Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
Lysosomes are highly dynamic organelles that serve antagonistic functions as terminal catabolic stations for the degradation of macromolecules and as central metabolic decision centers for anabolic growth signaling. Lysosome dysfunction is implicated in various human diseases. The physiological roles of lysosomes are linked to the control of lysosome position and dynamics via the activity of the kinesin-activating small GTPase ARL8. How the activity of ARL8 is regulated remains poorly understood. Here, we identify the GTPase-activating Tre-2/Bub2/Cdc16 (TBC) domain protein TBC1D9B as a critical negative regulator of ARL8B function. We demonstrate that TBC1D9B is associated with the lysosomal membrane protein TMEM55B, directly binds to ARL8B-GTP, and stimulates its GTPase activity. Knockout of TBC1D9B or its binding partner TMEM55B causes lysosome dispersion, defective autophagic flux, and impairs the adaptive degradative response of cells to limiting nutrient supply. These lysosomal phenotypes of TBC1D9B loss are occluded by concomitant depletion of ARL8 in cells. Collectively, our data unravel a key role for TBC1D9B in controlling lysosome function by serving as a negative regulator of ARL8 activity.

Introduction
Lysosomes are degradative organelles that mediate the hydrolytic breakdown of macromolecules delivered to their acidic lumen either by endocytosis or via autophagy processes1–3. Besides their central importance for the catabolic turnover of macromolecules, lysosomes also fulfill a critical anabolic function by serving as a recruitment platform for the multisubunit nutrient-sensing and signaling mechanistic target of rapamycin complex 1 (mTORC1), a major driver of anabolic programs, such as protein and lipid biosynthesis4,5. For example, amino acids derived from lysosomal proteolysis are exported from the lysosome lumen to provide building blocks for protein synthesis while concomitantly promoting the activation of mTORC1. Active mTORC1 represses catabolic programs, such as autophagy and lysosomal gene expression via transcription factor EB (TFEB) and its relatives1–3,6.

These janus-faced activities of lysosomes as catabolic endpoints and decision-making centers in metabolic signaling are intimately linked to lysosome dynamics and positioning7–9. Lysosomes can move bi-directionally along microtubule tracks via plus- (e.g., kinesin) and minus-end (e.g., dynein) directed motor proteins9–12, and their dynamics are further regulated by membrane contact sites (MCS) with other organelles, in particular with the endoplasmic reticulum (ER)12–14. Nutrient deprivation (e.g., amino acid starvation) triggers the perinuclear clustering of lysosomes7, the acidification of the lysosome lumen15, and concomitant elevation of lysosomal proteolysis, as well as the dissociation of mTORC1 from the lysosome surface5, resulting in the repression of mTORC1 signaling16. Conversely, in fed cells under conditions of active nutrient signaling, lysosome motility is promoted, and lysosomes become dispersed to the cell periphery via kinesin-based mechanisms7,16. A major driver of the anterograde motility of lysosomes is ARL8 (i.e., ARL8A and ARL8B in mammalian cells), an N-acetylated member of the ADP ribosylation factor family of small GTPases10,17,18. When bound to the lysosome surface, active ARL8-GTP directly or indirectly via association with the adaptor protein PLEKHM2/ SKIP recruits kinesins KIF1A19,20, and KIF5A-C10 to promote microtubule-dependent lysosome dispersal to the cell periphery. In addition to PLEKHM2/SKIP and KIF1A, other known ARL8 effectors include the hetero-hexameric tethering complex HOPS21,22 that mediates autophagosome-lysosome fusion and the RUN- and FYVE-domain-containing proteins RUFY123, RUFY3, and RUFY411,24. Despite the essential role of ARL8 in controlling lysosome dynamics and function in cells and tissues18,25, we know surprisingly little about the regulatory factors that control ARL8 localization and activity. It has been demonstrated that the recruitment of ARL8 to the surface of lysosomes and lysosome-related organelles is promoted by the evolutionarily conserved multisubunit BLOC-one-related complex (BORC)26,27, but the underlying mechanism remains uncertain. Importantly, no specific factors (e.g., guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs)) that control the nucleotide status and, thereby, the activity of ARL8 have been identified.

Here, we fill this knowledge gap by identifying TBC1D9B, a poorly characterized endo-lysosomal member of the Tre-2/Bub2/Cdc16 (TBC) domain protein family28, as a negative regulator of ARL8B function and an interaction partner of the multispanning lysosomal membrane protein TMEM55B29–31. TMEM55B is a 284-residue lysosomal protein and has been annotated as a phosphatidylinositol phosphatase (also named as PIP4P1)32. However, TMEM55B lacks sequence similarity to other lipid phosphatases, and in vitro activity as a lipid phosphatase could not be validated in a later study33. The crystal structure of TMEM55B also does not support its function as a phosphatase, but rather suggests a role as a protein-protein interaction platform34. In support of this, it was shown that TMEM55B promotes the perinuclear clustering of lysosomes via a mechanism that depends on the scaffold protein c-Jun N-terminal kinase (JNK)-interacting protein 4 (JIP4), which serves as an adaptor for the microtubule minus end-directed dynein-dynactin motor complex30,31,35. Other known interactors of TMEM55B include the phospho-RAB effector protein RILPL136 and members of the NEDD4-like family of ubiquitin E3 ligases30.

Here, we use affinity purification-mass spectrometry and co-immunoprecipitation to identify TBC1D9B as a binding partner of TMEM55B and a negative regulator of ARL8B activity. Knockout (KO) of either TMEM55B or TBC1D9B causes the ARL8-dependent dispersion of lysosomes to the cell periphery. We further demonstrate that loss of TBC1D9B impairs starvation-induced autophagic flux and lysosomal proteolysis via a mechanism that depends on its GAP activity and on the presence of ARL8B. These findings unravel a crucial role for TBC1D9B in regulating lysosome function by repressing ARL8B activity.

Results
TMEM55B directly interacts with TBC1D9B
Given the roles of TMEM55B in lysosome positioning31 and counteracting neurodegeneration36 and oxidative stress30, we searched for TMEM55B-interacting proteins. To this aim, we combined GFP nanobody-based affinity purification with mass spectrometric analyses (AP-MS) of material isolated from lysates of HEK293 cells expressing TMEM55B-eGFP or eGFP (Fig. 1a). Robust filtering of the candidate list for hits near undetectable in the negative controls revealed the bait TMEM55B as the top hit (Fig. 1b). JIP4, a previously identified interactor of TMEM55B31, was also among the top hits, validating our screen. Proteins functioning in “Golgi organization”, “localization to endoplasmic reticulum exit site”, and notably, “endomembrane organization” were enriched in our candidate list according to Gene Ontology terms (Fig. 1c). When we compared our top candidate hits with TMEM55B interaction partners identified in the BioPlex 2.0 dataset37, we found an overlap of four proteins of the top 25 (our AP-MS screen)/20 (BioPlex screen) hits, underscoring the high specificity and reproducibility of our screen. Among these proteins were JIP4, RNF213, BIRC6, and TBC1D9B (Fig. 1d). TBC1D9B was particularly interesting because of its putative function as a GTPase-activating protein (GAP) and its hypothesized role in endo-lysosomal trafficking, possibly by modulating the function of RAB proteins (i.e., RAB11A)38 and/or related small GTPases. We bidirectionally validated the interaction between TMEM55B and TBC1D9B by immunoprecipitation in HeLa cells. TMEM55B-GFP was robustly co-precipitated with TBC1D9B-HA (Fig. 1e). Conversely, TBC1D9B-GFP co-purified with TMEM55B (Fig. 1e). No co-immunoprecipitation was observed for GFP alone. To further examine the physical interaction between TMEM55B and TBC1D9B, we used CRISPR/Cas9 genome editing to establish knock-in cells expressing TBC1D9B carrying a C-terminal HA-tag from its endogenous locus (Fig. 1f and Supplementary Fig. 1a, b), enabling detection with high-affinity antibodies. Tagged endogenous TMEM55B was specifically associated with TBC1D9B-HA (Fig. 1g). These data reveal complex formation between endogenous TMEM55B and TBC1D9B proteins. We then tested whether TMEM55B directly binds to TBC1D9B. To this aim, we expressed and purified the cytoplasmic domain of TMEM55B fused to GST and incubated it with recombinant TBC1D9B-eGFP produced in insect cells. In line with our co-immunoprecipitation results, we found that recombinant, purified TBC1D9B-eGFP directly associates with GST-TMEM55B but not with GST alone (Fig. 1h). Hence, TMEM55B directly interacts with TBC1D9B.

TBC1D9B is a multi-domain protein comprising two N-terminal “glutamic acid (E)-rich repeat accumulated in membrane” (GRAM) domains, a TBC domain, an EF-hand, and a putative “α-interaction domain” (AID) domain. We generated and analyzed a series of deletion constructs to dissect the TBC1D9B domain(s) responsible for complex formation with TMEM55B (Fig. 1i). We then conducted co-immunoprecipitation experiments from lysates of cells co-expressing TMEM55B-eGFP with individual HA-tagged TBC1D9B-domains (C-terminus (aa 810–1250), N-terminus (aa 1–450), TBC-domain (aa 450–810), TBC+EF-hand domain (aa 450–950), TBC+GRAM domain (aa 301–810)) (Fig. 1j). Robust co-precipitation of TBC1D9B-domains with TMEM55B-eGFP was observed for the C-terminus, the N-terminus, and the TBC+EF-hand domains, but not for the TBC-domain alone. Hence, complex formation between TBC1D9B and TMEM55B is mediated by multiple sites but does not involve the TBC domain that harbors its GAP activity.

Finally, to gain initial insight regarding the interaction site for TBC1D9B within TMEM55B, we examined the AlphaFold2 model of TMEM55B (Supplementary Fig. 1c). This analysis, together with co-immunoprecipitation experiments, revealed that TMEM55B associates with TBC1D9B via a folded cysteine-rich domain starting from amino acid 65 (PDB: AF-Q86T03-F1-v4, AlphaFold), whereas the unstructured N-terminus of TMEM55B (i.e., amino acid residues 13–65) was dispensable for binding (Supplementary Fig. 1d, e).

Collectively, these data identify TBC1D9B as a binding partner of the integral lysosomal membrane protein TMEM55B.

Loss of TBC1D9B or its binding partner TMEM55B causes lysosome dispersion
Previous studies have implicated TMEM55B in lysosome positioning, e.g., overexpression of TMEM55B causes lysosomes to coalesce in the cell center31. We, therefore, hypothesized that TBC1D9B might control lysosome position akin to its binding partner TMEM55B. To test a possible physiological function of TBC1D9B in lysosome positioning, we used CRISPR/Cas9 to generate knockout (KO) HeLa cells lacking TMEM55B or TBC1D9B (Fig. 2a). Confocal imaging of wildtype vs TMEM55B KO cells stained for endogenous LAMP2 to mark lysosomes, paired with automated image analysis by OrgaMapper39 revealed the accumulation of lysosomes in the cell periphery in TMEM55B KO cells, in agreement with previous work using siRNA11,31 (Fig. 2b). Importantly, the loss of TBC1D9B also caused lysosomes to accumulate in the cell periphery, e.g., proximal to focal adhesion points, with a concomitant depletion of lysosomes within the perinuclear region (Fig. 2b–d), phenocopying TMEM55B KO in cells. TMEM55B and TBC1D9B KO cells, both displayed a trend towards increased cell size (Fig. 2b) and therefore we used cell size normalization in the quantification of lysosome positioning (see “Methods” section). Further, kinetic analysis of lysosome-movement by live-cell imaging of cells stained with LysoPrime Green revealed a significant increase in the fraction of motile lysosomes in both TMEM55B and TBC1D9B KO compared to wildtype cells (Fig. 2e). Moreover, the average speed of lysosome movement was increased in TBC1D9B KO cells, a phenotype that was rescued by re-expression of TBC1D9B-eGFP (Supplementary Fig. 2a). RAB11A-positive recycling endosomes were also partially redistributed to the cell periphery in TBC1D9B KO cells (Supplementary Fig. 2b), consistent with an earlier study and the known function of TBC1D9B as a GAP for RAB11A38. In contrast, the positioning of mitochondria (stained for cytochrome oxidase IV (Cox IV)), EEA1-positive early endosomes, and RAB8-positive endosomes was unaltered by the KO of either TMEM55B or TBC1D9B (Supplementary Fig. 2b). KO of TBC1D9, a TBC protein related to TBC1D9B, caused a mild accumulation of lysosomes in the cell periphery and a this phenotype was exacerbated by double loss of both TBC1D9 and TBC1D9B (TBC1D9/TBC1D9B DKO) (Supplementary Fig. 2c).

Given the physical association of TBC1D9B with lysosomal TMEM55B, we focused on the function of TBC1D9B at lysosomes. First, we examined the localization of TBC1D9B. TBC1D9B-HA expressed in TBC1D9B KO cells lacking the endogenous protein was found to partially colocalize with lysosomes marked by LAMP2 (Fig. 2f). Second, we tested whether the role of TBC1D9B in lysosome positioning and dynamics requires its GAP activity. To analyze this, we conducted rescue experiments by re-expressing wildtype TBC1D9B-HA or a GAP activity-defective point mutant with mutations in the arginine (R) and glutamine (Q) fingers38,40 (i.e., R559A/Y592A/Q594A (TBC1D9BRYQ/AAA)) (see also Fig. 4a) in TBC1D9B KO cells. Automated image analysis of the distribution of lysosomes marked by LAMP2 demonstrated that catalytically active wildtype TBC1D9B but not the inactive GAP mutant could rescue lysosome dispersion induced by loss of TBC1D9B (Fig. 2g, h).

Collectively, these data show that TBC1D9B and its binding partner TMEM55B control lysosome positioning and, thereby, autophagic flux and the cellular response to starvation.

TBC1D9B and TMEM55B are required for the cellular response to starvation
Previous work has shown that the cellular response to starvation involves the perinuclear clustering of lysosomes and elevated lysosomal proteolysis, e.g., increased cathepsin activity7,16. To probe whether loss of TBC1D9B or TMEM55B affects the cellular starvation response, we monitored the distribution of lysosomes in cells subjected to starvation. As expected, starvation induced a marked relocalization of lysosomes towards the microtubule-organizing center in wildtype cells. In contrast, lysosomes maintained their largely peripheral distribution in TMEM55B or TBC1D9B KO cells (Fig. 3a, b). Next, we tested the consequences of the altered distribution of lysosomes in TMEM55B- and TBC1D9B KO cells on lysosomal proteolysis probed by SiR-Lyso, a pepstatin A-coupled silicon rhodamine derivative that binds cathepsin D. TMEM55B- and TBC1D9B KO cells tended to display a slight, albeit statistically insignificant increase in cathepsin D activity under fed steady-state conditions (Supplementary Fig. 3b). Importantly, TMEM55B- and TBC1D9B KO cells—unlike wildtype cells—failed to upregulate cathepsin D activity in response to starvation (Fig. 3c, d). We challenged these results from cathepsin D activity-based probes by assaying proteolytic cleavage of DQ-BSA, a quenched fluorophore coupled to BSA taken up by endocytosis that emits fluorescence upon lysosomal proteolysis. As seen for SiR-Lyso, TMEM55B- and TBC1D9B KO cells revealed a significant defect in lysosomal proteolysis in response to starvation (Supplementary Fig. 3a); no significant differences were observed in the uptake of fluorescently labeled dextran, excluding the possibility that the observed differences are due to reduced DQ-BSA endocytosis (Supplementary Fig. 3b).

To functionally probe starvation, we employed a recently developed pulse-chasable reporter processing assay for mammalian autophagic flux that is based on a HeLa cell line stably expressing Halo-LC3B. In this assay, the autolysosomal cleavage of Halo-LC3B to yield free Halo ligand detected by in-gel fluorescence imaging, reflects autophagic flux41. We effectively depleted stable Halo-LC3B HeLa cells of endogenous TBC1D9B (Fig. 3e). We then subjected cells to starvation and monitored the ability of control vs. TBC1D9B-depleted cells to process Halo-LC3B to free Halo ligand. Whereas control and TBC1D9B-depleted HeLa cells displayed very low levels of free Halo ligand at steady state, TBC1D9B-knockdown cells suffered from a reduced ability to process Halo-LC3B to free Halo ligand in response to starvation compared to control cells (Fig. 3e), indicative of reduced autophagic flux. Reduced autophagic flux was also reflected by the partial accumulation of the autophagy adaptor and substrate p62, taken as a surrogate for autophagic flux, in TBC1D9B-depleted cells (Supplementary Fig. 3c, d).

Collectively, these data show that TBC1D9B and its binding partner TMEM55B control lysosome positioning and, thereby, autophagic flux and the cellular response to starvation.

Overexpression of TBC1D9B perturbs lysosomal positioning
Overexpression of TMEM55B causes perinuclear clustering of lysosomes31. We, therefore, tested the effect of TBC1D9B overexpression on lysosomal positioning. Ectopic expression of HA-tagged TBC1D9B in wildtype HeLa cells, followed by immunofluorescence staining, revealed a mostly cytoplasmic localization of TBC1D9B (Fig. 4a, b, d), in contrast to what was observed in TBC1D9B KO cells. Automated image analysis by OrgaMapper revealed the perinuclear clustering of lysosomes toward the cell center in cells overexpressing TBC1D9B (Fig. 4e), a phenotype similar, although less prominent, to that elicited by overexpression of TMEM55B. In contrast, overexpression of GAP-defective TBC1D9BRYQ/AAA did not affect lysosome positioning (Fig. 4c), consistent with the hypothesis that TBC1D9B functions as a GAP protein to control lysosome dynamics. Wildtype and GAP-defective TBC1D9BRYQ/AAA showed a similar degree of co-localization with LAMP2 (Fig. 4d).

TBC1D9B directly interacts with active ARL8B
The data presented thus far show that TBC1D9B controls lysosome positioning in various cell types by serving as a GAP protein. We tested the hypothesis that TBC1D9B might control lysosome position via its known role as a GAP for RAB11A. To this aim, we monitored lysosome position in HeLa cells depleted of endogenous RAB11. We found that knockdown of RAB11 alone had no effect on lysosome distribution. Moreover, co-depletion of RAB11 and TBC1D9B in double knockdown cells did not show additive effects compared to the single loss of TBC1D9B (Supplementary Fig. 4a, b). These data suggest that the effects of TBC1D9B on lysosome distribution and function are not mediated via its known regulatory role on RAB11.

Previous work had identified ARL8 as an important regulator of axonal transport of lysosomes and synaptic vesicle precursor organelles in human induced pluripotent stem cell-derived neurons (iN)20. In a parallel independent effort to identify regulators of ARL8 function, we used lentiviral constructs to express ARL8B-TurboID or TurboID tagged with a nuclear export signal as a negative control in iN and analyzed proteins captured on paramagnetic streptavidin beads by semi-quantitative mass spectrometry (Fig. 5a). This analysis, in addition to known interactors of ARL8B, such as the kinesin adaptor PLEKHM2 (SKIP)10, revealed the abundant presence of TBC1D9B (Fig. 5b). Given these findings and the fact that KO of either TMEM55B or TBC1D9B closely resembles ARL8 overexpression17,42, we hypothesized that TBC1D9B might control lysosome positioning by acting on ARL8, i.e., a small GTPase for which no GAP protein has been identified.

To test this hypothesis, we analyzed the association of TBC1D9B with ARL8B. First, we assayed the ability of both proteins to undergo complex formation in HeLa cells co-expressing TBC1D9B-HA together with wildtype, active GTP-locked (Q75L), or inactive GDP-locked (T34N) variants of ARL8B-eGFP. Immunoblot analysis of material captured via GFP anotrap beads revealed a vivid TBC1D9B association with wildtype or constitutively active GTP-bound ARL8B(Q75L) but not with the inactive GDP-locked ARL8B variant (T34N) (Fig. 5c). A GAP-activity-defective TBC1D9B mutant (TBC1D9BRYQ/AAA) retained the ability to interact with ARL8B-GFP (Q75L) (Supplementary Fig. 4c). Surprisingly, the ability of TBC1D9B to bind to ARL8 was restricted to the ARL8B isoform, as overexpressed wildtype or active GTP-locked (Q75L) ARL8A-eGFP failed to co-immunoprecipitate with TBC1D9B-HA (Supplementary Fig. 4d), despite >90% sequence identity between both isoforms (Supplementary Fig. 4e). Next, we tested whether TBC1D9B and ARL8B directly interact. To test this, we conducted affinity chromatography experiments using recombinant glutathione S-transferase (GST)-tagged ARL8B (Q75L) or (T34N) as a bait and incubated these with recombinant TBC1D9-eGFP. Only the GTP-locked (active) ARL8B variant (Q75L) showed a robust direct interaction with TBC1D9B-eGFP (Fig. 5d and Supplementary Fig. 5a). Moreover, we found that TMEM55B, TBC1D9B, and ARL8B are capable of forming a tripartite complex in vitro. GST-TMEM55B, but not GST alone, was able to capture a complex between recombinant TBC1D9B-eGFP and native untagged ARL8B (Fig. 5e). Finally, we found that the overexpression of active GTP-locked ARL8B-eGFP (Q75L) in HeLa was sufficient to facilitate the recruitment of TBC1D9B to lysosomes in the cell periphery marked by LAMP2, while inactive ARL8B-GFP (T34N) failed to affect TBC1D9B localization (Fig. 5f).

These results identify TBC1D9B as a specific direct binding partner of active ARL8B-GTP in vitro and in living cells and reveal the formation of a ternary interaction between TMEM55B, TBC1D9B, and ARL8B.

TBC1D9B facilitates GTP hydrolysis by ARL8B
Our collective results show that the TBC domain protein TBC1D9B directly binds to ARL8B in its GTP form, akin to the association of GAP proteins with their cognate active small GTPases. Moreover, KO of TBC1D9B (compare Fig. 3) or overexpression of ARL816,17,21,42 causes the peripheral dispersion of lysosomes, while conversely, the overexpression of TBC1D9B leads to perinuclear clustering of lysosomes similar to the loss of ARL8B16,21 (compare Fig. 4). Hence, TBC1D9B appears to act as a negative regulator of ARL8 function.

We used AlphaFold3 to obtain a structural model for the complex between TBC1D9B and ARL8B bound to GTP (Fig. 6a and Supplementary Fig. 5a). The interaction was predicted with very good confidence and revealed residues (e.g., R559, Y592, and Q594) critical for GAP activity40 to contact the GTP binding pocket of ARL8B (Fig. 6b). Based on this analysis, we hypothesized that TBC1D9B may facilitate GTP hydrolysis by ARL8B. We tested this by purifying recombinant His10-TBC1D9B wildtype or a GAP-inactive mutant (R559A, Y592A, and Q594A) from Sf21 insect cells (Supplementary Fig. 5c). Moreover, we expressed and purified recombinant GST-tagged RAB11A, a known substrate for TBC1D9B, as well as full-length GST-ARL8B (Supplementary Fig. 5b). We then conducted GDP Glo assays to analyze the ability of ARL8B (6 μM) to hydrolyze GTP in the absence or presence of a substoichiometric concentration (0.5 μM) of wildtype or GAP-defective mutant (i.e., TBC1D9BRYQ/AAA) recombinant TBC1D9B. We found that active wildtype TBC1D9B stimulated GTP hydrolysis by ARL8B in a time-dependent manner, whereas the GAP-defective mutant was inactive (Fig. 6c). Recombinant wildtype or mutant TBC1D9B did not display any measurable GTP hydrolyzing activity on their own (Supplementary Fig. 5d). GST-ARL8B alone was also nearly inactive (Fig. 6c). Importantly, the GAP activity of TBC1D9B towards ARL8B was comparable to its ability to stimulate GTP hydrolysis by its known substrate RAB11A (Supplementary Fig. 5e, f). These data show that TBC1D9B facilitates GTP hydrolysis by ARL8B. It is conceivable that the GAP activity observed in our in vitro assays may be further stimulated in vivo by interactions of TBC1D9B with other proteins (e.g., TMEM55B), by membrane lipids, and/ or via posttranslational modifications that could alter the conformational state and, thereby, the activity of TBC1D9B.

A prediction from the model that TBC1D9B serves as a negative regulator of ARL8B function (e.g., by serving as a GAP) is that loss of TBC1D9B and, possibly, of its lysosomal binding partner TMEM55B should facilitate its recruitment to lysosomes. Indeed, we observed that loss of TMEM55B or TBC1D9B in KO cells boosted ARL8B-eGFP recruitment to lysosomes marked by LAMP2 (Fig. 6d).

Collectively, these findings identify TBC1D9B as a negative regulator of ARL8B function in vitro and in living cells.

TBC1D9B controls lysosome function via ARL8B
Finally, we wanted to know whether its effects on ARL8B mediate the cellular effects of TBC1D9B loss. A prediction from our model is that the phenotypes caused by TBC1D9B loss should be occluded by the concomitant depletion of ARL8B. KO of TBC1D9B caused the dispersion of lysosomes to the cell periphery in control cells. In contrast, in cells depleted of ARL8B (Supplementary Fig. 5e), lysosomes remained perinuclearly clustered, irrespective of the presence or absence of TBC1D9B or its interactor TMEM55B (Fig. 7a, b). Hence, TBC1D9B controls lysosome positioning via a mechanism that depends on ARL8B.

Lysosomes are the degradative endpoint for autophagic cargo delivered to their lumen via fusion of autophagosomes (e.g., via the ARL8B-GTP effector HOPS22) under nutrient starvation conditions. Given the defects of TBC1D9B-depleted cells with respect to autophagic flux and the lysosomal starvation response (compare Fig. 3), we monitored the presence of LC3-positive autophagosomes in cells depleted of TBC1D9B, ARL8A/B, or both proteins. We observed that cellular depletion of TBC1D9B caused a significant reduction of autophagosomes marked by LC3 under nutrient starvation conditions. Conversely, we found autophagosomes to accumulate in double KO cells lacking ARL8A and ARL8B expression. This accumulation of LC3-positive autophagosomes persisted in ARL8A/B DKO cells depleted of TBC1D9B (Fig. 7c, d), suggesting that the partial depletion of LC3-containing autophagosomes in TBC1D9B-knockdown cells depends on the function of ARL8.

These collective data suggest that TBC1D9B controls lysosome function and autophagic flux via ARL8B (Fig. 7e).

Methods
Ethics statement
Experiments involving human iPSC-derived neurons (iNs) were performed using the iPSC line BIHi005-A. BIHi005-A cells were generated by Sendai virus–mediated reprogramming of human dermal fibroblasts. Subsequently, the transcription factor NGN2 (originally provided by laboratories at Stanford University, CA, USA) was introduced under the control of a doxycycline-inducible rtTA element via CRISPR/Cas9-mediated genome engineering into the AAVS1 safe harbor locus. The donor material was fully anonymized and cannot be traced back to the individual. The cell line is registered in hPSCreg, and registration details, including IRB approval and signed informed consent information, are available at [https://hpscreg.eu/cell-line/BIHi005-A].

Cell lines, antibodies, and reagents
Chemicals and antibodies. If not stated otherwise, analytical-grade chemicals were purchased from Sigma-Aldrich (MO, USA). The following primary antibodies were used:

Antibodies Source Identifier RRID
Actin Sigma Cat#A5441 RRID:AB_476744
Alexa Fluor 488 anti-rat probes Thermo/molecular probes Cat#A11006 RRID:AB_141373
Alexa Fluor 594 anti-rabbit probes Thermo/molecular probes Cat#A11037 RRID:AB_2534095
Alexa Fluor 488 anti-mouse probes Thermo/molecular probes Cat#A21202 RRID:AB_141607
Alexa Fluor 647 anti-mouse probes Thermo/molecular probes Cat#A21236 RRID:AB_2535805
Alexa Fluor 568 anti-rabbit probes Thermo/molecular probes Cat#A11036 RRID:AB_10563566
Alexa Fluor 647 anti-rabbit Invitrogen Cat#A21244 RRID:AB_2535812
ARL8A/B Abcam Cat#ab281997
ARL8B Proteintech Cat#13049-1-AP RRID:AB_2059000
Calnexin Proteintech Cat#10427-2-AP RRID:AB_2069033
CF647 anti-mouse Biotium Cat#20281 RRID:AB_10853311
CoxIV (clone 3E11) Cell signaling Cat#4850 RRID:AB_2085424
EEA1 (clone C45B10 Cell signaling Cat#3288 RRID:AB_2096811
GFP (clone 7.1+13.1) Sigma Aldrich Cat#11814460001 RRID:AB_390913
HA (clone 3F10) Sigma Aldrich Cat# 11867423001 RRID:AB_390918
Halo Promega Cat#G9211 RRID:AB_2688011
IRDye 800CW anti-mouse LI-COR Cat#926-32210 RRID:AB_621842
IRDye 800CW anti-rabbit LI-COR Cat#926-32211 RRID:AB_621843
LAMP1 Cell signaling Cat#9091 RRID:AB_2687579
LAMP2 Abcam Cat#ab18528 RRID:AB_775981
LAMP2 (clone H4B4) DSHB Cat#sc-18822 RRID:AB_626858
LC3 MBL Cat#M152-3 RRID:AB_10949134
TBC1D9B Bethyl laboratories Cat#A304-655A RRID:AB_2620850
TMEM55B Proteintech Cat#23992-1-AP RRID:AB_2879391
RAB8 Cell signaling Cat#6975 RRID:AB_10827742
RAB11A Invitrogen Cat#71-5300 RRID:AB_87868

Chemicals and reagents
Cell tracker green CMFDA Invitrogen Cat#C7025
Complete, mini protease inhibitor cocktail Roche Cat# 11836153001
Digitonin Invitrogen Cat# BN2006
FastDigest BpiI Thermo Cat#FD1014
GFP-Trap® magnetic agarose Proteintech Cat#gtma
Goat serum Gibco Cat# 16210072
Igepal Sigma-Aldrich Cat# CA-630
Intercept (TBS) blocking buffer LI-COR Cat# 927-60001
INTERFERin VWR Cat# 101000028
jetPrime VWR Cat# 101000001
Matrigel Corning Cat#356231
NucleoSpin tissue Macherey-Nagel Cat# 740952.250
Phosphatase inhibitor cocktail 2 Sigma-Aldrich Cat#P5726
Phosphatase inhibitor cocktail 3 Sigma-Aldrich Cat#P0044
Puromycin Gibco Cat# A1113803
Saponin Sigma Cat#47036
Shandon Immu-Mount Epredia Cat# 9990402

Oligonucleotides (siRNA)
siARL8B Dharmacon/Horizon Cat#L-031872-02
Sigma scr control Sigma Mission® siRNA universal negative control #1 Cat#SIC001
siTBC1D9B Dharmacon/Horizon Cat#L-020294-01

Cell lines. U2OS and HeLa cells were sourced from ATCC. The cell lines were cultured in DMEM medium containing 4.5g/ml glucose and L-glutamine (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 100U/ml penicillin and 100μg/ml streptomycin (Gibco). Cells were cultured in Dulbecco´s modified Eagle medium (DMEM, Gibco) with 10% (v/v) fetal calf serum (FCS, Thermo Fisher) and Penicillin/Streptomycin (PenStrep) at 37°C with a 5% CO2 atmosphere. Cell lines were routinely tested for Mycoplasma contamination, and all tests were negative.

Plasmids and cloning
hTBC1D9B-mCherry was a kind gift Drs. Xiao-Ming Yin and Yong Liao (Indiana University-Purdue University Indianapolis) and subcloned into the pcDNA3.1 Hygro (+) vector with a C-terminal HA-tag with HindIII and BamHI and into the pEGFP-N1 vector with a C-terminal GFP-tag. The cDNA encoding full-length TBC1D9B WT-GFP and TBC1D9B WT was subcloned from the mammalian expression vector (peGFP-N1-TBC1D9B) into pFL-His10-StrepII vector through Gibson assembly. Additionally, the GAP mutant region was subcloned from the mammalian expression vector (pcDNA3.1 Hygro (+)-TBC1D9B GAP mutant) into pFL-10His-StrepII-TBC1D9B WT vector through the KasI and SbfI sites within TBC1D9B.

Plasmids Backbone Insert gene Insert species
ARL8B-WT-GFP pEGFP-N1 ARL8B with GFP tag Human
ARL8B-QL-GFP pEGFP-N1 ARL8B Q75L with GFP tag Human
ARL8B-TN-GFP pEGFP-N1 ARL8B T34N with GFP tag Human
GST-ARL8B pGEX6P1 ARL8B with GST tag Human
GST-RAB11A pGEX-5X RAB11A with GST tag Canine
TMEM55B-HA pcDNA3.1 Hygro(+) TMEM55B with HA tag Mouse
TMEM55B-GFP pEGFP-N1 TMEM55B with GFP tag Mouse
TMEM55B-Δ13-65-HA pcDNA3.1 Hygro(+) TMEM55B with deletion of amino acids 13–65 with HA tag Mouse
TBC1D9B-HA pcDNA3.1 Hygro(+) TBC1D9B with HA tag Human
TBC1D9B-GFP pEGFP-N1 TBC1D9B with GFP tag Human
TBC1D9BRYQ/AAA-HA pcDNA3.1 Hygro(+) TBC1D9BRYQ/AAA-HA Human
TBC1D9B-TBC-HA pcDNA3.1 Hygro(+) TBC domain of TBC1D9B with HA tag Human
TBC1D9B-CT-HA pcDNA3.1 Hygro(+) C-terminal domain of TBC1D9B with HA tag Human
TBC1D9B-NT-HA pcDNA3.1 Hygro(+) N-terminal domain of TBC1D9B with HA tag Human
TBC1D9B-TBC+CT-HA pcDNA3.1 Hygro(+) TBC and C-terminal domain of TBC1D9B with HA tag Human
TBC1D9B-TBC+NT-HA pcDNA3.1 Hygro(+) TBC and N-terminal domain of TBC1D9B with HA tag Human

Mutagenesis of TBC1D9B
Mutations were inserted using mutagenesis or fusion-PCR. PCR mixtures were in both cases prepared with 0.2mM dNTPs, 1x Phusion GC buffer, 0.2µM of overlapping primers containing the desired mutation, 3% DMSO, and Phusion high-fidelity DNA polymerase. The PCR reaction was carried out in a FlexCycler2 PCR device (Analytik Jena) with 98°C denaturation, 45–55°C annealing, and 72°C elongation temperature. For the site-directed mutagenesis, the PCR product was incubated with DpnI at 37°C for 1h to digest the PCR template before transformation. Two sets of PCR were needed for the fusion-PCR. A first set to create at the mutation ending and beginning PCR products with an overlap, and another PCR to fuse these two PCR products.

CRISPR/Cas9-mediated gene editing
TMEM55B, TBC1D9, and TBC1D9B CRISPR/Cas9 mediated HeLa knockout cells were generated with CRISPR guide RNAs (“gene knockout kit”) from Synthego (Redwood City, CA, USA) with the following sequence: TBC1D9: CGGCGGCGGACUGGCGGGUA, ACCCAUACUUCAUCCUGCAG, CAUGUGGUGAACCCGGAGG, TBC1D9B: AGGGUGCCCACGAGAAGACC, CCUGUUCCCAGGUCUUCUCG. For the TMEM55B gene knockout, guides from the Synthego “gene knockout kit” with the following sequence were used: ACCGGCUCCGUACGGUGGUG, ACGUUUAGUGGGGCCCGGC, GUGGCGGCCAUGGCGGCAGA. Genomic HeLa Cells were generated with CRISPR/Cas9 mediated knockins using Alt-R sgRNA and ssODN Donors from IDT (Coralville, IA, USA) with the following sequences: Alt-R sgRNA: AGGCATCAGCCGGAAACTCC ssODN Donor: TGATGACACCTTGGGCCAGGCCTCACAGCTGCAGGCATCAGGCGTAGTCGGGGACGTCGTAGGGGTA GCCGGAAACTCCAGGCTGCTCATGGTCACTGGCGGTGCTGAACTGTC TC. CRISPR/Cas9-mediated knockout and knockin was performed using the Neon™ Transfection System (Thermo Fisher Scientific): Synthetic CRISPR guide RNAs, ssODN Donors and recombinant Cas9 (IDT) were electroporated according to the manufacturer’s protocol and recommendations (1005V, 35ms, 2 pulses). The medium was exchanged after reaching 90% confluence the next day, and diluted cells were seeded on 96-well plates to obtain single-cell clones. The single clones were expanded, and the editing was validated by immunoblot with specific antibodies against TBC1D9B and TMEM55B. The knock-in was additionally validated by Sanger sequencing and Synthego analysis tool (https://design.synthego.com/#/validate) analysis with the following Primers: amplification: CCGACTGGTGCATCTCCTTT and GAGAGCCTGTAATGCTAGGCAG sequencing: GAGAGCCTGTAATGCTAGGCAG.

U2OS ARL8 double knockout (DKO) cell line generation: pSpCas9(BB)-2A-Puro (PX459) V2.0 was sourced from Addgene (#62988). Oligonucleotides with gRNA sequences were annealed and cloned into the PX459 plasmid using the FastDigest BpiI (Thermo) restriction site. The following gRNA target sequences were employed to generate ARL8 DKO cell line: sgRNA for ARL8A: CGCGATCACGTTGACGAAGG, and sgRNA for ARL8B: AGAGATGGAGCTGACGCTCG20. The PX459 plasmids containing the corresponding sgRNAs were co-transfected into U2OS cells using jetPRIME, following the manufacturer’s instructions. Co-transfection was repeated after 48h. Two days after the second transfection, puromycin selection (2μg/ml) was applied to eliminate cells that had not been transfected with CRISPR plasmids. The selection process was maintained for 10 days, with medium changes every 2 days to replenish puromycin.

To generate clonal cell lines, limiting dilution cloning was conducted by plating cells into a 96-well plate at a density of 0.5–1 cell per well. Colonies derived from single cells were allowed to expand. Genomic DNA was then isolated with the help of NucleoSpin Tissue XS from the clonal cell lines and subjected to sequencing to identify frameshift mutations by amplifying the genomic regions with the following primers: for ARL8A KO, forward 5′- GATTCCGGGGCAGCGAGTCGT-3′ and reverse 5′-TACTAGGCCCCAAGCGCTCG-3′, and for ARL8B KO, forward 5′-TCATCTCCCGCCTGCTGGACT-3′ and reverse 5′-TCCAAATGCTGGCCCTCGG-3′. KO efficiency was assessed based on Sanger sequencing data using the Synthego analysis tool (https://design.synthego.com/#/validate). Additionally, DKO clones were verified by Western blot. Correct cell clones were expanded and cryopreserved in liquid nitrogen.

Transfection of cells with plasmid DNA
Cells were transfected using the proprietary cationic polymer TurboFectTM (Thermo Fisher). For transfection 1µL TurboFectTM per 500µg DNA was used and incubated with the DNA in serum-free media for 20min at room temperature. The mixture was dripped on adherent cells in DMEM with 10% FCS. The medium was changed after 4h.

siRNA-transfection
For ARL8B knockdown, cells were seeded into 6-well plates at a density of 2.5×105 cells per well and subjected to reverse transfection with 50nM siRNA using INTERFERin as per the manufacturer’s protocol. This transfection was repeated 24h later. For TBC1D9B KD, reverse transfection was similarly performed, with a subsequent repeat after 24h, utilizing 100nM siRNA and jetPRIME, following the manufacturer’s instructions. Cells were harvested and analyzed 72h post-initial transfection.

Starvation
Cells were washed three times with EBSS (Gibco) and incubated in EBSS for 6h to induce starvation.

Preparation of cell lysates and immunoblotting
To verify the knockdown of TMEM55B and TBC1D9B, cells were lysed on ice in PBS with 1% (v/v) Triton X-100 and 1x cOmplete Protease inhibitor cocktail (Roche) for 60min, followed by 3x 12s sonification with a Branson Sonifier 450 (Emerson Industrial Automation). Cell lysates were centrifuged at 12,000×g for 15min at 4°C. Protein concentration was measured with the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). For the SDS page of cell lysates and immunoprecipitation, the samples were denatured with Laemmli buffer at 95°C for 10min. Samples were separated via SDS-PAGE using 10% or 12.5% acrylamide gels and, in a semi-dry western blot, transferred to either nitrocellulose (GE Healthcare) or PVDF (GE Healthcare) membranes. Detection was carried out with the Lumigen ECL Ultra Kit (Beckman Coulter) in an Amersham Imager 680 (GE Healthcare).
To verify the knockdown of ARL8B and TBC1D9B, cells were lysed in a buffer containing 1% Igepal, 1M Tris pH 8, 150mM NaCl, and protease inhibitors cComplete mini, along with phosphatase inhibitor cocktails 2 and 3. Cell lysates were clarified by centrifugation, and the resulting supernatant was denatured with Laemmli buffer at 95°C for 10min. Lysates were resolved in 4–20% SDS-PAGE gels and transferred onto nitrocellulose membranes using transfer buffer (20% methanol). Membranes were blocked for 45min at RT in Intercept TBS Blocking Buffer, and further incubated with primary antibodies diluted in the same blocking buffer (overnight, at 4°C). After overnight incubation, membranes were washed 3 times with TBST, incubated with IRDye secondary antibodies (1h at RT), washed again 3 times again with TBST, and visualized using the LI-COR Odyssey FC 2800 imager. Quantification was performed using Fiji. Uncropped blots are presented in the Supplementary Information.

Proximity labeling proteomic analysis of ARL8B-TurboID-expressing iPSC-derived human neurons
Human iPSCs were differentiated into cortical neurons using an optimized method based on the rapid neuronal differentiation protocol described in refs. 20,60. WT iPSC-derived neurons were transduced with lentivirus expressing ARL8B-TurboID and TurboID-NES, respectively. A negative control group is also needed, in which no TurboID lentivirus was transduced. iNeurons were incubated with 50μM biotin for 8h to initiate labeling on DIV15. Then stop the labeling by moving cells onto ice and washing five times with DPBS. Neurons were lysed in RIPA lysis buffer, and biotinylated protein was enriched with streptavidin beads (Thermo Fisher Scientific, cat. no. 88817). Elute the enriched protein from the beads by boiling the sample in Laemmli sample buffer with 2mM biotin and 20mM DTT at 95°C for 10min, and resolved by SDS-PAGE. Two bands per lane were cut by hand, and in-gel tryptic digestion was performed. Tryptic peptides were analyzed by a reversed-phase capillary liquid chromatography system (Ultimate 3000 nanoLC system; Thermo Scientific) connected to an Orbitrap Fusion mass spectrometer and Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). Identification of proteins was performed using MaxQuant (version 1.6.26) software. Data were searched against the UniProt human protein database. The initial maximum mass deviation of the precursor ions was set at 20 ppm, and the maximum mass deviation of the fragment ions was set at 0.35 Da. Methionine oxidation was used as the variable modification, Cys carbamidomethyl was used as the fix modification. False discovery rates were <1% based on matches to reversed sequences in the concatenated target-decoy database. Proteins were considered if at least two sequenced peptides were identified (at least one unique peptide and at least two razor + unique peptides).

Affinity purification mass spectrometry (AP-MS)
For GFP-immunoprecipitation, HEK293 cells were transfected using x-tremeGeneTM (Roche, Rotkreuz, Switzerland) with eGFP-TMEM55B or GFP as a negative control. After 2 days of expression, cells were harvested in ice-cold PBS and subsequently lysed in AP-MS buffer (10mM Tris/Cl pH 7.5, 150mM NaCl, 10% Glycerol, 0.5mM EDTA, 0.5% Nonidet™ P40) supplemented with protease inhibitor cocktail (Roche, Rotkreuz, Switzerland) by sonification, followed by centrifugation (20,000×g for 10min. at 4°C) to remove cellular debris. Supernatants were subjected to immunoprecipitation using GFP-Trap beads (Proteintech, Planegg-Martinsried, Germany) blocked with 5% BSA in AP-MS buffer for 1h at 4°C. Beads were collected by centrifugation at 4°C and washed three times with AP-MS buffer and two times with AP-MS buffer without detergent. After washing, on-bead trypsin digestion was performed, following the GFP-Trap protocol, using sequencing-grade modified trypsin (Promega, Walldorf, Germany); see below.

Sample preparation for AP-MS
Protein pellets were resuspended in 50µL of 8M urea dissolved in 50mM ammonium bicarbonate (ABC) buffer. The same volume of 0.2% ProteaseMAX (Promega, Cat# V2072) solution in ABC buffer was added and incubated for one hour with vortexing. The disulfide bonds in proteins were reduced with 5mM Tris (2-carboxyethyl) phosphine (TCEP) for 20min at RT, followed by alkylation with 10mM iodoacetamide (IAA). Tubes were incubated in the dark for 15min and immediately quenched with excess (25mM) of TCEP prepared in ABC. Subsequently, proteins were digested overnight at 37°C using MS-grade trypsin (Promega, Cat# V5280). The next morning, the digestion reaction was stopped by acidification using 1% formic acid (FA). Desalting using C18 spin columns (Thermo Scientific, Cat# 89,870) was performed according to the manufacturer’s instructions. Peptide solutions were dried down in a refrigerated speed vac and stored at −80°C.

Tandem mass spectrometry analysis of affinity-purified samples
Three micrograms of each fraction or sample were auto-sampler loaded with an UltiMate 3000 HPLC pump onto a vented Acclaim Pepmap 100, 75μm×2cm, nanoViper trap column (Thermo Fisher Scientific, Cat# 164535) coupled to a nanoViper analytical column (Thermo Fisher Scientific, Cat# 164570, 3 μm, 100Å, C18, 0.075mm, 500mm) with stainless steel emitter tip assembled on the Nanospray Flex Ion Source with a spray voltage of 2000 V. An Orbitrap Fusion (Thermo Fisher Scientific) was used to acquire all the MS spectral data. Buffer A contained 94.785% H2O with 5% ACN and 0.125% FA, and buffer B contained 99.875% ACN with 0.125% FA. Each sample was analyzed in a single-shot analysis lasting 150min.
We used a CID-MS2 method. Briefly, ion transfer tube temp=300°C, Easy-IC internal mass calibration, default charge state=2, and cycle time=3s. Detector type set to Orbitrap, with 60K resolution, with wide quad isolation, mass range=normal, scan range=300–1500m/z, max injection time=50ms, AGC target=200,000, microscans=1, S-lens RF level=60, without source fragmentation, and data type= positive and centroid. MIPS was set as on, included charge states=2–6 (reject unassigned). Dynamic exclusion enabled with n=1 for 30s and 45s exclusion duration at 10ppm for high and low. Precursor selection decision=most intense, top 20, isolation window=1.6, scan range=autonormal, first mass=110, collision energy 30%, CID, Detector type=ion trap, OT resolution=30K, IT scan rate=rapid, max injection time=75ms, AGC target=10,000, Q=0.25, injections for all available parallelizable time.

AP-MS data analysis and quantification
Protein identification/quantification and analysis were performed with Integrated Proteomics Pipeline-IP2 (Bruker, Madison, WI) using ProLuCID,61,62, and DTASelect233,63. Spectrum raw files were extracted into MS1, MS2, and MS3 (For TMT experiments) files using RawConverter (http://www.scripps.edu/yates/Software.html). The tandem mass spectra were searched against the UniProt human protein database, including isoforms (downloaded on 01-01-2014), and matched to sequences using the ProLuCID/SEQUEST algorithm (ProLuCID version 3.1) with 5ppm peptide mass tolerance for precursor ions and 600ppm for fragment ions. The search space included all fully and half-tryptic peptide candidates within the mass tolerance window with no-miscleavage constraint, assembled, and filtered with DTASelect2 through IP2. To estimate peptide probabilities and false-discovery rates (FDR) accurately, we used a target/decoy database containing the reversed sequences of all the proteins appended to the target database (UniProt). Each protein identified was required to have a minimum of one peptide of minimal length of six amino acid residues within 10 PPM of the expected m/z; however, this peptide had to be an excellent match. After the peptide/spectrum matches were filtered, we estimated that the protein FDRs were ≤1% for each sample analysis. Resulting protein lists include subset proteins to allow for consideration of all possible protein forms implicated by at least two given peptides identified from the complex protein mixtures.

Protein expression and purification
His10-tagged TBC1D9B wildtype and TBC1D9BRYQ/AAA GAP mutant proteins were expressed in Sf21 insect cells using SF900-II serum-free medium (Thermo Fisher Scientific). Briefly, Sf21 cells (800ml) were grown to a density of approximately 1.5×10⁶ cells per ml and infected with 8ml of amplified baculovirus. Cells were harvested 48h post-infection. For purification, cell pellets were resuspended in 35ml of lysis buffer (50mM Tris, pH 7.5, 300mM NaCl, 10mM imidazole, 1mM dithiothreitol (DTT), 0.5% Triton X-100, and one tablet of protease inhibitor cocktail per 50ml), sonicated for 1min (1s on, 5s off), and centrifuged at 87,000×g for 20min. The supernatant was collected and incubated with 0.5ml of Ni-NTA agarose beads (Sigma) on a rotating wheel for 1h at 4°C. The beads were washed once with 10ml of lysis buffer, followed by three washes with 10ml of wash buffer (50mM Tris, pH 7.5, 300mM NaCl, 20mM imidazole, 1mM DTT). Proteins were eluted with 4ml of elution buffer (20mM Tris, pH 7.5, 300mM NaCl, 300mM imidazole, 5mM DTT). The Ni-NTA eluate was further purified by size-exclusion chromatography (SEC) using a buffer containing 20mM Tris, pH 7.5, 300mM NaCl, and 5mM DTT at 4°C. Purified proteins were concentrated to approximately 0.4mg/ml for TBC1D9B WT and 3mg/ml for the GAP mutant. Proteins were either used immediately for biochemical assays or flash-frozen in liquid nitrogen and stored at −80°C for future use.
For the expression of GST-ARL8B, the plasmid was transformed into E. coli BL21 DE3 cells and induced with 0.5mM IPTG overnight. The cells were lysed in 20mM Tris pH 7.5, 150mM NaCl, 5mM EDTA, 0.1% Triton-X, 5mM DTT, and one protease inhibitor tablet (Roche) by ultrasonication. The lysate was centrifuged at 27,000 rpm for 20min. The supernatant was incubated with pre-washed glutathione-coupled beads from Novagen on a rotation wheel for 1.5h. For GST-ARL8B, the beads were washed with 20mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, and eluted with 50mM reduced glutathione.

GST-pulldown assay
The GST-pulldown assay was performed with immobilized GST, GST-fused ARL8B Q75L (a constitutively active mutant), and ARL8B T34N (a dominant-negative mutant). Fifteen micrograms of immobilized GST-fused ARL8B Q75L and ARL8B T34N were complexed with 0.2mM GTPγS or GDP in 500µl binding buffer (50mM Tris pH 7.5, 150mM NaCl, 5mM DTT, 5mM MgCl2, 0.02% Triton X100) overnight at 4°C. Binding assays were started by adding 10µg purified His10-TBC1D9B-GFP. Samples were incubated on a rotation wheel for 1h at 4°C. Unbound material was washed out by 3x washes with binding buffer, and bound proteins were eluted with 2x SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and followed by GFP in-gel fluorescence or Coomassie blue staining.

In vitro GTPase-Glo assay
In vitro GTPase-Glo assay was performed using Promega’s GTPase-Glo Assay kit (V7861). His10-tagged TBC1D9B wildtype (WT) and TBC1D9BRYQ/AAA GAP mutant proteins were diluted in GAP buffer containing 5μM GTP and 1mM DTT in the absence or presence of GST-ARL8B or GST-RAB11A protein. Reaction mixes were incubated at 37°C for the indicated times. Once the GTPase reaction was completed, 5μl of reconstituted GTPase-Glo reagent was added to all the wells at room temperature. The plate was incubated at room temperature for 30min with gentle shaking. At the end of the incubation period, 10μl of the Detection reagent was added to all the wells. The luminescence signal was measured using a TECAN (Infinite MPLEX) plate reader after the plates were further incubated for 10min at room temperature.

Immunofluorescence microscopy
Cells were grown on glass coverslips and fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 20min at room temperature and permeabilized with 0.2% (w/v) saponin in PBS for 5min at room temperature. PFA-derived fluorescence was quenched with 0.12% (w/v) glycin in 0.2% saponin-PBS for 10min at room temperature. Blocking was carried out in a 10% (v/v) FCS in 0.2% (w/v) saponin-PBS blocking solution for 1h at room temperature. Coverslips were incubated overnight at 4°C with the primary antibody diluted in blocking solution and subsequently incubated at room temperature for 1h with the fluorophore-coupled secondary antibody (Alexa Fluor 488, 594, and 647).
For quantitative imaging and automated image analysis, cells were seeded at a density of 5×104 on 12 mm coverslips coated with Matrigel in 24-well plates. The following day, the cells were treated with 10μM CMFDA for 1h, then fixed in 4% paraformaldehyde/4% sucrose in PBS (15min at RT). After three washes with PBS, the cells were incubated with primary antibodies (1:100 in 10% goat serum/0.05% saponin in PBS for 1h, at RT). The cells were then washed three times with PBS and incubated with fluorophore-conjugated secondary antibodies (1:500 in 10% goat serum/0.05% saponin/PBS, with DAPI for 1h at RT). After the incubation, the cells were washed three times with PBS and mounted using Shandon Immu-Mount.
For LC3 staining, the permeabilization step was additionally included, performed with a buffer containing 20μM digitonin (in 10% goat serum in PBS for 10min, at RT). The cells were then washed three times with a buffer consisting of 10% goat serum and PBS. From this point onward, the detergent was excluded from the protocol, while all other steps were carried out as described above.
Finally, the coverslips were mounted on glass slides and embedded in Mowiol containing 1mg/ml 4′, 6-diamidino-2-phenylindol (DAPI). Fixed cells were analyzed with a Zeiss LSM980 fluorescence microscope equipped with an automated stage and the ZEN 3.1 (blue edition) software (Carl Zeiss Microscopy GmbH) or with Nikon CSU Spinning Disk using a 40x air objective (NA=0.95), hardware autofocus, and semi-automatic multi-position image acquisition: Random fields of view were selected based on DAPI signal, saved in the software, and imaged all at once.

Analysis of lysosome positioning
Lysosome positioning analysis in fixed cells was carried out using the OrgaMapper Fiji Plugin39. Briefly, the Plugin segments individual nuclei, filters them by size, excludes nuclei touching the edges of the field of view, segments single cells using background subtraction and seeded watershed segmentation, and detects lysosomes using the Laplacian of Gauss (LoG) filter and the Find Maxima command. The distance of each lysosome detection within the individual cell mask is then measured based on a Euclidean distance map from the edge of the nuclei mask. R scripts were used to compile all measured parameters into single Excel files for further analysis. The percentage of cells with peripherally lysosomes was calculated as previously39 to cancel out possible biases of cell size on lysosome positioning measurements. Briefly, the average distance of lysosomes to the nucleus, as well as the average distance normalized by the cell’s Feret’s diameter, was computed for the entire cell population of each experiment. Cells with above-average distance as well as above-average distance normalized by the cell’s Feret’s diameter were regarded as cells with a true peripheral lysosome phenotype. The percentage of cells with true peripheral lysosomes in each sample was plotted.

Lysosomal motility measurements
HeLa cells were grown on ibidi μ-Slide 8-well high dishes and grown to approximately 70% confluency. The cells were loaded with Lysotracker red, and imaged on a Zeiss LSM980 Airyscan 2 microscope with a 63× oil objective equipped with environmental control (37°C, and 5% CO2). Images were recorded in a 0.1s interval with 10 images in the Z-direction (0.5μm per image) over 3min. For starved conditions, HeLa wildtype and knockout cells were grown to near confluency in Greiner Bio-One 10-well glass-bottom dishes, starved in EBSS, loaded with Lysotracker red, and imaged live on a Nikon CSUW1 spinning disc microscope equipped with environmental control (humidity, 37°C, and 5% CO2) using a 60x oil objective. Time-lapse sequences were recorded with a 0.5-s interval for 15s. Analysis was performed per field of view using the TrackMate Fiji plugin.

DQ-BSA degradation assay
For degradation analysis, 100.000 HeLa cells were seeded on glass coverslips in a 12-well plate and incubated overnight (37°C, 5% CO2). The cells were subsequently incubated with 10µg/ml DQ-BSA (Thermo Fisher Scientific) in either DMEM with 1% FCS, 1% NEAA (Gibco), and 1% 1M HEPES (Gibco) or EBSS for 6h. Microscopy samples were prepared as described previously. Corrected total cell fluorescence (CTCF) measurement was carried out in ImageJ.

Dextran endocytosis assay
HeLa cells were prepared for equal to DQ-BSA degradation measurements. After overnight incubation, the cells were incubated for 30min with 0.5mg/ml dextran-Texas Red® (70.000 MW, Life Technologies) in DMEM supplemented with 1mg/ml BSA, washed 3 times with PBS, and cultivated in DMEM with 10% FCS or EBSS for 6h. The cells were fixed and imaged as described previously and analysed using ImageJ.

LC3 and SiR-lysosome intensities
All measurements were performed in the OrgaMapper Fiji plugin39. Single cells were segmented, LC3 or SiR-lysosome punctae were detected, and mean intensities on the detections were calculated per cell.

Measurement of p62 levels
Cells seeded on Matrigel-coated coverslips were washed 5 times in EBSS, then supplemented with full media or EBSS, and treated with bafilomycin A1 as indicated. Cells were incubated for 3h, and then supplemented additionally with 1µM CMFDA. Cells were finally washed once in PBS, fixed in 4% PFA/4% sucrose in PBS for 20min, washed twice in PBS, and stored in PBS.
For immunostainings cells were blocked for 30min in blocking buffer (10% GS, 0.3% Triton-X-100, 20mM HEPES pH 7.4), then incubated with anti-p62 antibody (1:200, BD Bioscience, 610832), washed three times for 10min in PBS, incubated with AF568-labeled goat-anti-mouse antibody, washed again three times in PBS for 10min, and finally mounted using ShandonTM Immu-MountTM containing Hoechst 33342.
Imaging was performed on a Nikon TiE2 (Nikon) equipped with a confocal spinning-disk unit (CSU-W1, Yokogawa) and a PL APO λ 40x/0.95 NA air objective, without additional magnification. The microscope was equipped with two sCMOS cameras (pco.edge, 4.2bi, 6.5μm/pixel, 2048×2048 pixel) and controlled with NIS-Elements software (Nikon). Images were taken with the following sequential fluorophore settings: Hoechst 33342 (Ex.: 405nm; Em.: 420–460nm), CMFDA (Ex.: 488nm; Em.: 500–550nm), AF568 (Ex.: 561nm; Em.: 574–626).
Image processing and quantitative analysis were performed with the open-source software Fiji (ImageJ). Automated single-cell segmentation, p62 detection, and intensity measurements were carried out using the custom-made ImageJ plugin OrgaMapper39.
All data are presented as mean ± SEM, and were obtained from ≥3 independent experiments with total sample numbers provided in the figure legends. Statistical significance was evaluated using Student’s t test or one-way ANOVA and Dunnett´s multiple comparisons test. Significant differences were marked as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Measurement of autophagic flux using Halo_LC3 stable cell lines
Cells were seeded into six-well dishes and treated as indicated. Media were supplemented with 100nM Halo ligand CA-JFX650. Cells were washed once in PBS, harvested by trypsinization, washed once in PBS, and resuspended in 75µl RIPA buffer containing protease inhibitors. Upon 15min incubation on ice, cell lysates were cleared by centrifugation for 15min at 10,000×g and subjected to SDS-PAGE and Western blot analysis. For each experiment, samples were loaded as technical duplicates. The incorporated Halo-ligand was detected by in-gel fluorescence on a Biorad ChemiDoc Imaging System controlled by Image Lab software, version 6.1.0. Halo-tag was detected by quantitative Western blotting using anti-Halo primary antibody (Promega, #G9211, 1:1000) and a fluorescently labeled secondary antibody. The fluorescent signal was detected on a LICOR bio detection system controlled by Image Studio Lite software.

Data and reproducibility
All statistical analyses were performed using GraphPad Prism version 7.04 or later. The statistical tests used are indicated in the figure legend. It is also indicated whether the error bar represents the SD or SEM. No sample-size calculation was performed, but statistical methods were used to calculate standard deviation as noted in the figure legends. Experiments were performed with at least n=3; n-numbers are provided in the figures/figure legends. Data were only excluded if obvious technical problems occurred during the experiments. Generally, no data were excluded. Unless otherwise stated, experiments with representative results were repeated at least three times. The number of independent experiments is provided in the figure legends. Statistical significance is represented with the following symbols: *p≤0.05; **≤0.01; ***p≤0.001; ****p≤0.0001; ns=no statistical difference. For all treatments, cultured cells of the required genotype/treatment were randomly allocated to the control and genotype/treatment groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript identifies the GTPase-activating protein (GAP) TBC1D9B as a crucial negative regulator of the lysosome-associated small GTPase ARL8B. The authors show that TBC1D9B binds directly to active ARL8B-GTP, stimulates its GTPase activity, and thereby modulates lysosome positioning and the cellular autophagic response. TBC1D9B interacts with the lysosomal membrane protein TMEM55B, which appears to support its localisation and function. Knockout and overexpression studies demonstrate that TBC1D9B and TMEM55B are necessary for proper lysosome dynamics and adaptation to nutrient starvation. This study offers novel mechanistic insight and proposes TBC1D9B as the first known GAP for ARL8B, marking a significant advance in understanding lysosomal regulation. The dataset is of high quality and convincing overall, however the following issues need to be addressed before the manuscript can be recommended for publication.\n1. Supplementary figures referenced throughout the text were not available in the submission system and full evaluation of critical claims (e.g., structure predictions, GTPase assays, and proteolysis assays) was impossible.\n2. The mechanism underlying the TMEM55B–TBC1D9B interaction requires clarification. Provide amino acid boundaries for the TBC1D9B fragments used, and reconcile the discrepancy where the N-terminal fragment interacts experimentally but is not predicted to bind by AlphaFold2.\n3. The structural models generated by AF2 should include confidence scores. The key residues mutated in the GAP-inactive TBC1D9B mutant (R559, Y592, Q594) must be clearly annotated and visualised. Triple mutation is likely to affect the structure of the protein (this could also be modelled by AF2). Can authors test single mutants of TBC1D9B to clarify the key residue in GAP function?\n4. The authors should attempt to investigate interactions between endogenous TMEM55B, TBC1D9B, and ARL8B.\n5. It is unclear whether the GAP-deficient TBC1D9B mutant localises to lysosomes to the same extent as the wild-type protein. This should be assessed and quantified.\n6. No statistical analyses are evident in some figures, e.g. in the starvation-induced changes in wild-type cells in Fig. 3b or in cathepsin activity in Fig. 3d. Can authors provide clarification? Significance markers and confidence intervals should be made clearer.\n7. ARL8B GTPase activity in the absence of TBC1D9B should be shown as a baseline control in Fig. 6c.\n8. The observed specificity of TBC1D9B for ARL8B and not ARL8A warrants further exploration, ideally through structural modelling or sequence comparison to elucidate the molecular basis of this selectivity.\n9. Given the high-confidence structural prediction, the authors may consider supporting their model further with molecular dynamics simulations to provide insight into the TBC1D9B– ARL8B interface and its catalytic implications.\n10. Is complex between TBC1D9B, TMEM55B and ARL8B possible? Can this be tested using AF2, MD and experimentally? How does TMEM55B affect GAP function of TBC1D9B?\n11. The authors are encouraged to include autophagy flux assays using reporters such as mRFP-GFP-LC3 or Halo-GFP-LC3, or use bafilomycin A1 treatment to assess LC3-II/p62 turnover.\nMinor:\n12. Human gene and protein names should be capitalised consistently throughout the manuscript (e.g., TBC1D9B, ARL8B).\n13. A few figure references are confusing or mislabelled (e.g., \"Figure 5a below\" in the main text), which should be corrected for clarity.\n14. Domain abbreviations (e.g., GRAM, EF-hand, AID) should be defined at first mention in the text for clarity.\n15. JIP4/TMEM55B introduction/discussion requires additional relevant reference: PMID: 36394115.", "reviewer_2": "This paper investigates the function of the known lysosomal membrane protein TMEM55B by looking for interaction partners by immunoprecipitation. They find a wide range of proteins enriched in precipitates, including a previously reported interactor JIP4. Amongst the other hits they select TBC1D9B, a member of large family of TBC proteins that act as GAPs on the Rab family of GTPases. They show that varying the levels of TBC1D9B or of TMEM55B alters the distribution of lysosomes. A GTPase Arl8 has been previously shown to be involved in lysosomal positioning and so they test a role for TBC1D9B in regulating Arl8B and find that it can bind the GTP-Arl8B and stimulate its GTPase activity. Finally, they show that the effects of TMEM55B and TBC1D9B on lysosomal positioning require Arl8 to be present.\nThere is a lot of interest in lysosomal positioning as it controls important aspects of lysosomal function and autophagy. The data in this paper are clearly presented and the text is well written. However, the authors do not make a very compelling case for TBC1D9B either binding TMEM55B or being a major regulator of Arl8. In addition, aspects of the work seem hard to reconcile with previously reported data on TMEM55B or TBC1D9B, some of which is not cited. As such the work does not seem suitable for publication. Below I have explained the major issues with the study, and hope that they will be of use for the authors to revise the paper for resubmission elsewhere.\nA) Paralogues. TMEM55B has a paralog TMEM55A that arose during vertebrate evolution and so is likely to have role that is at least partially redundant. Likewise, TBC1D9B has three closely related paralogs, TBC1D9, TBC1D8 and TBC1D8B. Again, these could well be partially redundant as this is often seen with paralogs that arose during vertebrate evolution. However, none these paralogs are mentioned in the paper. It may be that they are expressed at low levels in the cells under investigation, but they should at least have been mentioned, especially as the authors do mention the fact that Arl8 activity is shared by two paralogs Arl8A and Arl8B. Likewise, the authors do not mention that TMEM55B has the gene name of PIP4P1, reflecting reports that it has phosphatase activity on phosphatidylinositol phosphates. They may not believe this activity is correct, but again the gene name and this past work should have been mentioned and a brief reason stated for why they feel it can be ignored.\nB) Mouse KOs. The International Mouse Phenotyping Consortium (IMPC) has generated KO mice lacking TMEM55B and TBC1D9B. The mice are viable and fertile and show only a small number of subtle phenotypes, such as reduced startle reflex or hyperactivity. This seems very hard to reconcile with these two proteins being major regulators of Arl8 activity given that mice lacking Arl8 activity (through loss of BORC) show embryonic lethality. This is not mentioned or discussed in the paper.\nC) TMEM55B binding TBC1D9B. The authors identify TBC1D9B as an interactor of TMEM55B by doing an IP of over-expressed GFP-TMEM55B. This brought down a wide range of proteins mostly from other organelles such as ER exit sites. Of these >25 proteins TBC1D9B was selected for follow up. The authors show that over-expressed HA-tagged TBC1D9B co-precipitates with over-expressed GFP-TMEM55B. IPs of two over-expressed proteins are notoriously unreliable as both may be a sticky due to lacking normal interaction partners, and so usually one or other is examined at native levels using antibodies to the endogenous protein. The authors attempt to map the region of TBC1D9B that binds to GFP-TMEM55B, and are unable to map it to a specific region which is again indicative of non-specific binding. Finally, AlphaFold does not predict that the two proteins interact, and the interaction was not detected when GFP-TMEM55B expressed at native levels was precipitated by the OpenCell project.\nEven if the co-ip of TMEM55B and TBC1D9B is physiologically relevant, the authors do not show that it is direct, nor do they discuss the relationship of the interaction with the various others reported for TMEM55B (JIP4, RILPL1, NEDD4 etc) – ie are mutually exclusive.\nD) Effect of TBC1D9B on lysosomal positioning. The authors examine the effect on lysosomal localization of over-expressing or removing TMEM55B and/or TBC1D9B. Previous studies have already shown the TMEM55B has an effect, and the authors argue that this is mediated through TBC1D9B, presumably by inactivating Arl8B via its GAP activity. However, this is hard to interpret at Arl8B is known to mediate the recruitment to lysosomes of both kinesin (via PLEKHM2) and dynein (via RUFY3/4). Thus, altering levels of Arl8B activity might be expected to affect the speed rather than the overall distribution of lysosomes.\nE) TBC1D9B GAP activity on Arl8B. To determine if Arl8B is involved in the effects of TMEM55B and TBC1D9B, the authors use TurboID on Arl8A to look to see what proteins are close to it. The problem with this approach is that this will label proteins that are not directly binding to Arl8B but are simply on the lysosomal surface with Arl8B. The control that the authors use of a cytosolic TurboID is not suitable as it is not on the lysosomal surface. Having found that the known lysosomal protein TMEM55B is close to the lysosomal GTPase Arl8B, along with TBC1D9B, they then test direct binding and find evidence for TBC1D9B binding to the GTP-bound form of TBC1D9B. However, this could simply reflect it being an effector of Arl8B rather being its GAP. Moreover, the interaction is only seen with Arl8B rather that the closely related Arl8A, which is surprising, especially as there is good evidence that the two Arl8 paralogs are redundant and so any major regulators would be expected to act on both. They test directly GAP activity of TBC1D9B using GST-Arl8B and detect what they refer to as “low GAP activity” which “may be stimulated in vivo by membrane lipids or post-translational modifications” but this latter is just speculation.\nTBC1D9B having Arl8B GAP is also hard to reconcile with Arl8B-GFP being still on lysosomes when TBC1D9B-HA is over-expressed (Figure 5F). Indeed, there seems to be more Arl8B-GFP on lysosomes in the presence of TBC1D9B overexpression than without (compare Figure 5F with Figure 6D).\nOverall, the data in the paper do not support many of the conclusions that the authors wish to make. It may be that Arl8B recruits TBC1D9B to lysosomes as an effector – perhaps to inactivate its known substrate Rab11, but the data do not support the conclusion that TMEM55B binds directly to TBC1D9B, nor the conclusion that TBC1D9B has GAP activity on Arl8B.", "reviewer_3": "The manuscript ‘Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B’ by Duhay and Tian et al, describes a number of interesting findings with regards to the role of the protein TBC1D9B in lysosomal function. The study identifies TBC1D9B as a key negative regulator of the lysosomal small GTPase Arl8b, showing that it binds Arl8b-GTP and promotes its inactivation. In addition, the study shows that loss of TBC1D9B, or its partner TMEM55B, disrupts lysosome positioning. The authors conclude that the loss of TBC1D9B affects autophagic flux, an effect that is dependent on Arl8b.\nThe finding that TBC1D9B has GAP activity towards Arl8b, is novel, and will be of interest to a broad community of researchers interested in organelle biology, membrane trafficking, and cellular nutrient sensing. Generally, the manuscript is well-written, the methodology is appropriate and most of the conclusions drawn are supported by the data. However, some conclusions are not supported by sufficient evidence and would require additional experiments to be conducted. It is recommended that the authors revise the current manuscript to address a few main concerns, as well as a list of minor items, appended below.\nMain Items\n1) Figure 3c-d shows an impaired increase in Cathepsin D activity in response to starvation in TBC1D9B and TMEM55B KO lines. However, it is not clear why this is, or even how the increased activity in WT cells is working. The paper cited (which is from one of the corresponding authors of this manuscript), Ebner M. et al. 2023, showed a lipid switch at lysosomes that alters lysosome functions including proteolytic activity, and blocking this lipid switch alters lysosome position. Another study also cited here, Johnson D.E. et al. 2016, shows that peripheral lysosomes are more alkaline – this could also stand to reduce Cathepsin D activity. Or, do the authors think that 6h starvation is causing significant transcriptional changes that boost Cathepsin D activity?\nThe rationale and result should be discussed in more detail.\n2) In the text relating to Figure 4, I would hesitate to conclude anything about lysosome “dynamics”. All the data is based on images of fixed cells. Dynamics implies that one would be measuring lysosomal speed or velocity, motility tracking to determine if they are undergoing motor-based transport, or lysosomal morphology changes/membrane remodeling such as tubulation, fusion, fission.\nLive-cell imaging experiments would need to be performed to compute some of the above parameters and make conclusions about lysosome dynamics. Indeed, such additional data would strengthen the manuscript.\nHowever, at the very least, Figure 4 (and the corresponding section in the text) should be labeled and discussed as the effects on lysosome clustering/positioning upon overexpression of TBC1D9B and TMEM55B.\n3) What is the knockdown efficiency of siTBC1D9B in Figure 7c-d? Western blot or, if there is no suitable antibody, qPCR, should be conducted and shown.\n4) The single immunostaining experiment for LC3 is not sufficient to suggest that the lower levels of LC3 observed in the siTBC1D9B condition is due to increased autophagic flux as flux wasn’t measured. An autophagy flux assay (for example, Halo-Tag Flux assay PMID: 36095089 ) should be performed, if the authors wish to make these conclusions.\nOn that note, how does this result (if the authors do suggest there is increased autophagic flux in siTBC1D9B cells) compare to the findings from Liao et al. 2018 that concluded TBC1D9B promoted autophagic flux and it’s silencing reduced autophagic degradation?\nMinor Items\n-Regarding the callout of Figure 1f in the text: “Conversely, TBC1D9B-GFP was co-immunoprecipitated with TMEM55B (Figure 1f).” This second co-IP is in Figure 1e (right).\n-Figure 1f – I would recommend moving all the red labels to one side (i.e. right) of the drawn constructs as it is a bit difficult, at first, to determine which label goes with which fragment\n-Supplemental Figure 1c: What is the asterisk in the HA blot referring to? Legend says the asterisk shows cleaved GFP, but it is also shown in the HA blot.\n-Since OrgaMapper is used throughout to measure lysosome positioning, the authors should give a brief description (a couple of sentences) about how it works, in addition to the reference to the bioRxiv preprint that is already there. For example, readers will wonder if cell size is taken into consideration in this analysis since the TMEM55B and TBC1D9B KOs are obviously larger than WT. On this note, this phenotype should be mentioned (if it is real), and followed up by a statement on how OrgaMapper normalizes organelle positioning to cell size (i.e. diameter?) and is therefore a suitable method. It would help the readers as they could have doubts about this and them having to go into the OrgaMapper preprint (as I did) to see whether this method controls for cell size, is a minor inconvenience.\n-The statement: “Rab11-positive recycling endosomes were also partially redistributed to the cell periphery in TBC1D9B KO cells, consistent with an earlier study 34” should be accompanied by a callout to Supplemental Figure 2 (Rab11 staining)\n-Figure 2d should have a small legend for the schematic, as shown in Figure 4b.\n-Callout of Figure 2e in the text is missing a parenthesis\n-Figure 2e – The authors should show the individual channels in the whole-cell image. It looks, to this reviewer, like TBC1D9B-HA may have an ER localization, as well as some puncta that overlap with LAMP2. This ER localization is somewhat corroborated in Figure 2f, yet there is no discussion of this additional subcellular localization in cells. Is it also in the ER? If so, do the authors think this is real/expected? Or possibly a virtue of overexpression? An IF in which the ER is co-stained, would determine if any TBC1D9B signal is there. It is good that the overexpression is done in the background of the KO cells rather than WT to reduce overexpression-induced artefacts, however, TBC1D9B may be in the ER and this is not discussed. It is concluded that it has a cytosolic localization (and on some lysosomes) but the cytosolic localization is not convincing.\n-It is not obvious by eye in Figure 2f that the GAP activity-deficient TBC1D9B mutant is unable to rescue the lysosome dispersal phenotype. On the contrary, it looks like a partial rescue. Is this a matter of choosing a more representative image?\n-Figure 2g quantification should have the WT condition quantified as well because the experiment is measuring a “rescue” of lysosome positioning. This rescue can be best assessed by seeing the quantification of WT cells.\n-In this statement on pg 6: “As expected, starvation-induced marked relocalization of lysosomes towards the microtubule organizing center in wildtype cells.” there should be no hyphen, just “starvation induced…”\n-Is the TBC1D9B KO (fed) image in Figure 3a representative? The lysosomes do not look as peripheral as in Figure 2b and Figure 2f.\n-Regarding Figure 3 c-d: The SiR lysosome signal in the fed condition, at least from the images in Figure 3c, doesn’t seem to be the same between the genotypes (i.e. TMEM55B KO seems to have very diminished signal). Is this due to the extreme dispersal of lysosomes just making it difficult to see this signal by eye? SiR lysosome signal should be quantified in this condition alone across the different genotypes (fed) and perhaps placed into Supplemental.\n-Supplemental Figure 3: Was the DQ-BSA allowed to co-endocytose with a pH- and protease-insensitive fluorescent probe, such as Alexa555-dextran to normalize for amount of endocytic uptake? It seems from the figure legend that cells were given DQ-BSA following the treatments (fed/starved). Thus, the difference in fluorescence intensity of DQ-BSA in the KOs could also be due to altered endocytosis. Authors are advised to either perform this experiment with an endocytosis control (as suggested above), or at the very least, mention this limitation of their current approach in the text.\n-Figure 4c should be accompanied by the Pearson’s correlation coefficient in TBC1D9B KO + TBC1D9B-HA staining with LAMP2. Since this result is not discussed, it is unclear if the Pearson’s value shown in Figure 4c actually means loss of TBC1D9B from lysosomes and into the cytosol in the o/e condition. The result therefore seems arbitrary.\n-It is not clear why Figure 5 a/b was done in iNeurons? It is also not clear why an “unbiased” approach needed to be taken here. The authors had just identified TBC1D9B as a regulator of lysosome positioning and it’s GAP activity was important for this. The next logical step would be to look, in a targeted manner, at the lysosomal small GTPases known to couple to motors - Arl8 and Rab7. An explanation about the experimental rationale here would be useful.\n-In the statement “A GAP-activity-defective TBC1D9B mutant (TBC1D9BRYQ/AAA) (see Fig. 5a below) retained the ability to interact with Arl8b-GFP (Q75L) (Figure 5d).” What is the first callout \"(see Fig 5a below)\" referring to?\n-Why does the overexpression of WT Arl8b (Figure 5f) show a lower Pearson’s correlation coefficient for Lamp2/TBC1D9B? From the images it looks like there’s good LAMP2/TBC1D9B overlap in the WT Arl8b condition.\n-Figure 6d – The images for TMEM55B KO do not seem to show any more increase in Arl8b recruitment to LAMP2 compartments than WT. Are these images representative?\n-In this statement on pg 9: “ Given the defects of TBC1D9B-depleted cells with respect to the lysosomal starvation response (compare Figure 2), we monitored the presence of LC3 positive autophagosomes in cells depleted of TBC1D9B, Arl8a/b, or both proteins.” In the callout, do the authors mean compare Figure 3?\n-I am assuming from the discussion of Figure 7, that Figure 7c and d are showing cells under starvation condition? If so, this is not labeled in the micrographs (Fig 7c), the quantification (Fig 7d) or the figure legend. Is the quantification in Figure 7d referring to fold change in starved condition over fed condition? Is the WT siTBC1D9B quantification significantly different from WT scr? Also, check the labels in Figure 7d, they say TBC1B9D. And, as stated in main item #3, KD efficiency needs to be shown here.\n-With regards to this statement on pg 9: “Third, we show that TBC1D9B directly and selectively associates with active Arl8b-GTP in living cells and in vitro (Figure 4a-e).” Are the authors referring to Figure 5?\n-With regards to this statement on pg 10: “In preliminary experiments in HeLa cells overexpressing TMEM55B, we failed to detect any overt effects on TBC1D9B localization (Supplementary Figure 6a).”\nSupplemental Figure 6a doesn’t show TBC1D9B staining, only LAMP2 and the TMEM55B-GFP expression in WT and TBC1D9B KO cells.\n-In methods, change “Mutagenesis of TBCD9B” to \"Mutagenesis of TBC1D9B”", "reviewer_author_response": "Response: We thank the reviewer for the lucid summary and these highly positive comments and her/ his support to publish our study in Nat. Commun.\n1. Supplementary figures referenced throughout the text were not available in the submission system and full evaluation of critical claims (e.g., structure predictions, GTPase assays, and proteolysis assays) was impossible."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments sufficiently and the manuscript can be recommended for publication.", "reviewer_2": "This is a revised version of a paper that investigates the lysosomal membrane protein TMEM55B and finds an interaction with TBC1D9B which the authors then argue acts as a GAP for the lysosomal GTPase Arl8B, in addition to is previously reported role as a GAP for Rab11.\nMy review of the original version of the manuscript I noted the potential interest in the regulation of lysosomal positioning but expressed concerns about the strength of the evidence for TMEM55B binding directly to TBC1D9B and for the latter having GAP activity on Arl8B. In their rebuttal the authors state on several occasions that they “respectfully disagree” with my comments, but nonetheless they have added a significant amount of new data to strengthen these central conclusions of the paper. Overall, this has been done to a high standard and the paper seems significantly improved. As such I am happy to “respectfully” recommend acceptance. There are a couple of minor issues that would need addressing before publication:\na) The authors cite a recent study describing the binding between TMEM55B and a different partner, a study which synergises well with their own work. However, this paper is cited as Waschbusch et al 2025, but it is in fact from 2026.\nb) As the authors discuss, there are likely to be multiple regulators of Arl8B and its paralog Arl8A, and in this context they should cite a recent preprint that reports that DENND6A also has GAP activity toward ARL8B (PubMed ID 41542567).", "reviewer_3": "Overall, I believe the additional experiments and the corrections the authors have implemented have greatly improved the manuscript and I am satisfied with the authors' response. However, please correct all of the following:\n1) The manuscript file contains a duplication of the manuscript. Pages 1-36 is one manuscript without figures and pages 37 (labeled as page 1)-79 is another copy/version of the manuscript with figures. I assume this was a copy/paste error, but I am not sure if the two manuscripts are different in any way?\n2) Figure 2e and Supp Fig 2a – It is nice to see that lysosome speed has been computed. However, units of the speed are either incomplete (Figure 2f currently says “Speed>0.1 um”, do the authors mean speed > 0.1 μm/sec?) or missing (Supp Figure 2 has no units of speed on y-axis or in the legend). Speed should be presented as distance/time.\n3) Supp Fig 3d –The significance stars appear shifted to the left of the graph. The authors should check that this is moved to the right place. The legend for Supp Fig 3d doesn’t correspond to TBC1D9B KD experiment.\n4) Supp Fig 3e – “TBC1D9B fed plus +BafA1”, the authors may want to remove the “plus”? Again, the legend needs to be corrected for this panel.\n5) Supp Fig 3b – Legend needs correction – refers also to a dextran experiment.\n6) Overall for Supp Fig 3: Fix the entire legend as it also mentions a panel (f) and there is no panel f in the figure."}, "author_response": "Response: We thank the referee for these positive remarks and his/ her enthusiastic recommendation to publish our study in Nat. Commun.\na) The paper has appeared in Structure in November 2025 as an “ahead of print” paper. It was now assigned to an issue (on February 5th 2026). We have updated the reference accordingly.\nb) We added the mentioned preprint in the discussion:\n“A prediction from our work, therefore, is that other GAP proteins must exist that act to physiologically control ARL8 activity in cells and tissues. Such GAP proteins could conceivably act selectively on ARL8A or both ARL8 isoforms, or exhibit cell- and tissue-specificity. Candidates are DENND6A, for which GAP activity was demonstrated in vitro (Vignogna & Fromme, bioRxiv 2026), or other family members of the TBC family, including the TBC1D9B paralogs TBC1D9, TBC1D8, and TBC1D8B.”\nResponse: We would like to thank the reviewer for his/her positive feedback, careful reading and keen eye.\n1) This problem did not occur in the final version of the manuscript document. We assume that the submission system appended the manuscript versions with and without “track changes” to each other. In any case, the problem should no longer exist in the version that has now been uploaded.\n2) Thank you! We have updated the figure and corresponding legend accordingly.\n3) We thank the reviewer for noting this mistake, which has been corrected.\n4) We would like to thank the reviewer for their careful reading and keen eye. The error has been corrected.\n5) The legend for the Supplemental Figure 3 has been revised and corrected.\n6) The legend for the Supplemental Figure 3 has been revised and corrected."}]

Decision letter