the controversy and the data presented by Richards\net al. and Pechincha et al. in our manuscript. Specifically, we refer to the two science\npapers in the following context:\n- As mentioned above, we give an introduction to the two proposed models and\nacknowledge that the processing model does not account for the reduction of total\nGNPTAB in LYSET KO cells (Lines 82-94).\n- In lines 442-448 we mention that we could not see the mislocalization of GNPTAB\nto the lysosome via immunostaining as seen by Richards et al., but specifically\nmention that it could be due to the antigen regions being digested. In this\nparagraph we mentioned that Richards et al., observed relocalization of GNPTAB\nto the lysosomes in HAP1 cells (lines 445-448). Please note that we do not dispute\nthe point that GNPTAB is mislocalized to the lysosome, since, as discussed\nabove, we confirmed this via western blot.\n- We have, as per the previous rebuttal letter, changed the wording and included\nRichards et al., when noting the fact that BafA1 stabilizes GNPT in the lysosome\n(lines 227-229 and 445-448)\n- We mentioned that Pechincha et al., proposed that GNPT is unstable due to its\nrelatively hydrophilic transmembrane domain (lines 452-453). Our data support\nthis hypothesis by using alanine scanning to identify multiple critical regions of\nGNPTAB, including the transmembrane regions (Figure 5C).\nWe thank the reviewer for all the thoughtful input to help improve the manuscript so that\nwe more accurately express our data and contribute to the field.\n“We thank the reviewer for pointing this out. This result strongly supports our conclusion\nthat LYSET facilities S1P-mediated GNPTAB cleavage. All three papers confirmed that\nthe mRNA level of GNPTAB is not affected in LYSET KO cells. With the degradation of\nthe precursor, one should expect to see reduced processing.“\nThe authors again misinterpret the conclusion. While both Richards et al. and Pechincha\net al. focus on the degradation of the cleaved subunits, they do not exclude that the\nprecursor can equally be lysosomaly degraded. Pechincha et al., however, demonstrate\nthat the kinetics of GNPTAB cleavage are not altered in the absence of LYSET. Of course,\nthe cleavage of the precursor is reduced if it is degraded. This is a trivial conclusion which\ndoes not support the authors’ interpretation that LYSET facilitates the S1P-mediated\ncleavage. The authors seem uncertain to confirm their originally proposed role for LYSET\nas a cleavage-activating factor.\nTo answer the reviewer, we discussed this point in depth in both our manuscript and the\nprevious rebuttal letters. We used the beginning of this manuscript to resolve the\ncontroversy between the models proposed by us (Zhang et al.,) and Richards et al. and\nPechincha et al. We acknowledge in lines 230-233 that LYSET is necessary to keep both\nfull length and uncleaved GNPTAB in the Golgi, unlike what we initially proposed in Zhang\net al. Instead, we suggest that LYSET facilitates S1P-mediated cleavage by maintaining\nGNPT in the Golgi. Without LYSET, S1P cleavage efficiency is reduced since the\nGNPTAB precursor concentration is lowered (lines 428-429). In line 425-426, we mention\nthat LYSET can pull down both S1P and GNPT as shown in Zhang et al. which supports\nthe processing model. However, we also mention that without LYSET, the precursor\nrapidly leaves the Golgi which could also affect S1P cleavage of GNPTAB (lines 427-\n429). Moreover, our data show that overexpressing either GNPTAB or S1P bypasses the\nneed for LYSET (lines 429-431, Figure 2C-D).\nWe would like to respectfully disagree with the Reviewer, as all these experiments and\ndiscussion do not just lead to a trivial conclusion. It instead allows us to resolve the\ncontroversy that arose between the papers. We believe that when differences arise it is\nin the scientific community’s best interest to clarify, and we have done so extensively as\ndiscussed above.\n“WCL stands for whole cell lysate, which is the starting material used for lysosome\npurification. There might be two reasons for the many bands in LAMP2 blots: 1) LAMP2\nis a heavily glycosylated protein. It always migrates as a smear or several bands. 2) It is\nnot uncommon for antibodies to recognize additional, non-specific bands. In the “LYS”\nfraction, however, the two non-specific bands, were removed by lysosome purification,\nconfirming lysosome purification can remove non-specific bands from the WCL.“\nThere are several excellent LAMP2 antibodies (e.g. from DSHB) that detect no unspecific\nbands, therefore the reviewer is surprised by the poor quality of the western blot.\nThank you for the suggestion. We used the DSHB antibody to probe for LAMP2 in our\nWCL and Lysosome fractions and subsequently replaced the blots in Figure 3D and\nFigure 7C.\nResponse to Reviewer\nWe would like to thank the reviewer for taking the time to read through and assisting us\nin improving our manuscript titled “Molecular Insights into the Regulation of GNPTAB by\nLYSET”.\nPlease see below for a detailed response to the reviewers’ concerns:\nReviewer #1 (Remarks to the Author):\nThe manuscript by Yang and colleagues describes the recently identified LYSET protein\nas a two-pass Golgi membrane protein that stabilizes GNPT, promotes its S1P-\ndependent cleavage, and prevents its degradation by protecting it from mislocalization to\nlysosomes. The authors suggest that LYSET achieves this function by interacting with\nGOLPH3 and the retromer—through distinct LYSET motifs—to retain the LYSET–GNPT\ncomplex in the Golgi, thereby ensuring proper operation of the M6P pathway.\nAs stated, multiple times in my earlier reviews, I acknowledge and appreciate certain new\nfindings and contributions to the field. However, I find that the overall scientific\nadvancement and impact compared to previously published studies is not as substantial\nas claimed. A journal such as Communications Biology or a comparable publication would\nlikely be more appropriate for this work.\nWe are regretful that we could not convince the Reviewer of the suitability of this\nmanuscript for Nature Communications despite our best efforts to address the\nnumerous critiques of the reviewer. Nevertheless, we hope that our manuscript\ndoes contribute to furthering the M6P field and we are grateful to the editors for\nallowing us to share our research.\nUpon re-examining the manuscript, I also noted that, despite my previous comments,\nsome data presentations still rely on only two measurements (e.g., Fig. 2E and the activity\ndeterminations in Supplementary Fig. S1). Moreover, no error bars or standard deviations\n(STDEVA) are provided.\nAs the reviewer requested we have performed an additional repeat for Figure S1C\nand added error bars to the values.\nUnfortunately, due to our collaborator’s passing, with his lab closed down, and the\nmaterials no longer available we cannot repeat Figure 2E. However, Figure 2E is\nindependently supported by Figure 2F, which demonstrates a dose-dependent\nGNPTAB-V5 processing using a different experimental approach. We respectfully\nargue that this independent validation in Figure 2F provides stronger support than\nsimply adding an additional replicate to Figure 2E."}]

Decision letter