K48-ubiquitin-dependent proteases cut-up post-ER proteins

Domain: biology
Published: 2026-01-13
Editorial outcome: minor_revision

Abstract
Polyubiquitin chains, linked via K48 or K63 of ubiquitin, direct membrane proteins in the secretory system to distinct degradative fates. However, it’s unclear whether these linkage isomers are functionally interchangeable. Here we show that for post-endoplasmic reticulum (ER) proteins, K63-linked polyubiquitination induces sorting into multivesicular bodies (MVBs) and lysosomal degradation. In contrast, K48-linked polyubiquitination induces shearing from the membrane and proteasomal degradation. This process involves two ubiquitin-dependent proteases: Ddi1, a conserved cytosolic ubiquilin that generates fragments from soluble and membrane proteins, and Rbd2, an intramembrane rhomboid protease that produces lumenal fragments from membrane proteins at Golgi/endosomes and the vacuolar membrane. Ddi1’s catalytic core, the HDD-RVP domain, is sufficient for ubiquitin-dependent proteolysis. It binds ubiquitin directly and its activity is enhanced by auxiliary ubiquitin binding domains: an atypical UBL domain and a UBA domain. These findings demonstrate that polyubiquitin chains linked by different residues encode distinct degradative fates for post-ER proteins, and reveal two proteases that target ubiquitinated integral membrane proteins in a process we call CUT-UP (Cleavage of Ubiquitinated Targets by Ubiquitin-dependent Proteases).

Introduction
Ubiquitin (Ub) marks membrane proteins for degradation in the secretory system. Degrading these proteins is challenging since they span the cytosol, lipid bilayer, and lumen. ER-associated degradation (ERAD) and endosome and Golgi associated degradation (EGAD) overcome this by retrotranslocating proteins into the cytosol for proteasomal degradation1,2. Another pathway, multivesicular body (MVB) sorting, uses the ESCRT apparatus to sort post-ER membrane proteins into intralumenal vesicles (ILVs), which are destroyed upon MVB fusion with lysosomes3. A third mechanism may degrade membrane proteins by clipping them into smaller fragments for lysosomal and/or proteasomal degradation4. Although clipping membrane proteins is a recognized cell signaling mechanism, its role in Ub-dependent degradation is poorly understood.

How substrates are assigned to each pathway may depend not only on their localization but also on the type of ubiquitination. A feature of Ub is that it can be attached to proteins in various configurations: as a monomer or polymer, where additional Ubs are linked to one of the seven lysine residues or the M1 residue of the preceding Ub5. These distinct configurations may act as a “Ub code” that specify different cellular processes. For membrane proteins, K48- vs K63-linked polyUb chains correlate with different degradation routes: ERAD substrates are K48-polyubiquitinated, whereas MVB sorting substrates are mono-ubiquitinated and K63-polyubiquitinated. Yet, it remains unclear whether these specific Ub configurations actively direct cargo into distinct pathways or merely correlate with them. For instance, does K48-polyUb direct post-ER cargo toward the MVB pathway like K63-polyUb, or does it signal a separate fate as predicted by the Ub code model? This question is especially intriguing because both K48- and K63-polyUb can bind ESCRT proteins, the Ub receptors of the MVB pathway6,7.

Here, by directing synthetic K48- or K63-specific ligases to post-ER membrane proteins, we demonstrate a functional Ub code and an alternative degradative route involving a series of Ub-dependent proteases. While K63-polyubiquitination mediated MVB sorting, K48-polyubiquitination directed substrates into a distinct pathway we term CUT-UP (Cleavage of Ubiquitinated Targets by Ubiquitin-dependent Proteases), involving the Ub-dependent proteases Ddi1, Rbd2, and the proteasome.

Results
K48- and K63-specific Ub-ligases target a post-ER protein for degradation via different pathways
To investigate the effect of K48- or K63-linked polyubiquitination on post-ER proteins, we targeted synthetic Ub-ligases to Vps10, a stable post-ER type I membrane protein that cycles between the TGN and late endosomes, and that can undergo MVB sorting when fused to mono-Ub8–11.

The synthetic Ub-ligases included HECT-, RING- and SCF-types (Fig. 1A–B). The HECT and RING type Ub-ligases consisted of a HECT- or RING- catalytic domain fused to an HA-tagged α-GFP nanobody, which targeted them to a GFP-tagged Vps10 (Fig. 1A)12. A modified TetOn3G system was used to produce these ligases within 30 min of doxycycline addition. For K63-linked polyubiquitination, we used the HECT domain of Rsp513 or the RING domain of Pib114,15. For K48-linked polyubiquitination we used a RSP5-E6AP chimeric HECT domain or a dimer of the RNF4 RING domain16. The chimera, created by swapping the C-lobe of Rsp5 HECT domain for that of E6AP, was previously shown to switch specificity to K48-polyUb17. The SCF-type Ub-ligase was based on the TIR1-AID system. TIR1, a plant F box protein, combines with endogenous proteins to form a Skp1-Cullen-Fbox (SCFTIR1) K48-polyUb ligase complex (Fig. 1B)18–21. SCFTIR1 targets proteins tagged with the auxin-inducible degron in the presence of auxin, but because it exhibited low activity even in the absence of auxin, we placed TIR1 under a copper-inducible (CUP1) promoter. To implement this system, Vps10 was tagged with NeonGreen (NG) and an auxin-inducible degron (AID)22, and CUP1-TIR1-Skp1 was integrated at the PDR5 locus to also disrupt PDR5.

By microscopy (Fig. 1C–D) and immunoblot analysis (Fig. 1E–F), we observed that the Ub-ligases induced Vps10 degradation through two distinct routes. The Rsp5-HECT and Pib1-RING Ub ligases degraded Vps10-GFP via the MVB pathway. This was evident by the stabilization of full-length Vps10-GFP in cells in activated of vacuolar proteases by pep4Δ mutation, and by the PEP4-dependent generation of a GFP fragment (Vac-GFP)23, a remnant of GFP produced by vacuolar proteases. Similarly, we found that Rsp5-HECT directed sorting of Mup1-GFP from the plasma membrane into the MVB pathway and vacuolar lumen (Fig. 1G). In contrast the K48-Ub ligases Rsp5-E6AP HECT and RNF4-RING induced Vps10-GFP degradation in both PEP4 and pep4Δ cells, without increasing Vac-GFP (Fig. 1E). Likewise, SCFTIR1-induced Vps10-NG-AID degradation in PEP4 and pep4Δ cells, without increasing Vac-NG (Fig. 1F).

These data indicate that K48- and K63-Ub ligases engaged distinct degradative pathways, demonstrating a functional Ub code for post-ER proteins and that the ESCRT/MVB pathway selectively excludes K48-ubiquitinated cargo.

K48-ubiquitination shears post-ER Vps10 into fragments targeted for proteasomal and vacuolar degradation
Next, we investigated how the post-ER reporter protein Vps10 was degraded by K48-Ub ligases, focusing on SCFTIR1-mediated degradation of Vps10-NG-AID. We constructed a strain that maximally inhibited not only vacuolar proteases, via pep4Δ, but also the proteasome, by targeting all three of the proteasome’s proteolytic sites. The trypsin-like activity of the Pup1/β2 subunit and caspase-like activity of Pre3/β1 subunit were inactivated by installing pup1-T30A and pre3-T20A alleles24,25. The chymotrypsin-like activity of the Pre2/β5 subunit was inhibited by treating cells with MG132, following deletion of PDR5 to prevent drug efflux26. Inhibiting all three of the proteasome’s proteolytic sites was necessary to inhibit overnight cell growth or degradation of a cytosolic reporter protein (NeonGreen fused to AID) (Supplementary Figs. 1A–B). Importantly, MG132 did not affect the growth of pup1-T30A pre3-T20A mutants over the course of our assays, allowing comparison of conditions with and without acute treatment (Supplementary Fig. 1B).

Immunoblotting for the NG-tagged C-terminus of Vps10-NG-AID revealed that inhibiting the proteasome and vacuolar proteases did not prevent its degradation in response to SCFTIR1-mediated ubiquitination (Fig. 2A). However, C-terminal fragments of Vps10-NG-AID accumulated upon proteasome inhibition that migrated between 40 kDa and 70 kDa by SDS-PAGE and were consistent with cleavage of Vps10-NG-AID in the juxtamembrane region of the cytosolic domain (~52 kDa). This indicated that the cytosolic portion of Vps10-NG-AID was cleaved by a Ub-dependent protease and was degraded by the proteasome.

We next tracked the fate of the N-terminal lumenal domain of Vps10-NG-AID, which spans residues 1-1372, using an antibody raised against residues 307-696 of Vps109. The antibody detected a truncated lumenal fragment of Vps10-NG-AID that accumulated in a pep4Δ strain, but not in a PEP4 strain (Supplementary Fig. 1C). The fragment size, between 140 kDa marker and the 250 kDa full-length Vps10-NG-AID, was also consistent with cleavage of Vps10-NG-AID in the juxtamembrane region. These data indicated that the lumenal portion of Vps10-NG-AID, was delivered to and degraded in the vacuole.

Because the α-Vps10 antibody detected non-specific bands, we inserted mCherry (designated as RFP) between the signal sequence and lumenal domain of Vps10-NG-AID. Immunoblots (α-RFP) of RFP-Vps10-NG-AID after SCFTIR1-dependent ubiquitination, confirmed production of a lumenal fragment between 140 kDa and full-length RFP-Vps10-NG-AID (Fig. 2B). Fluorescence microscopy showed that the RFP-tagged lumenal fragment relocated from endosomes to the vacuole lumen, while the NG-tagged cytosolic domain was lost (Fig. 2C).

The localization of the RFP-tagged lumenal domain to the vacuole is consistent with degradation of the cytosolic domain, which carries retrieval signals recognized by Retromer9,27. However, loss of retrieval alone should redirect Vps10 to the limiting vacuolar membrane rather than the lumen in pep4Δ mutants8,9,28. We confirmed this in a vps35Δ mutant lacking Retromer activity (Fig. 2C, Supplementary Fig. 1D). In vps35Δ mutants, RFP-Vps10-NG-AID indeed localized to the vacuolar limiting membrane, but following SCFTIR1-mediated ubiquitination, RFP was released into the lumen, while NG was lost. Fractionation by high-pressure lysis, high-salt extraction, and ultracentrifugation confirmed production of a soluble lumenal fragment upon SCFTIR1-mediated ubiquitination (Fig. 2D).

Together, these results demonstrated that Vps10 ubiquitinated by SCFTIR1 is cleaved by Ub-dependent proteases. Its cytosolic domain is released and degraded by the proteasome, and the lumenal domain is severed from the membrane and degraded in the vacuole.

Ddi1 is a Ub-dependent protease that cleaves the cytosolic domain of K48-polyubiquitinated Vps10
To identify the protease responsible for Vps10-NG-AID shearing, we screened ten candidates listed in the MEROPS database29 with potential to function in endosomal compartments: Yps7, Ape4, Ydr415c, Ste24, Mca1, Rim13, Yps5, Ypf1, Rbd2, and Ddi130–33. In parallel we screened components of DSC retrotranslocation complex (Tul1, Vld1, Gld1, Dsc3, Ubx3, Dsc2 and Cdc48), which operate the EGAD pathway, the only known route for proteasomal degradation of endosomal and Golgi substrates in S. cerevisiae2,34. (Supplementary Fig. 2). Only ddi1Δ exhibited a defect in Vps10-NG-AID degradation. In these cells Vps10-NG-AID still degraded, but cleavage of the cytosolic domain was impaired (Fig. 3A). This phenotype was more pronounced in the pup1-T30A, pre3-T20A pep4Δ strain, in which lysosomal and proteasomal proteases were inactivated. In this strain, proteasome inhibition with MG132 caused high levels of cleaved cytosolic fragments to accumulate, yet these were nearly eliminated in a ddi1Δ mutant. These data indicated that Ddi1 is responsible for forming the clipped cytosolic fragment of Vps10-NG-AID. However, since full-length Vps10-NG-AID was not stabilized in a ddi1Δ mutant, additional degradation mechanisms act on Vps10-NG-AID, which we explored in the next subsection.

Ddi1 is one of three conserved ubiquilin proteins, along with Rad23 and Dsk2, that are thought to shuttle ubiquitinated substrates to the proteasome. These cytosolic proteins contain a Ub-associated (UBA) domain that binds ubiquitinated substrates35, and a Ub-like (UBL) domain that binds Ub receptors on the proteasome36. Ddi1 is distinguished from other ubiquilins by a retroviral aspartyl protease-like (RVP) domain and a helical domain, dubbed the helical domain of Ddi1 (HDD). Little is known about the proteolytic substrates of Ddi1, however, in vitro, Ddi1 was shown to cleave substrates linked to K48-polyUb37. Human DDI2 has two known substrates: the integral membrane proteins Nrf1 and AMOT, which it cleaves after they are ubiquitinated to generate transcription factors from their cytosolic Ub-tails38,39.

We next tested whether Ddi1-dependent cleavage was driven by K48-polyUb and if other Ub linkages were required by using Ub mutants expressing only a single lysine-to-arginine substitution. Because K48 is essential40, the K48R strain carried one wild type allele, resulting in ~20% wild type Ub41. In the K48R strain, both Vps10-NG-AID degradation and formation of the Ddi1-dependent cytosolic fragment were reduced, confirming that these processes are K48-polyUb-dependent (Fig. 3B, Supplementary Fig. 3A). In contrast, strains carrying K6R, K11R, K27R, K29R, K33R, or K63R as their sole form of Ub were all capable of degrading Vps10-NG-AID and forming the Ddi1-dependent clip, indicating that these linkages are not required. However, Vps10-NG-AID degradation was slightly impaired in the K63R mutant, as was Ddi1-dependent fragment formation in the K6R mutant. These minor effects may reflect indirect roles, as reduced Vps10-NG-AID expression, consistently observed in the K63R strain, correlated with reduced degradation (Supplementary Figs. 3B, C).

To examine the mechanism of Ddi1 action, we tested which of its features are necessary for Ub-dependent cleavage of Vps10-NG-AID. Inactivation of the Ddi1 catalytic domain (D220A) completely abolished activity, indicating Ddi1 functioned as a protease (Fig. 3C, Supplementary Fig. 4A). We also assessed the contribution of its Ub-binding domains: a UBA domain and an atypical UBL domain, which unlike other UBL domains can bind Ub32,42. Loss of the UBL, but not the UBA domain, impaired Ddi1 activity; however, loss of both domains caused a more severe defect, indicating that the UBA contributes to Ub-dependent processing but is dispensable when the UBL is present. We investigated whether the UBL contribution could be attributed to Ub- or proteasome-binding. This was done by replacing the UBL with variants defective in Ub-binding: one construct carried E8A/I13A substitutions to prevent Ub-binding42, and in two other constructs the UBL was swapped for those of Dsk2 or Rad23, which bind the proteasome but not Ub. These constructs expressed well, except for the Rad23 fusion, which was less abundant than wild type Ddi1 (Supplementary Fig. 4). All UBL replacements compromised Ddi1 activity (Fig. 3C), indicating that the UBL domain contributes to Ddi1-activity by binding Ub rather than the proteasome. The capacity to bind Ub is conserved across species but achieved through distinct domains. Human DDI1 and DDI2 have N-terminal UBL domains and C-terminal UIM (Ub-associated interaction motif) domains that bind Ub43, whereas S. pombe Ddi1 (Mud1) has a C-terminal UBA domain that binds K48-polyUb44. All orthologs complemented ddi1Δ cells (Fig. 3C), although the cleavage pattern differed slightly, suggesting that variation in the architecture of Ddi1 may influence substrate positioning within the proteolytic site (Supplementary Fig. 4A).

Interestingly, Ddi1 lacking both UBL and UBA domains, retained some Ub-dependent activity unlike the catalytically inactive mutant (D220A). This indicated that the central HDD-RVP domain has intrinsic Ub-dependent proteolytic ability, consistent with the prior in vitro study of Ddi137. To test whether it could bind Ub directly, we performed NMR HSQC experiments with 15N-Ub, which showed that the HDD-RVP domains of ScDdi1 and human DDI2 bind mono-Ub with a Kd of ~100 µM through the canonical hydrophobic patch surrounding I44 used by a variety of Ub-binding proteins45 (Fig. 3D–E, Supplementary Fig. 5). While the RVP domain harbors the catalytic site, the function of conserved HDD domain is less clear. Ub-binding was lost when the RVP domain either from S. cerevisiae Ddi1 or human DDI2 alone was tested, indicating that the HDD is required for this interaction. Furthermore, alanine substitution of conserved residues I183 or D184 in the HDD domain of ScDdi1, inhibited proteolysis to a similar extent as the catalytically inactive D220A mutant (Supplementary Fig. 4B).

We also found that Ddi1 could target a soluble ubiquitinated substrate (Fig. 3F). SCFTIR1-dependent ubiquitination of cytosolic NeonGreen-AID, induced both proteasome- and Ddi1-dependent proteolysis. Full-length NeonGreen-AID (~35-40 kDa) was cleaved Ddi1-dependently into two ~25 kDa products after ubiquitination. In cells lacking both Ddi1 and proteasome activity, NeonGreen-AID accumulated as high-molecular-weight ubiquitinated species, which resolved into smaller discrete ubiquitinated forms upon ATP-depletion prior to lysis, which halts ubiquitination and promotes deubiquitination by endogenous enzymes.

Collectively, these data demonstrate that Ddi1 is a K48-Ub dependent protease that can operate outside of the proteasome to target membrane proteins as well as cytosolic proteins. It utilizes a central HDD-RVP catalytic core as its Ub-sensitive catalytic center, which can be enhanced by additional auxiliary Ub-binding domains.

Rbd2 is a Ub-dependent protease that cleaves the lumenal domain of K48-polyubiquitinated Vps10
Inhibiting Ddi1- and proteasome-dependent proteolysis did not prevent loss of full-length Vps10 after ubiquitination, which prompted us to examine processing of the lumenal domain (Fig. 2C, D). Immunoblotting RFP-Vps10-NG-AID for RFP revealed two distinct RFP-tagged cleavage products, which migrated between full-length RFP-Vps10-NG-AID and the 140 kDa marker (Fig. 4A). The larger band arose from proteasomal cleavage of the cytosolic domain, as its formation was inhibited by MG132 and blocked in cells with additional pup1-T30A, pre3-T20A mutations. In contrast, the smaller band still formed in a pup1-T30A, pre3-T20A ddi1Δ strain, and was thus produced by another protease. Rbd2, a predicted rhomboid protease localized to endosomes and the Golgi33,46, was a strong candidate, and indeed its deletion eliminated the smaller RFP-fragment (Fig. 4A). In pup1-T30A, pre3-T20A rbd2Δ cells treated with MG132, formation of both RFP-tagged fragments was blocked (Fig. 4B). Furthermore, fractionation experiments demonstrated that Rbd2 was required for producing the soluble lumenal fragments following ubiquitination (Fig. 4C).

As a Rhomboid protease, Rbd2 belongs to a broader family that includes pseudoproteases with functions such as retrotranslocation of membrane proteins47. Rbd2 has six transmembrane domains and shares a topology with the bacterial GlpG protease48,49, whose membrane-embedded catalytic cleft is open to the lumenal side48, allowing it to cleave both transmembrane and lumenal substrates50 (Fig. 4D). Mutation of Rbd2’s serine–histidine dyad active site (S124, H179) in TMD4 and TMD6 (S124S) abolished lumenal cleavage (Fig. 4B), demonstrating that Rbd2 functions as a protease in Vps10 degradation. Imaging of Rbd2-GFP confirmed its localization to endosomes and Golgi/TGN compartments by colocalizing with Vps10 and Sec7 (Fig. 4E). Furthermore, when RFP-Vps10-NG-AID was relocalized to the vacuolar limiting membrane in a vps35Δ mutant (Fig. 4A), it still underwent Rbd2-dependent cleavage, showing Rbd2 was also active at this location and suggesting Rbd2 may have a broad itinerary throughout the secretory pathway.

Combined inhibition of the vacuolar proteases, the proteasome, Ddi1, and Rbd2 rescues K48-polyubiquitinated Vps10 from degradation
Four components contributed to degradation of K48-polyubiquitinated RFP-Vps10-NG-AID: the proteasome, vacuolar proteases, Ddi1, and Rbd2. Because loss of any single component did not block degradation, we tested whether their combined inactivation would. Cells with inactivated vacuolar proteases (pep4Δ), inactivated proteasome subunits (pup1-T30A, pre3-T20A) and, sensitized to MG132 (pdr5Δ), were deleted for DDI1, RBD2, or both. Combined inhibition of these pathways blocked formation of all truncated fragments and led to the accumulation of high molecular weight (high MW) forms of RFP-Vps10-NG-AID, that likely corresponded to highly ubiquitinated intermediates (Fig. 5A). This parallelled a global increase in high MW K48-polyubiquitinated proteins ddi1Δ and ddi1Δ rbd2Δ strains (Supplementary Fig. 6A), consistent with previous studies of ddi1Δ cells37,38.

To determine whether combined loss of these proteolytic components blocked degradation, we compared RFP-Vps10-NG-AID levels before and after SCFTIR1-mediated ubiquitination. Because diffuse banding and poor gel transfer limited precise quantification of the high-MW smear, we depleted ATP prior to lysis to promote deubiquitination, which collapsed the smear into compact bands (Fig. 6A). Quantification of these bands confirmed that proteasome inhibition in ddi1Δ rbd2Δ pep4Δ cells stabilized RFP-Vps10-NG-AID. Furthermore, NeonGreen fluorescence measured by flow cytometry was largely preserved, although RFP fluorescence levels were too low for reliable measurement (Supplementary Fig. 6B). These results demonstrate that the proteasome, vacuolar proteases, Ddi1, and Rbd2 comprise the entire K48-polyUb degradative pathway for this substrate. We then applied the ATP-depletion method to evaluate Ddi1 and Rbd2 individually. ATP-depletion did not alter the pattern of cytosolic Ddi1-dependent cleavage products, indicating that multiple species arise from distinct cleavage sites rather than differential ubiquitination. In addition, when only Rbd2 was active, lumenal RFP-fragment levels were not fully retained, likely because some cleaved fragments were secreted. This is consistent with Rbd2 acting throughout the Golgi–endosomal system, including the TGN, where Vps10 cycles and the secretory and vacuolar routes diverge.

Having defined the essential proteolytic steps required for substrate degradation, we termed this protein shearing mechanism the CUT-UP pathway, which stands for Cleavage of Ubiquitinated Targets by Ubiquitin-dependent Proteases. We tested whether the CUT-UP pathway also mediated degradation of Vps10 following ubiquitinated by one of the α-GFP K48-Ub ligases, specifically RNF4-RING Ub-ligase (Fig. 5B). For these experiments, we replaced the C-terminal GFP-tag on Vps10 with superfolder GFP (sfGFP)51 and E. coli DHFR and AID52, because GFP alone proved highly sensitive to proteasomal degradation upon K48-ubiquitination (Supplementary Figs. 6C, D). RNF4-RING-mediated ubiquitination of RFP-Vps10-sfGFP-DHFR-AID produced both an Rbd2-dependent lumenal fragment and a Ddi1-dependent cytosolic fragment. Furthermore, full loss of the CUT-UP pathway (pup1-T30A, pre3-T20A, pdr5Δ, pep4Δ, rbd2Δ, ddi1Δ and treated with MG132) inhibited degradation of RFP-Vps10-sfGFP-DHFR-AID, causing it to accumulate as a high MW species at levels comparable to the initial protein before ubiquitination.

We verified that the CUT-UP pathway acts on post-ER Vps10 by preventing new synthesis during the assay. A tetracycline-sensitive riboswitch was placed upstream of RFP-Vps10-sfGFP-DHFR-AID, preventing translation upon tetracycline binding52,53. Tetracycline pre-treatment alone slightly reduced RFP-Vps10-sfGFP-DHFR-AID levels, indicating there was some background turnover (Fig. 5C). Nonetheless, both Ddi1-dependent and Rbd2-dependent fragments were produced upon SCFTIR1-mediated ubiquitination and loss of both Ddi1 and Rbd2 caused accumulation of high molecular weight products upon proteasome inhibition.

We next examined the division of labor within the CUT-UP pathway. Our data indicated that Ddi1, Rbd2, and the proteasome act in parallel, since only combined inhibition of all three stabilized Vps10 (Fig. 5A, B). Furthermore, loss of Rbd2 did not affect formation of the Ddi1-dependent cytosolic fragment, and loss of Ddi1 did not affect formation of the Rbd2-dependent lumenal fragment. However, under proteasome inhibition with MG132 in ddi1Δ cells, the Rbd2-dependent lumenal fragment was diminished following RNF4-RING- or SCFTIR1-mediated ubiquitination. While this could suggest that Rbd2 requires Ddi1 activity, Rbd2 remained active since full-length and high-MW Vps10 reporters were not stabilized as they were in ddi1Δ rbd2Δ cells; instead, this decrease might reflect increased secretion of the lumenal fragment in the absence of Ddi1. We next asked whether each pathway targets a distinct Vps10 pool or acts on the same molecule. If these pathways acted on distinct pools of Vps10, blocking one should increase processing by the other. Instead, we observed no increase in Rbd2- or Ddi1-derived fragments when the other was deleted (Fig. 5A, B), indicating that both pathways can act on the same molecule. This raises a mechanistic puzzle: cleavage and degradation of the ubiquitinated cytosolic tail by Ddi1/proteasome should preclude recognition by Rbd2. We propose that Rbd2 either acts first, outcompeting Ddi1, or can also recognize substrates partially processed by Ddi1/proteasome.

Having established that CUT UP components cooperate to degrade K48-polyUb cargo, we next asked whether K48-polyub cargo stabilized in a CUT-UP mutant are redirected, even inefficiently, into the MVB route. To test this, we introduced PEP4 in a CUT-UP mutant and assayed Vac-NG and Vac-RFP formation. Vac-RFP was unsuitable as a vacuolar sorting reporter because it did not form in a positive control (Supplementary Fig. 6B). In contrast, Vac-NG formed from a known MVB substrate, Mup1-NG-AID, but was not detected for RFP-Vps10-NG-AID targeted by SCFTIR1 (Fig. 6B, Supplementary Fig. 6E, F). These results indicate that the MVB pathway excludes K48-polyUb cargo. Additionally, Vps10 remained stabilized as high-MW species in PEP4 CUT-UP mutants, indicating that vacuolar degradation occurs downstream of Rbd2, Ddi1 and proteasomal activity.

Previous experiments showed that Cdc48 is not required for overall CUT-UP-mediated degradation (Supplementary Fig. 2B). We performed further experiments that showed it is also unnecessary for Ddi1-dependent cleavage or proteasomal degradation of the K48-ubiquitinated Vps10 cytosolic domain (Supplementary Figs. 6G, H). Ddi1-dependent fragments were still formed in cells carrying cdc48-ts alleles grown at the restrictive temperature. Further, by omitting MG132 in rbd2Δ ddi1Δ mutants, we found that the proteasome was capable of degrading the NG-tagged cytosolic tails of RFP-Vps10-NG-AID following SCFTIR1-mediated ubiquitination in cdc48-3 mutants at the restrictive temperature. These data indicate that, at least for this substrate, the proteasome can degrade membrane protein cytosolic tails without prior cleavage and independently of Cdc48.

Together, these data show that Rbd2, Ddi1, the proteasome, and vacuolar proteases comprise the CUT-UP pathway, which cleaves and degrades K48-polyubiquitinated Vps10 in post-ER compartments. They further support a model in which K48-polyubiquitination commits substrates to CUT-UP rather than the MVB pathway, which fails to engage even when CUT-UP is disabled.

Alternative processing of the lumenal domain of RFP-Vps10-NG-AID
During experiments tracking degradation of the RFP-tagged lumenal domain in response to either SCFTIR1 or RNF4-RING Ub-ligases, production of a ~55 kDa RFP fragment (RFPp55) was consistently observed (Fig. 7A–D, Figs. 4–6). Its levels were equivalent to only 1–4% of the full-length precursor, however, its production increased 3.3–6.1 fold in rbd2Δ cells (Fig. 7A). The size of RFPp55 indicated that it was a product of cleavage within the lumenal domain. Yet, unlike other lumenal fragments, it persisted in PEP4 expressing cells with active vacuolar proteases. Instead, it was stabilized by proteasome inhibition and required Ddi1 for its production (Fig. 7B–D).

These observations suggest that the lumenal domain became exposed to the cytosol, possibly via ERAD or EGAD-mediated retrotranslocation1,2. However, we ruled out ERAD of newly synthesized RFP-Vps10-sfGFP-DHFR-AID because RFPp55 was still produced when synthesis of Vps10 was suppressed by a tetracycline riboswitch (Fig. 7E). Furthermore, unlike ERAD and EGAD, which require Cdc48/p97 and, in the latter case, the DSC complex, RFPp55 production persisted in cdc48-2 or cdc48-3 mutants at the non-permissive temperature of 37°C (Fig. 6C), as well as in dsc2Δ cells (Fig. 7F). These results reveal a cryptic retrotranslocation-like pathway by which the lumenal domain of TGN/endosomal membrane proteins access the cytosol for Ddi1-dependent cleavage and proteasomal degradation when Rbd2 activity is blocked.

Ddi1 and Rbd2 cleave various integral endosomal proteins targeted by K48-Ub ligases
We also examined whether other single pass integral membrane proteins in the secretory/endosomal system degraded if they were targeted by K48 Ub-ligases, and if so, whether Ddi1 or Rbd2 targeted these substrates. To monitor for protein shearing, we tagged the N- and C-termini of several proteins with RFP and NG-AID, respectively. The proteins occupied different areas of the secretory endomembrane system, but were not always at their reported native localization (Fig. 8A). Within 1 h of SCFTIR1-mediated ubiquitination, all examined proteins degraded, and were partially stabilized by the combined inactivation of Rbd2, Ddi1, the proteasome and vacuolar proteases (Fig. 8B). Ylr001c, which was localized to the vacuolar limiting membrane, and Ynd1, which was localized to endosomes and the vacuole lumen, both produced a Rbd2-dependent degradation product. They also produced a high molecular weight product upon combined inactivation of Ddi1 and Rbd2. Pex3 and Fet5, which were localized to the ER and vacuole limiting membrane, produced a Ddi1-dependent fragment. Pex3, but not Fet5, produced a high-MW product upon Ddi1 inactivation. This indicates that Rbd2 and Ddi1 can cleave multiple ubiquitinated integral membrane substrates throughout the secretory pathway.

Methods
Yeast Strains and plasmids
Yeast strains, plasmids, and antibodies are described in Supplementary Table 1, 2 and 3, respectively, and their use throughout this work is listed in Supplementary Table 4. DNA cassettes used for genome engineering yeast and constructing plasmids were sourced from the yeast genome, plasmids or synthesized (IDT, Coralville, IA). DNA constructs were assembled using Gibson assembly cloning (NEB BioLabs, Ipswich, MA). Site-directed PCR-based mutagenesis was performed using Pfu Ultra polymerase (Quick Change, Agilent Technologies, Santa Clara, CA). DNA constructs were verified by Sanger or Oxford nanopore sequencing (UIowa Genomics core (Iowa City, IA) and Plasmidsaurus (Eugene, OR)). Yeast genomes were edited using homologous recombination. Annotated maps of key plasmids and engineered loci within the yeast strains are publicly available (https://doi.org/10.6084/m9.figshare.28678325).

All yeast strains were confirmed for their ability to respire on ethanol/glycerol as the carbon source prior to use and storage. All pep4 mutants were passaged until Pep4-dependent maturation of CPY was no longer detected as described79. Strains carrying temperature-sensitive cdc48-2 (A547T, T803A) and cdc48-3 (P257L, R387K) alleles in BY4742 (as described previously80) were confirmed to be inviable at 37°C. To introduce these alleles into other strains, an allelic replacement cassette was constructed by cloning a PCR-amplified mutant locus containing a XhoI site upstream of the mutations into pRS306. The integrated XhoI-linearized cassette resulted in a tandem locus consisting of the cdc48-ts allele and a disrupted CDC48 gene with a large open reading frame deletion. Strains with defined Ub K>R mutations were described previously41. Briefly, in these strains all four Ub encoding genes (UBI1-UBI4) are modified to ensure a given Ub K>R mutant is the sole source of Ub, with the exception of the Ub K48R strain. In Ub K48R strain, one of the three tandem copies of Ub in the UBI3 locus encodes wild type Ub, resulting in ~80% replacement of the Ub pool with Ub K48R. The proteasome mutant strains were as described in Arendt and Hochstrasser24, in these strains PRE3 and PUP1 were deleted and in control strains wild type alleles of PRE3 and PUP1 were reintroduced, and in catalytic inactive strains pre3-T20A and pup1-T30A were introduced. For SCFTIR1 assays, Skp1-TIR1 fusion protein (S. cerevisiae Skp1 and O. sativa TIR1) under the control of a CUP1 promoter was integrated into the PDR5 locus, thereby eliminating the Pdr5 drug efflux pump and sensitizing cells to MG132 treatment26. Cargo targeted for SCFTIR1 degradation were tagged with Auxin-Inducible Degron (AID: residues 63-111 of A. thaliana IAA17: ADB93635). Endogenous Vps10 was modified for degradation assays. Vps10 was C-terminally tagged with NeonGreen-AID, GFP, or sfGFP-DHFR by integrating the corresponding coding sequences into the VPS10 locus. To N-terminally tag Vps10 with mCherry, the native promoter was replaced either with the MET25 promoter or with a modified TEF1 promoter followed by tetracycline-binding riboswitches that block translation when bound to tetracycline. For nanobody (α-GFP)-Ub-E3 ligase assays, the E3 ligase was encoded in a tetracycline/doxycycline-inducible low-copy plasmid. The bicistronic plasmid encoded the TetOn3G protein81, constitutively expressed from a modified TEF1* promoter82. Transcription was terminated by both DEG1 and ADH terminator sequences to prevent read-through of the second ORF83. The second ORF expressed the E3 ligases from a synthetic low-background promoter derived from PDR3 and 8 tandem tetO sites. The E3 ligases were fused C-terminally to four tandem HA epitopes and the VHH-domain cABGFP4 nanobody84.

Cell growth and assays
Yeast growth was monitored using optical density at 600 nm (OD600). Cells were inoculated from a starter culture and grown overnight to an OD600 of 0.3–0.6 at 29°C, on an orbital shaker at 190 RPM unless otherwise stated. Synthetic complete media contained 2% glucose, yeast nitrogen base without amino acids and ammonium sulfate (DifcoTM Becton Dickinson, Franklin Lakes, NJ, or Y1251 Sigma Aldrich), 0.5% ammonium sulfate (Research Products International, Mount Prospect, IL), and complete supplement dropout mixture that allowed for appropriate selection (Formedium, Norfolk, UK).

For SCFTIR1 degradation assays, degradation was triggered by addition of 10 μM CuCl2, to induce expression of Skp1-TIR1, and auxin to induce interaction of AID and TIR1. The auxin used in these studies was indole-3-acetic acid (IAA, Sigma-Aldrich, St. Louis, MO) at 1 mM. Cells were harvested 60 min later. Skp1-TIR1 was placed under an inducible CUP1 promoter, since TIR1 partially induced degradation of AID tagged cargo in the absence of auxin.

For α-GFP-Ub-E3 ligase degradation assays, E3 ligase expression from a yeast TetOn3G plasmid was induced by 100 μg/ml doxycycline (Sigma-Aldrich, St. Louis, MO), and cells were harvested 60 min later. To inhibit proteasome activity, cells were treated with 100 μM MG132 (UBPbio, Dallas, TX) 30 min prior to inducing degradation. For these experiments, PDR5 was disrupted by insertion of the CUP1pr-Skp1-TIR1 fusion gene.

Expression of mCherry-Vps10-NeonGreen-AID fusion protein was controlled by the MET25 promoter. To avoid overexpression and mis-targeting to the vacuole, 10 mg/ml methionine (Alfa Aesar, Ward Hill, MA) was included in the growth medium.

Expression of mCherry-Vps10-sfGFP-DHFR-AID was controlled by a modified TEF1 promoter (TEF1*pr) and tetracycline-inducible riboswitch53,85. Expression was inhibited with the addition of 0.5 mM tetracycline (Research Products International, Mount Prospect, IL).

For experiments with ATP depletion, cells were pelleted, washed, and incubated for 30 min in PBS containing 0.1 M NaF, 0.1 M NaN3, 20 mM 2-deoxyglucose, and 100 μM MG132 prior to lysis.

Immunoblotting
All antibodies were validated using extracts from cells with and without the protein or epitope to which the antibody was directed. Whole-cell extracts were prepared by resuspending pelleted (11,000g for 30s) cells in 0.2 M NaOH, repelleting, and resuspending in SDS-lysis buffer (8 M urea, 5% SDS, 10% glycerol, 50 mM Tris-HCl pH 6.8, 2.5% 2-mercaptoethanol, 0.02% bromophenol blue) at a concentration of 10 OD600 units/ml. The samples were heated at 95°C for 1 min immediately before being loaded onto 4–12% gradient gels (ExpressPlus™, Genscript, Piscataway, NJ) for SDS-PAGE, followed by transfer to 0.22 μm nitrocellulose membranes. To detect the high molecular weight forms of Vps10 (Figs. 6–8) the entire gel including the loading lanes was transferred. Primary antibodies and HRP-conjugated secondary antibodies (Supplementary Table 4) were used to generate chemiluminescent signals with SuperSignalTM West Femto or Pico PLUS Substrate (Thermo Fisher, Waltham, MA), that were recorded with a FluorChemTM 8800 (Alpha Innotech, San Leandro, CA) or an iBrightTM Imaging System (Thermo Fisher, Waltham, MA). Densitometry analysis was performed in Fiji78. The following primary antibodies were used. For NeonGreen detection, we used a rabbit polyclonal antibody from Cell Signaling Technology (CST; Cat. 53061S; discontinued) and, preferentially, the rabbit monoclonal mNeonGreen Tag antibody (CST; Cat. 41236), each at 1:1000–1:2000. GFP was detected using rabbit monoclonal α-GFP D5.1 (CST; Cat. 2956S; 1:1000–1:2000). HA-tagged proteins were detected using mouse monoclonal α-HA (BioLegend; 1:1000–1:2000). PGK was detected with mouse monoclonal α-PGK (Molecular Probes; Cat. A-6457, RRID: AB_22154; 1:1000–1:2000). For mCherry, we used a rabbit polyclonal antibody (Sigma Aldrich; Cat. AB356482, Lot 3536102) that was pre-absorbed with pep1Δ pep4-3 lysate to reduce batch-dependent non-specific binding; the effective dilution was ~1:1000. The rabbit monoclonal α-mCherry E5D8f (CST; Cat. 43590S) was used at 1:1000–1:2000. Vps10 was detected using a rabbit polyclonal antibody raised against aa 306–6969. maintained as serum and preabsorbed against vps10Δ pep4Δ extract; the effective dilution was 1:1000. CPY and ALP were detected using rabbit polyclonal antibodies from Tom Stevens (University of Oregon), each at 1:1000–1:2000. K48-linked polyUb was detected using rabbit monoclonal α-K48Ub (CST; Cat. 8081; 1:1000). Secondary antibodies (α-mouse, CST #7076; α-rabbit, CST #7074S) were used at a dilution of 1:2000.

Microscopy
Yeast cells were concentrated by centrifuging at 2,000g for 2 min, transferred to microscope slides, and imaged at room temperature. Images were acquired using an Olympus fluorescence BX60 microscope equipped with UPlanSApo 100×/1.40 oil objective and Hamamatsu Orca-R2 digital camera (Hamamatsu, JP) controlled by iVision software (BioVision Technologies).

Flow cytometry
Flow cytometry data were acquired on a BD LSRII or Cytek Aurora equipped with a 488 nm laser. The median fluorescence intensity (MFI) of cells without RFP-Vps10-NG-AID was subtracted from the MFI of each sample to remove background signal. Data are expressed as percentage of MFI in untreated samples, which were used to measure baseline RFP-Vps10-NG-AID expression. The forward and side scatter gating strategy used is shown in Supplementary Fig. 6B.

Subcellular fractionation
Cell fractions were collected from pelleted yeast that were washed twice in ice cold PBS containing protease inhibitors (cOmplete EDTA-free and Pefabloc (Roche, Basel, CH) and lysed using a One Shot cell disruptor (Constant Systems, Daventry, UK) at 35 kPsi. Lysate was centrifuged at 5,000g for 10 min at 4°C, to generate a post-nuclear supernatant, that was subsequently centrifuged at 200,000g for 1 h, generating a soluble cytosolic fraction and crude membrane pellet.

NMR titration studies of Ub and HDD-RVP binding
Interactions between Ub and ScDdi1 or human DDI2 HDD-RVP domains were monitored using a series of NMR titration experiments. 15N-ubiquitin was produced as previously described86. Recombinant 6xHis tagged ScDdi1 or human DDI2 HDD-RVP were expressed in E. coli BL21 (DE3) cells upon induction with 0.5 mM IPTG at 18°C for 20 h in LB media supplemented with antibiotic and 0.1% glucose. Cells were suspended in ice cold PBS containing protease inhibitors (cOmplete EDTA-free and Pefabloc; Roche, Basel, CH) and lysed using a One Shot cell disruptor (Constant Systems, Daventry, UK) at 25 kPsi. The 6xHis-tagged proteins were purified using Talon Co2+ affinity resin (Takara Bio, USA), eluted with 150 mM Imidazole in PBS (pH 7.8), and equilibrated to 50 mM NaCl, 40 mM NaPO4, pH 6.95 (NMR buffer). NMR samples contained 10% D2O in NMR buffer. Data were collected at 25°C on a Bruker Avance II 800 MHz NMR spectrometer equipped with a TCI cryoprobe and analyzed with SPARKY and NMRView87,88. 15N/1H HSQC spectra of 15N-Ub (30 μM) were collected in the presence of increasing concentrations of recombinant ScDdi1 or human DDI2 HDD-RVP domains, up to molar ratios of 9.4 and 16.8 fold, respectively. 1H chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). 15N chemical shifts were referenced indirectly to DSS as described in Bruker software. Chemical shift perturbations in the [1H, 15N]-HSQC spectra of Ub were calculated using Eq. (1).

Δδ = sqrt(ΔδH^2 + (0.2ΔδN)^2) (1)

Residues at or near the binding surface were defined as those with CSPs greater than 0.5σ above the mean Δδ. To quantify the binding affinity of HDD-RVP domains for 15N-Ub, changes in the Ub L71 resonance were monitored during titration experiments. Residue L71 was selected because it lies within the binding interface and exhibits both larger chemical shift perturbations and peak intensity changes. The normalized change in peak intensity (ΔI) of Ub L71 was assumed to reflect the fraction of Ub bound to Ddi1 ([PL]/[P]t). ΔI was calculated using Eq. (2), where Ifree and Ibound are the peak intensities of Ub L71 in the absence and presence of Ddi1.

ΔI = (Ifree - Ibound) / Ifree (2)

The resulting ΔI values were used to fit the single-site binding model described in Eq. (3)89,90 using a least-squares in GraphPad Prism (GraphPad Software) to obtain Kd and the maximal bound fraction ([PL]max).

[PL]/[P]t = [PL]max * (Kd + [L]t + [P]t - sqrt((Kd + [L]t + [P]t)^2 - 4[L]t[P]t)) / 2[P]t (3)

where [P]t is the total concentration of 15N-Ub (held constant during titration); [L]t is the titrating ligand Ddi1 concentration; and [PL] is the concentration of Ub–Ddi1 complex. In Fig. 3D, E, [PL]/[P]t is plotted on the Y-axis (bound fraction) as a function of [L] on the X-axis.

Software use
Graphpad Prism 10.4.2 (GraphPad Software, Inc.) was used for statistical analysis and graphing. Fiji/NIH Image (ImageJ2, v2.14.0, imageJ.net) was used to quantify immunoblot data. Adobe Illustrator (v27.8.1, Adobe Inc., San Jose, CA, USA) was used to assemble figures. Protein structures were drawn using ChimeraX91 and Pymol (v2.5.5, Schrödinger, LLC). NMR spectra were graphed using the NMRpipe suite92. Alphafold multimer93 was used to model the HDD-RVP domain of Ddi1 as a dimer.

Statistics and reproducibility
Statistics were calculated on data collected from at least three independent biological replicates of the yeast strains listed in Supplementary Tables 1 and 4. The exact sample size (n) is reported in figure legends. Investigators were not blinded to samples. Data were expected to have normal distributions and equal variances, and statistical significance was determined at p < 0.05. Data are presented as mean ± SD with individual data points shown as semi-transparent grey filled circles. The type of statistical test and exact p-values are reported in Supplementary Information (Supplementary Note: Statistical Outcomes).

For experiments showing only representative data, each was repeated with similar results as follows: 1C (3 times), 1D (3 times), 1G (2 times), 2C (3 times), 2D (3 times), 3D and 3E (Ub-binding by HDD–RVP was reproduced in two independent NMR experiments: first using one protein batch at a single HDD–RVP: ¹⁵N-Ub ratio, and then with a second batch used for the titration analysis shown), 4A (3 times), 4E (2 times), 6B (1 time), 7F (1 time), 8A (1 time), 8B (1 time); Supplemental Figures: 1B (1 time), 1C (5 time), 1D (3 times), 2A (1 time), 2B (2 times), 4B (1 time), 6A (1 time), 6C (1 time), 6D (1 time), 6E (1 time), 6F (1 time), 6G (twice as shown), 6H (twice as shown).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript submitted by Minard and co-workers focuses on post-endoplasmic reticulum proteins and how K48-linked\npolyubiquitination and K63-linked polyubiquitination affect lysosomal degradation (in the case of K63-linkage) as well as\nshearing from the membrane (in the case of K48-linkage). Two ubiquitin-activated proteases have been studied in detail:\nDdi1 and Rbd2. The data of the authors support the established fact that the site used for polyubiquitination (K48 and K63 as\nused here) encode the fate for proteins affected. The authors have made use of a repertoire of different experimental\nmethodologies belonging to molecular biology, biochemistry, and biophysics to address the scientific objective of activating\nproteases by the ubiquitin machinery.\nI will focus in my review on the results obtained by high-resolution NMR spectroscopy in the following as it represents my\nexpertise regarding the experimental repertoire used by the authors.\nThe NMR data obtained in this study are presented in Figure 3D, E in the main part of the manuscript as well as in\nSupplemental Figure 4.\nOverall, I have to say that I am surprised about the superficiality of the presentation of the NMR data, especially when\nsubmitting the work to such a respective Journal like Nature Communications.\nSpecifically:\nThe data presented in Figure 3D, E (left) aim to report on the interaction between 15N isotopically enriched ubiquitin and\nHDD-RVB domains from Sc (D) and human (E).\n- The manuscript does not comprise the correct information about the analytical function the authors have used to fit\n(continuous line in the plots) the experimental data. The statement “… fitted to a standard quadratic equation …” is not only\nsuperficial but also wrong.\n- A reliable analysis of an NMR spectroscopic titration experiment relies on accurate referencing of the dimensions used for\ndata acquisition. Which efforts have the authors made in this respect?\n- Which cross peaks arising in 2D 1H-15N HSQC spectra have been used for the two individual plots shown in panel D\nand E? What is the rationale for the specific color coding used here (note: color coding used in Supplemental Figure 4 cannot be\nidentified; see also below)?\n- Labeling of y-axes misses completely, labeling of x-axes is incorrect. In fact, to quantitatively analyze an NMR titration\nexperiment, the concentration of the ligand (here: HDD-RVB domains) has to be presented, the concentration of the target\nprotein (here: ubiquitin) is contained in the fitting function. Additionally, neither the parameter nor the unit is presented on the\nx-axes. Which information is additionally encoded in using circles as triangles in panel D, E (no information found in the\nlegend accompanying this Figure)?\n- The legend of Figure 3 states “residues of Ub that underwent the largest chemical shift perturbation are mapped in red onto\nthe structure of Ub”. Which specific cut-off value of the perturbation of the chemical shift has been used? Additionally, a\nseparate plot presenting residue-specific perturbations of chemical shifts is highly recommended.\n- Data presented in Figure 3 report on an NMR titration experiment. The authors have solely analyzed changes in chemical\nshifts upon adding of an HDD-RVB domain to ubiquitin. Unfortunately, line width and signal heights of resonance signals\narising in 2D 1H-15N HSQC have not been analyzed by the authors.\n- The authors have not mentioned the binding model (e.g. one-to-one binding, …) they have used for interpreting the NMR\nspectroscopic data. The stoichiometry represents an important parameter when performing an interaction study.\n- The title of Supplemental Figure reads as “HSQC analysis of 15N-Ub in the presence of the HDD-RVP domains from\nScDdi1 and human DDI2” even panel A presents a SDS-PAGE.\n- Panel B of Supplemental Figure 4 presents rather small sections of the entire spectral ranges the 2D 1H-15N HSQC NMR\nspectra comprise. It is highly recommended to show the entirely acquired data set this is, if required, followed by smaller\nsections. Additionally, cross peaks originating in 2D NMR correlation spectra are shown as contour lines. Unfortunately, the\nauthors present border strips only.\n- General: I am not convinced that the authors have not used D2O in their NMR experimental work.", "reviewer_2": "In this paper, authors report that K63-linked, but not K48-linked, polyubiquitination induces MVB sorting and lysosomal\ndegradation of membrane proteins in S. cerevisiae. They also report that K48-polyUb substrates are degraded after leaving\nthe ER via a pathway, termed CUT-UP (Cleavage of Ubiquitinated Targets by Ubiquitin-activated Proteases), which induces\nshearing of membrane proteins. They also report that K48-polyUb substrates are processed by the proteasome and by the\ntwo Ub-activated proteases Ddi1, a conserved cytosolic ubiquilin that generates cytosolic fragments and Rbd2, a rhomboid\nprotease of Golgi/endosomes but also acting on the vacuole, which produces lumenal fragments. Finally, they report that the\ncatalytic core of Ddi1, consisting of the HDD-RVP domain, is sufficient for Ub-dependent proteolysis, and that its activity is\nenhanced by auxiliary ubiquitin binding domains (atypical UBL domain and a UBA domain). They conclude that polyUb\nchains linked by different residues lead to different fates for post-ER proteins, and that there are two proteases that target\nubiquitinated integral membrane proteins.\nThe topic is interesting, and the paper provides novel insights into our understanding of the mechanisms responsible for the\ndegradation of membrane proteins. The paper combines elegant strategies with solid biochemical and morphological data.\nIn general, I find the data convincing, with some relatively minor reservations below.\nMy main comment – or concern – is that it would be appropriate and helpful to illustrate conditions where native candidate\nproteins follow these Ub-dependent (Rbd2- or Ddi1-dependent) CUT-UP, protein shearing degradation pathways (see\nmodel in Fig 7).\nOther comments\n1) Nowhere in the text (Methods, etc.) is it stated that anti-GFP E3 ligases are HA-tagged, as seen on the blots Fig 1E. It\nshould be mentioned at least in the Fig legend.\n2) It is stated that strains carrying K6R, K11R, K27R, K29R K33R, or K63R as their only form of Ub, all degraded Vps10-NG-\nAID and formed Ddi1-dependent cytosolic fragments (Fig 3B), but that Vps10-NG-AID degradation and formation of Ddi1-\ndependent fragments were slightly impaired in the K63R mutant strain. However, the data are not clear-cut and K63R\ndoes not appear to be that different from other strains.\n3) The authors mention that the loss of both Rbd2 and Ddi1 together with MG132 treatment prevent the formation of\ntruncated fragments, and that a HMW product then forms that remains in the SDS-PAGE stacking gel. The HMW product,\nwhich I assume ran at the position of the top arrowhead in Fig 5A, is barely visible, and the total amount of this high MW\nproduct does not seem to be roughly equivalent to the starting amount of full-length protein. Where is the boundary between\nstacking and separating gels?", "reviewer_3": "This study establishes a role for the intramembrane protease Rbd2 and the ubiquilin Ddi1 in processing ubiquitinated\ntransmembrane proteins at post-ER compartments (Golgi, endosomes, vacuole/lysosomes). The authors further demonstrate\nthat K48-linked chains are the preferred signal for engaging these Ub-dependent proteases, while K63-linked chains funnel\nthe same transmembrane protein substrate into ESCRT-dependent MVB formation and consequently lysosomal/vacuolar\ndegradation. The direct comparison of the signal on the same substrate type is key to compare the effect of the Ub chain\ntypes.\nThese data have important implications for the proteostasis and ubiquitin field.\nMajor points\nPlease check for correct terminology i.e. when to use the term Ub-activated protease. To my understanding ubiquitination\nfacilitates substrate targeting for Rbd2, however does not activate the protease activity per se. Similarly, the term ‘amplified’\nin the abstract, when referring to the effect of the UBA and UBL domains on the activity of Ddi1 should be reconsidered.\nLine 256 – in this study K63 and K48 chains were compared. Selectivity of the ESCRT machinery for K63 cannot be\nconcluded from these data alone. Other chain types may be processed/engaged by the ESCRT machinery. Please clarify.\nLine 269 – which data show effects on cell growth? Is cell growth affected under the applied conditions and if so, how was\nthis taken into consideration when interpreting degradation data?\nLine 269 – the degradation of the construct is not blocked but rather strongly impaired. Please rephrase.\nLine 381/Figure 3C – Based on supplemental figure 3, the expression level of the Rad23-DDI1 chimera is not at all\ncomparable to wt. Based on the presented data, quantification of the clip under these conditions should be omitted. The\nrescue with pTEF-WT shows high variability. Could the authors please comment on as to why that could be the case?\nDo the authors expect all constructs tested in Figure 3C to have K48 chain type specificity?\nFigure 4B – what does the fragment, which appears between 50-70 kDa in the absence of RBD2 activity correspond to? Is\nthis fragment also seen on the gels shown in panel A? The fragment also appears in Figure 5.\nIn Figure 4C – which difference was quantified here? How do negative values arise in the quantification? Could this be\nrelated to the Western blot signal being out of the linear range?\nLine 485 – Did the authors attempt to quantify the levels of the model substrates by other methods than Western blot?\nImaging- or flow cytometry-based quantification of the fluorescence signal of the model substrate or a deubiquitination prior\nto SDS-PAGE may help to clarify the question whether the degradation of the model substrate completely blocked.\nLines 498-504 – please rephrase this paragraph for clarity. In what way was the Rbd2-lumenal product variable? How do\nyou interpret this effect?\nFigure 6 – why does the Figure legend refer specifically to endocytic substrates? The pathway could equally target secretory\nsubstrates.\nCould the authors comment on whether a single K48 modified substrate protein is processed by both proteases DDI1 and\nRBD2? Do they expect the different proteases/degradation pathways to act in parallel on a single substrate protein?\nIs p97 required for the proteasome to engage the model substrate or does the proteasome act directly on the membrane?\nMinor points\nFor clarity please refer to indole-3-acetic acid by IAA OR Aux in Figures and text.\nLines 242-252 duplicate the previous paragraph.\nLine 289 – was mCherry or RFP inserted? Please clarify.\nLine 348 – which additional mechanisms could underlie the degradation of the construct? Please elaborate.\nLine 358 – should be Nrf1.\nFigure 3B – please refer to the strain that was used to generate data in panel B in the figure legend.\nLine 379 – should be HA-tagged DDI1 variants.\nLine 444 – Cdc48/p97 is not a Ub-adaptor protein. The AAA unfoldase cannot bind Ub on its own but requires adaptor\nproteins (UFD1/NPL4) to engage with ubiquitinated substrates. Cdc48/p97 works with a host of adaptors, some of which are\nthe SHP box adaptors. Please clarify in the text.\nPlease highlight the vacuole/delimiting membrane, ER, and other cellular organelles where appropriate in fluorescence\nmicroscopy images for people not familiar with imaging studies in yeast.\nThe comprehensive literature review in the Supplement is a great resource. Possibly consider presenting in Table format in\nthe main text."}, "author_response": "Response to Reviewer’s suggestions and comments\nReviewer 1\nThe manuscript submitted by Minard and co-workers focuses on post-endoplasmic\nreticulum proteins and how K48-linked polyubiquitination and K63-linked\npolyubiquitination affect lysosomal degradation (in the case of K63-linkage) as well as\nshearing from the membrane (in the case of K48-linkage). Two ubiquitin-activated\nproteases have been studied in detail: Ddi1 and Rbd2. The data of the authors support the\nestablished fact that the site used for polyubiquitination (K48 and K63 as used here)\nencode the fate for proteins affected. The authors have made use of a repertoire of\ndifferent experimental methodologies belonging to molecular biology, biochemistry, and\nbiophysics to address the scientific objective of activating proteases by the ubiquitin\nmachinery.\nI will focus in my review on the results obtained by high-resolution NMR spectroscopy in\nthe following as it represents my expertise regarding the experimental repertoire used by\nthe authors.\n1. The NMR data obtained in this study are presented in Figure 3D, E in the main part of the\nmanuscript as well as in Supplemental Figure 4. Overall, I have to say that I am surprised\nabout the superficiality of the presentation of the NMR data, especially when submitting\nthe work to such a respective Journal like Nature Communications.\nResponse: We thank the reviewer for their critical feedback. In response, we have\nexpanded the presentation of the NMR data, which demonstrate that the central part of\nyeast and human Ddi1 (HDD-RVP domains) binds mono-ubiquitin in HSQC experiments\nwith 15N-Ub. These findings provide useful mechanistic insight into Ddi1’s-ubiquitin-\ndependent activity. At the same time, we would like to emphasize that the NMR analysis\nconstitutes a secondary focus of the manuscript, whereas the central message is to\ndemonstrate the existence of an unexpected Ub-linkage-specific mechanism for degrading\npost-ER membrane proteins by two underappreciated enzymes.\n2. The data presented in Figure 3D, E (left) aim to report on the interaction between 15N\nisotopically enriched ubiquitin and HDD-RVB domains from Sc (D) and human (E). The\nmanuscript does not comprise the correct information about the analytical function the\nauthors have used to fit (continuous line in the plots) the experimental data. The statement\n“… fitted to a standard quadratic equation …” is not only superficial but also wrong.\nResponse: In the methods section, we now fully describe how the data in Figure 3D, E were\nfitted to solve for the dissociation constant of the interaction of 15N-Ub and the HDD-RVP\ndomain of yeast Ddi1 and human DDI2. The change in peak intensity of Ub L71 was\nassumed to reflect the fraction of ubiquitin bound to Ddi1 (Fielding, 2007). The change in\npeak intensity (ΔI) was calculated as the difference in peak intensity in the absence (I )\nfree\nand presence (I ) of Ddi1, then divided by the peak intensity in the absence of Ddi1 (see\nbound\nequation (1)).\n𝛥𝐼 = $\n!\n!\"##\n\"!$%&'(%\n≡ [PL]/[𝑃 ] (1)\n#\n!\n!\"##\n.\nThe changes in peak intensity were fitted to the single-site binding model described in\nequation (2), using a least-squares fit calculated with GraphPad Prism to find the\ndissociation constant K and maximal concentration of bound protein [PL ].\nd max\n[PL]/[𝑃 ] = [PL ]\n((\n)\n)[+])[-*])\"(/((\n)\n)[+])[-*])+(\n0[+][-* ])\n(2)\n# $%&\n1[-*]\nWhere [P], [L], and [PL] refer to:\n• the total molar protein concentration of the protein 15N-Ub, which had a fixed\nconcentration during titration.\n• the titrating ligand Ddi1 concentration.\n• the bound ubiquitin-Ddi complex.\nIn Figure 3D, E [PL]/[P] is plotted on the Y axis (Bound Fraction) and [L] is on the X axis (µM).\nt\n3. A reliable analysis of an NMR spectroscopic titration experiment relies on accurate\nreferencing of the dimensions used for data acquisition. Which efforts have the authors\nmade in this respect?\nResponse: This is now stated in the NMR methods section. The 1H chemical shifts are\nreferenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The 15N chemical shifts were\nindirectly referenced to DSS using the Bruker software protocol.\n4. Which cross peaks arising in 2D 1H-15N HSQC spectra have been used for the two\nindividual plots shown in panel D and E? What is the rationale for the specific color coding\nused here (note: color coding used in Supplemental Figure 4 cannot be identified; see also\nbelow)?\nResponse: We used Ub L71 peak intensity data for the two individual plots. We chose L71\nbecause it is located in the binding interface and exhibits relatively large peak intensity\nchanges. Originally, the color coding corresponded to the concentration of the HDD-RVP\ndomain and matched the contours of the HSQC 15N-Ub spectra shown in the\nSupplemental Figure 4. We have now changed the figures in response. Full contour plots\nof 15N-Ub HSQC (-/+) HDD-RVP are now shown in Supplemental Figure 5. The titration\ncurves are now simplified without color in Figure 3D,E\n5. Labeling of y-axes misses completely, labeling of x-axes is incorrect. In fact, to\nquantitatively analyze an NMR titration experiment, the concentration of the ligand (here:\nHDD-RVB domains) has to be presented, the concentration of the target protein (here:\nubiquitin) is contained in the fitting function. Additionally, neither the parameter nor the\nunit is presented on the x-axes. Which information is additionally encoded in using circles\nas triangles in panel D, E (no information found in the legend accompanying this Figure)?\nResponse: The new figures (3D and 3E) are labelled as: Y axis is ‘bound fraction’; X-axis is\nconcentration of the HDD-RVP domain in µM.\n6. The legend of Figure 3 states “residues of Ub that underwent the largest chemical shift\nperturbation are mapped in red onto the structure of Ub”. Which specific cut-off value of\nthe perturbation of the chemical shift has been used? Additionally, a separate plot\npresenting residue-specific perturbations of chemical shifts is highly recommended.\nResponse: The cut-off values of chemical shift perturbations quantified by ((0.2ΔN2 +\nΔH2)1/2) that were used to map the largest CSPs onto the Ub structure are now designated\nin Supplemental Figure 5 and explained in the Methods section that specified we used a\n0.5 SD above the mean CSP.\n7. Data presented in Figure 3 report on an NMR titration experiment. The authors have\nsolely analyzed changes in chemical shifts upon adding of an HDD-RVP domain to\nubiquitin. Unfortunately, line width and signal heights of resonance signals arising in 2D\n1H-15N HSQC have not been analyzed by the authors.\nResponse: We now clarify this in the NMR methods section. We have analyzed the data\nusing both chemical shift perturbation and peak intensities (heights), knowing peak height\nis inversely proportional to the linewidth.\n8. The authors have not mentioned the binding model (e.g. one-to-one binding, …) they\nhave used for interpreting the NMR spectroscopic data. The stoichiometry represents an\nimportant parameter when performing an interaction study.\nResponse: As mentioned in response to point 2, we now clarify that the binding model\nused was a stoichiometry of 1:1 in the NMR methods section\n9.\n• The title of Supplemental Figure reads as “HSQC analysis of 15N-Ub in the presence of the\nHDD-RVP domains from ScDdi1 and human DDI2” even panel A presents a SDS-PAGE.\n• Panel B of Supplemental Figure 4 presents rather small sections of the entire spectral\nranges the 2D 1H-15N HSQC NMR spectra comprise. It is highly recommended to show the\nentirely acquired data set this is, if required, followed by smaller sections. Additionally,\ncross peaks originating in 2D NMR correlation spectra are shown as contour lines.\nUnfortunately, the authors present border strips only.\nResponse: Thank you for the suggestion. These data are now presented in Supplemental\nFigure 5 that shows the full spectra, a zoomed in section where more conventional contour\nplats are presented, and the magnitude of chemical shift perturbations for residues of 15N-\nUb when bound to HDD-RVP.\n10. General: I am not convinced that the authors have not used D2O in their NMR\nexperimental work.\nResponse: In the NMR methods section, we now clearly state that all NMR samples\ncontained 10% D2O.\nReviewer 2\nIn this paper, authors report that K63-linked, but not K48-linked, polyubiquitination\ninduces MVB sorting and lysosomal degradation of membrane proteins in S. cerevisiae.\nThey also report that K48-polyUb substrates are degraded after leaving the ER via a\npathway, termed CUT-UP (Cleavage of Ubiquitinated Targets by Ubiquitin-activated\nProteases), which induces shearing of membrane proteins. They also report that K48-\npolyUb substrates are processed by the proteasome and by the two Ub-activated\nproteases Ddi1, a conserved cytosolic ubiquilin that generates cytosolic fragments and\nRbd2, a rhomboid protease of Golgi/endosomes but also acting on the vacuole, which\nproduces lumenal fragments. Finally, they report that the catalytic core of Ddi1, consisting\nof the HDD-RVP domain, is sufficient for Ub-dependent proteolysis, and that its activity is\nenhanced by auxiliary ubiquitin binding domains (atypical UBL domain and a UBA domain).\nThey conclude that polyUb chains linked by different residues lead to different fates for\npost-ER proteins, and that there are two proteases that target ubiquitinated integral\nmembrane proteins.\nThe topic is interesting, and the paper provides novel insights into our understanding of the\nmechanisms responsible for the degradation of membrane proteins. The paper combines\nelegant strategies with solid biochemical and morphological data. In general, I find the\ndata convincing, with some relatively minor reservations below.\n1. My main comment – or concern – is that it would be appropriate and helpful to illustrate\nconditions where native candidate proteins follow these Ub-dependent (Rbd2- or Ddi1-\ndependent) CUT-UP, protein shearing degradation pathways (see model in Fig 7).\nResponse: We thank the reviewer for their interest in our work. We agree that an important\nnext step is to identify the native substrates of this pathway, the proteotoxic conditions\nunder which they engaged, and the biological consequence of their loss. We highlight this\npoint in the Discussion ‘Further studies are needed to identify the natural substrates of\nCUT-UP pathway and physiological conditions which activate it.’ However, pursuing this\ndirection with the same rigor as the present work would require substantial groundwork\nand, in effect, constitute a new and distinct chapter of investigation. Notably the targeted\ndiscovery of protease substrates is a technically challenging undertaking, as reflected by\nthe limited number of known substrates for Ddi1 and Rbd2 and more generally for\nrhomboid proteases. Technical advances such as using substrate-traps (PMID: 35173328)\nmay accelerate progress, but these techniques would still need to be adapted and\nvalidated in our hands before we could apply them to the CUT-UP pathway.\nIn the current study, our aim is to provide a clear example of the “ubiquitin code” in\naction and to identify two underappreciated enzymes with newly defined roles in protein\ndegradation. We provide the first characterization of Ddi1 and Rbd2 enzymatic activity in\nyeast, with evidence supporting a general role for these enzymes in processing K48-linked\nubiquitinated proteins. Our newly included data further demonstrate that Ddi1 can\nprocess cytosolic proteins and that, in the absence of Ddi1 and Rbd2, the cytosolic\ndomain of our reporter is processed by the proteasome in a manner independent of Cdc48.\nWe also addressed reviewer 3’s question regarding the additional Ddi1-dependent product\nthat appears when Rbd2 is absent. Finally, using CUT-UP pathway mutants, we extended\nour findings to show that the ubiquitin code is strict for MVB sorting, since it is not engaged\nby K48-ubiquitinated Vps10, even when the alternative CUT-UP pathway is disrupted.\n2. Nowhere in the text (Methods, etc.) is it stated that anti-GFP E3 ligases are HA-tagged,\nas seen on the blots Fig 1E. It should be mentioned at least in the Fig legend.\nResponse: This omission has been corrected. The methods are revised to state “The E3\nligases were fused C-terminally to four tandem HA epitopes and the VHH-domain\ncABGFP4 nanobody (Saerens et al., 2005).”; and the Results are revised to state “The Ub-\nligases (Figure 1A-B) included HECT- and RING- catalytic domains fused to an HA-tagged\nα-GFP nanobody …” . The legend for Fig. 1 now reads: “A) K63-Ub-ligases were engineered\nby fusing an HA-tagged GFP nanobody to the RING domain of Pib1, two tandem RING\ndomains fo RNF4, the HECT domain of Rsp5, and a chimera of the Rsp5 and E6AP HECT. …\nImmunoblots (α-GFP, α-HA) of Vps10-GFP and the indicated HA-tagged GFP nanobody-Ub\nligases after 1h of doxycycline treatment in PEP4 and pep4Δ cells.”\n3. It is stated that strains carrying K6R, K11R, K27R, K29R K33R, or K63R as their only form\nof Ub, all degraded Vps10-NG-AID and formed Ddi1-dependent cytosolic fragments (Fig\n3B), but that Vps10-NG-AID degradation and formation of Ddi1-dependent fragments were\nslightly impaired in the K63R mutant strain. However, the data are not clear-cut and K63R\ndoes not appear to be that different from other strains.\nResponse: Thank you for pointing this out as an issue to call out to the reader. The yeast\nstrains carrying single Ub K>R mutants were obtained from David Toczyski’s lab, who\noriginally published them in eLife (doi.org/10.7554/eLife.42955). The K63R strain, while not\ndescribed in detail in that work, came to us with the caveat that it may have growth defects\nand is unusually canavanine-sensitive even when CAN1 is deleted. Upon reviewing the\ndata we found that this strain consistently expressed lower levels of the Vps10-NG-AID\n(Supplemental Figure 3B), and for this dataset overall (across all samples for all strains) a\nlow level of Vps10-NG-AID in any K>R mutant was associated with less degradation\n(Supplemental Figure 3C). Given the strain’s phenotypes and low expression, we think the\ndegradation defect is most likely an indirect effect, and have therefore presented these\nobservations without overstating their importance. The main question we wished to\naddress was whether any non-K48 linkage was required for degradation, and none were.\n4. The authors mention that the loss of both Rbd2 and Ddi1 together with MG132 treatment\nprevent the formation of truncated fragments, and that a HMW product then forms that\nremains in the SDS-PAGE stacking gel. The HMW product, which I assume ran at the\nposition of the top arrowhead in Fig 5A, is barely visible, and the total amount of this high MW\nproduct does not seem to be roughly equivalent to the starting amount of full-length\nprotein. Where is the boundary between stacking and separating gels?\nResponse: We used SurePAGE commercial gradient gels for these studies, which have a\nshort “stacker” portion whose position we did not record. In earlier work, we routinely\ntrimmed the top of the gel just below the wells; however, we found that omitting this\ntrimming allowed detection of the HMW species. Accordingly, we can only indicate the\nposition of the top MW marker rather than a stacking/separating boundary. We have\nrevised the Methods text to clarify: “To detect the high molecular weight forms of Vps10\n(Figure 6-8) the entire gel including the loading lanes was transferred.”\nTo make the high-MW species more visible, we reconfigured Figure 5, 8 and Supplemental\nFigure 6 to include two exposures: a lighter one showing overall degradation and a darker\none showing the high-MW species.\nWe also now demonstrate that a cytosolic NG-AID fusion protein is cleaved not only\nby the proteasome but also by Ddi1, and that combined inhibition of Ddi1 and the\nproteasome results in a high-MW smear with laddering consistent with poly-Ub chain\nattachment (Figure 3F).\nTo better determine whether the starting protein is fully recovered when Ddi1, Rbd2,\nand the proteasome are absent/inhibited, we performed a deubiquitination assay to\ncollapse the high-MW band. We reasoned that the high-MW forms diluted the signal by\nspreading across a broad size range, which could have made quantification and transfer\nfrom the gel unreliable. To address this, we deubiquitinated Vps10 to collapse the high MW\nproducts and improve detection. To deubiquitinate Vps10, we used an ATP-depletion\nprotocol (2-deoxyglucose, fluoride, and azide) to halt ATP-dependent processes such as\nubiquitination while allowing deubiquitination by endogenous ubiquitin-peptidases to\nproceed. This collapsed the high-MW smear for both cytosolic NG-AID and RFP-Vps10-NG-\nAID (Figure 3F and 6A). Quantification of western blots of RFP-Vps10-NG-AID revealed that\nNG signal was fully recovered, and RFP signal was fully recovered after ATP depletion\n(Figure 7A).\nWe further assessed the extent of recovery of RFP-VPS10-NG-AID using flow\ncytometry. This revealed that Neon Green fluorescence was largely recovered in the ddi1Δ\nrbd2Δ mutant upon proteasome inhibition (Supplemental Figure 6B), however, mCherry\nexpression was too low to be detected by this method. Together, these approaches\nsupport the conclusion that loss of Ddi1 and Rbd2, combined with proteasome inhibition,\nleads to robust stabilization of the parent protein.\nIn addition to the above findings, the ATP depletion assay also revealed that Ddi1\ngenerated multiple cleavage products. Ddi1 generated multiple clipped products and\nsince their sizes were unaffected by ATP depletion, the sizes were unlikely due to\ndifferences in polyubiquitin chain length, but instead from cleavage at multiple sites.\nReviewer 3\n1. Please check for correct terminology i.e. when to use the term Ub-activated protease. To\nmy understanding ubiquitination facilitates substrate targeting for Rbd2, however does not\nactivate the protease activity per se. Similarly, the term ‘amplified’ in the abstract, when\nreferring to the effect of the UBA and UBL domains on the activity of Ddi1 should be\nreconsidered.\nResponse: We agree with the reviewer that this is a point of biochemical precision, with\n“ubiquitin-activated protease” indicating that the catalytic domain of a protease is\nactivated by ubiquitin binding, whereas “ubiquitin-dependent” could encompass the\nformer model as well as a model where ubiquitin-binding targets the substrate to an\nenzyme with an already active catalytic domain. In the Discussion we consider both\nmechanisms. In the case of Ddi1, in vitro studies show that the recombinant catalytic\ndomain can only act on ubiquitinated substrates. Therefore, some mechanism must exist\nto activate its proteolytic RVP domain, which we hypothesize involves direct ubiquitin\nbinding given that the HDD-RVP domain binds ubiquitin. In the case of Rbd2, the lack of in\nvitro data and the unknown mechanism of its interaction with ubiquitin make it unclear\nwhether ubiquitin facilitates substrate binding, activation of Rbd2, or both.\nWe also appreciate the reviewer’s point about the term “amplify” and now use\n“enhance” to describe the effect of the UBA and UBL domains on Ddi1 activity.\n2. Line 256 – in this study K63 and K48 chains were compared. Selectivity of the ESCRT\nmachinery for K63 cannot be concluded from these data alone. Other chain types may be\nprocessed/engaged by the ESCRT machinery. Please clarify.\nResponse: We agree this is the wrong conclusion. The text has been changed to “ These\ndata indicate that K48- and K63-Ub ligases engaged distinct degradative pathways,\ndemonstrating a functional Ub code for post-ER proteins and that the ESCRT/MVB pathway\nselectively excludes K48-ubiquitinated cargo.”\nWe also have additional data demonstrating this point in the revised manuscript.\nSince it was possible that rather than the ESCRT/MVB pathway actively rejecting K48-\nubiquitinated cargo, the cargo was simply more efficiently engaged by the CUT-UP\npathway, we tested whether disrupting the CUT-UP pathway caused K48-Ub cargo to\nundergo MVB sorting. As shown in Figure 6B, even when Ddi1 and Rbd2 are absent and\nproteasome activity is inhibited, RFP-Vps10-NG-AID does not undergo MVB sorting. We\ndemonstrated this by activating lumenal vacuolar proteases, which generate a processed\nform of NeonGreen (Vac-NG) from NG-tagged MVB substrates. K48-ubiquitinated RFP-\nVps10-NG-AID did not produce a vacuolar-processed form of NeonGreen and instead\naccumulated in high-molecular-weight forms.\n3. Line 269 – which data show effects on cell growth? Is cell growth affected under the\napplied conditions and if so, how was this taken into consideration when interpreting\ndegradation data? Line 269 – the degradation of the construct is not blocked but rather\nstrongly impaired. Please rephrase.\nResponse: We have clarified these points in the revised manuscript.\n• For conditions using MG132 inhibitor, we now use the term “inhibited” rather than\n“blocked.”\n• We added Supplementary Figure 1B, which shows that overnight growth is strongly\ninhibited by MG132 only in the pup1T30A pre3T20A pdr5Δ strain, but not in the PUP1 PRE3\npdr5Δ strain.\n• This figure also shows that in the short term (2 h), the pup1T30A pre3T20A pdr5Δ strain\nincreased its OD₆₀₀ to the same extent whether MG132 is present or not. This 2 h window\nencompasses all experiments reported here. Since no short-term growth defect was\nobserved from MG132 treatment, there was no need to adjust degradation measurements\nfor potential reduced cell numbers. These data demonstrate that inhibiting all three\ncatalytic sites was necessary to achieve meaningful inhibition of proteasome activity. By\ninhibiting the proteasome thoroughly, we were able to reveal other proteases in CUT-UP\npathway.\nWe also investigated whether degradation of a cytosolic substrate occurred independently\nof the proteasome via Ddi1. Fig 3F demonstrates that Ddi1 deletion prevented clipping of a\ncytosolic NeonGreen-AID and led to the accumulation of high molecular weight species of\nNeonGreen-AID. These data reveal that Ddi1 cooperates with the proteasome to clip\ncytosolic substrates as well as membrane substrates.\n4. Line 381/Figure 3C – Based on supplemental figure 3, the expression level of the Rad23-\nDDI1 chimera is not at all comparable to wt. Based on the presented data, quantification of\nthe clip under these conditions should be omitted. The rescue with pTEF-WT shows high\nvariability. Could the authors please comment on as to why that could be the case?\nResponse: We now note in the text that the expression level of the Ubl –Ddi1 chimera is\nRad23\nlower than that of wildtype Ddi1. We have retained the quantitation in Figure 3, as the\nconstruct was still expressed and its activity was measured with multiple independent\nexperiments. By explicitly stating the difference in expression, we allow readers to interpret\nthe data with this caveat in mind.\nRegarding the variability in the pTEF-WT rescue, we agree with the reviewer that it is\nnotable. This construct overexpresses Ddi1, and was made in case orthologous Ddi1\nproteins expressed poorly in yeast. pTEF-WT overexpressed Ddi1-HA in all five replicates;\nin two replicates it exhibited no change in Ddi1 activity and in three replicates it exhibited\nincreased activity. However, our assay was not optimized to detect increases in Ddi1\nactivity since we assessed activity at a single time point (60 min after SCFTIR1 induction)\nwhen a large proportion of Vps10 is degraded (Figure 1F). It’s unclear why in some\nreplicates more Ddi1 fragments were recovered, there might be unanticipated secondary\nproteostasis adaptions in response to Ddi1 overexpression, or this could just reflect\ntechnical and biological variability. A more detailed study would be required to assess\ndose dependence of Ddi1 on its biological activity. Importantly, with the exception of the\nUbl –Ddi1 chimera, the variants expressed to similar levels as their wildtype control.\nRad23\n5. Do the authors expect all constructs tested in Figure 3C to have K48 chain type\nspecificity?\nResponse: This is an interesting question that we think relates two other questions, to what\nextent is Ddi1 (or Rbd2) specifically activated by Ub linkages other than K48, and what\ndomains confer K48-Ub specificity. We think these questions are best addressed in future\nstudies focused on the biochemical mechanisms of Ddi1 and Rbd2.\nWhile our current data provide strong evidence that K48 linked ubiquitination does\nnot support MVB sorting, even in the absence of the CUT UP pathway, the reciprocal\nquestion, whether other ubiquitin chain types might serve as signals for Ddi1 or Rbd2\ndependent cleavage, remains unresolved. Our preliminary experiments however, indicate\nthat ubiquitinated Mup1 GFP in an ESCRT mutants is highly stabilized, and does not divert\ninto Ddi1 or Rbd2 dependent degradation.\nIt is also unclear how K48 ubiquitinated substrates are recognized by Ddi1, given\nthat the ubiquitin binding elements it employs, including the HDD RVP core domain\ncharacterized here, bind mono ubiquitin in vitro. As noted by the reviewer, a key\nmechanistic question is whether this apparent chain type preference is conferred by the\nHDD RVP domain, or other Ub binding modules. Addressing this will require improved\nassays sensitive enough to detect the low level of activity associated with the HDD RVP\ncore and with alternative polyUb chains. This assay should perhaps use a cytosolic\nsubstrate, that is not subject to MVB, ERAD, or EGAD pathways. Although these\nmechanistic questions are beyond the scope of the current study, they represent an\nimportant direction for future work to define how Ddi1 achieves its ubiquitin dependence\nand any specificity toward ubiquitin chain topology.\n6. Figure 4B – what does the fragment, which appears between 50-70 kDa in the absence of\nRBD2 activity correspond to? Is this fragment also seen on the gels shown in panel A? The\nfragment also appears in Figure 5.\nResponse: We thank the reviewer for this astute observation. Additional experiments, now\nincluded in Figure 7, focus on this extra band, termed RFPp55, which is derived from the\nlumenal domain of the RFP-Vps10-NG-AID reporter protein. As shown in Figure 7A, it\naccounts for only ~1% of the total parental protein after ubiquitination and proteasome\ninhibition are triggered, but this proportion increases when Rbd2 is deleted. This suggests\nthe existence of a backup pathway that can be engaged when Rbd2 is absent.\nRFPp55 is not observed in the absence of Ddi1 (either in ddi1Δ alone or in the rbd2Δ\nddi1Δ double mutant treated with MG132). This indicates that the lumenal domain either\ngains access to the cytosol to become a Ddi1 substrate, or that Ddi1 acts indirectly on the\nmachinery processing RFPp55. We considered the Tul1/Dsc complex as a candidate for\ncytosol export activity, given recent studies suggesting it can act as a translocation\nchannel for some post-ER ubiquitinated proteins. However, RFPp55 was still produced in a\ndsc2Δ mutant. Likewise, we replaced the wild-type CDC48 allele in our reporter strain with\neither cdc48-2 or cdc48-3, simultaneously inactivating the endogenous CDC48, to assess\nany dependence on Cdc48; at the restrictive temperature (37 °C), RFPp55 production\npersisted in cdc48-ts mutants. This makes it unlikely that the fragment is generated via ER\nretrotranslocation through known Cdc48-dependent machinery. Finally, we used a\ntetracycline-sensitive riboswitch to shut off translation of newly synthesized reporter for 1\nh before inducing ubiquitination to ensure RFPp55 was generated from a post-ER\npopulation rather than a newly synthesized one in the ER. Overall, these findings suggest\nan interesting backup or parallel processing pathway to Rbd2, which becomes more\napparent in its absence.\n7. In Figure 4C – which difference was quantified here? How do negative values arise in the\nquantification? Could this be related to the Western blot signal being out of the linear\nrange?\nResponse: Negative values arose because the data are expressed as the difference in\nsoluble RFP between the “copper and auxin treated” and “untreated” conditions, with the\n“untreated” value subtracted from the “treated” value. In some rbd2∆ samples, slightly\nlower levels of soluble RFP were detected in the treated condition compared to the\nuntreated condition, resulting in a negative value. Overall, in rbd2∆ samples no increase in\nsoluble fragments were observed, and thus the difference between “untreated” and\n“treated” condition was zero with minor biological and technical variation between\nreplicates.\n8. Line 485 – Did the authors attempt to quantify the levels of the model substrates by\nother methods than Western blot? Imaging- or flow cytometry-based quantification of the\nfluorescence signal of the model substrate or a deubiquitination prior to SDS-PAGE may\nhelp to clarify the question whether the degradation of the model substrate completely\nblocked.\nResponse: Thank you for this suggestion. Reviewer 2’s fourth question also asked whether\nRFP-Vps10-NG-AID degradation was completely blocked by inhibiting the proteasome in\ncells lacking Rbd2 and Ddi1. We were unable to successfully isolate high–molecular\nweight forms of RFP–Vps10–NG–AID under native lysis conditions in order to add\nrecombinant deubiquitinating enzymes. Instead, we rapidly depleted ATP using sodium\nazide (electron transport inhibitor), sodium fluoride (enolase inhibitor), and 30 mM 2-\ndeoxyglucose (glycolysis inhibitor that consumes ATP) to exhaust ATP stores. This\napproach was intended to prevent new ATP-dependent ubiquitination and degradation\nwhile allowing endogenous deubiquitinating enzymes to remove ubiquitin chains. Under\nthese conditions, the high–molecular weight smear collapsed, and quantification of these\nblots showed near-complete preservation of RFP–Vps10–NG–AID immunoreactivity\nfollowing SCFTIR1-mediated ubiquitination when MG132 was present in rbd2Δ ddi1Δ\nmutants (Figure 6A).\nWe also performed flow cytometry to complement these observations\n(Supplementary Figure 6B). These data showed degradation of NG fluorescence was\nstrongly inhibited in MG132-treated rbd2Δ ddi1Δ mutants comparable to that observed in\nimmunoblots of ATP-depleted cells. Unfortunately, the RFP signal was too low to measure\nby flow cytometry.\n9. Lines 498-504 – please rephrase this paragraph for clarity. In what way was the Rbd2-\nlumenal product variable? How do you interpret this effect?\nResponse: We now clarify that the production of the Rbd2-dependent lumenal fragment is\nreduced when Ddi1 is lacking and cells are treated with MG132. Although this may be\ninterpreted as indicating that Rbd2 processing occurs after the cytosolic domain is\nprocessed, we call attention to the observation that in fact full-length Vps10 is still\ndegraded in an Rbd2-dependent manner, indicating that Rbd2 activity does not rely on\nproteasomal or Ddi1 activity. A potential explanation for this trend is that perhaps loss of\nDdi1 increases the ability of Rbd2 to process RFP–Vps10–NG–AID in the TGN, which could\nlead to more secretion of the lumenal domain and a decrease in what is recovered in\nimmunoblots. However, given that the effect is small, we do not think it warrants detailed\nspeculation or additional experimentation.\n10. Figure 6 – why does the Figure legend refer specifically to endocytic substrates? The\npathway could equally target secretory substrates.\nResponse: We agree. The text in the results and the figure legends (previously those for 6\nand 7) reference the broader secretory/endocytic endomembrane system.\n11. Could the authors comment on whether a single K48 modified substrate protein is\nprocessed by both proteases DDI1 and RBD2? Do they expect the different\nproteases/degradation pathways to act in parallel on a single substrate protein?\nResponse: Our data demonstrate that Rbd2 and Ddi1 act in parallel since neither are\ndependent on each other for cleaving Vps10. We considered that if they acted on separate\nmolecule loss of one should increase substrate availability for the other, resulting in\nelevated product levels. However, neither Rbd2 or Ddi1 deletion stimulated an increase in\neach other’s cleavage products, indicating that both proteases acted on the same single\nmolecule.\nConsideration must also be given to the order of action. If Ddi1 acts first to remove the\nubiquitinated C-terminus, this should prevent recognition of the substrate by Rbd2,\nunless\nRbd2 can also recognize other features e.g. partially cleaved tails. Conversely, if Rbd2 acts\nfirst, the ubiquitinated C-terminus is still intact for Ddi1 processing and would not affect\nDdi1 activity. Thus, we propose two models that explain our observations. 1) a flux-driven\nsequential model, in which Rbd2 outcompetes Ddi1 and proteasomes, cleaving the\nsubstrate first. Or 2) a parallel processing model, which is possible if Rbd2 could also\nrecognize other features of Vps10, e.g. partially cleaved tails.\n12. Is p97 required for the proteasome to engage the model substrate or does the\nproteasome act directly on the membrane?\nResponse: This is an intriguing question that we now address in Supplemental Figure 6E.\nWe moved the cdc48-2 (A547T, T803A) and the cdc48-3 alleles (P257L, R387K) into our\nreporter strain system and found that at 37°C there was still degradation of the NeonGreen\ncontaining cytosolic tail upon SCFTIR1-mediated ubiquitination. Thus, Cdc48 is not\nrequired, and we propose that the proteasome is acting directly on the membrane\nembedded substrate.\nFor clarity please refer to indole-3-acetic acid by IAA OR Aux in Figures and text.\n> We now clarify in the methods section that the type of Auxin we use for these studies is\nexclusively IAA. And exclusively use Auxin in the figures and text unless explaining what IAA\nis.\nLines 242-252 duplicate the previous paragraph.\n> Thank you for pointing out this error\nLine 289 – was mCherry or RFP inserted? Please clarify.\n> We have clarified in the text that mCherry, a red fluorescent protein (RFP) variant, was\ninserted. The text now states “mCherry (designated as RFP)” to indicate that mCherry is\nthe specific RFP used in these experiments.\nLine 348 – which additional mechanisms could underlie the degradation of the construct?\nPlease elaborate.\n> We now state ‘These data indicated that Ddi1 is responsible for forming the clipped\ncytosolic fragment of Vps10-NG-AID, however, since full-length Vps10-NG-AID was not\nstabilized, additional degradation mechanisms act on Vps10-NG-AID.’ This section is in\nthe Results and explains the motivation to discover the additional degradation\nmechanisms, which we reveal is mediated by Rbd2.\nLine 358 – should be Nrf1.\n> Thank you. Corrected.\nFigure 3B – please refer to the strain that was used to generate data in panel B in the figure\nlegend.\n> All strains used are explicitly called out in the supplementary strain table that describes\nthe strains genotype and which figures and subpanels where it is used.\nLine 379 – should be HA-tagged DDI1 variants.\n> Yes, it should be and has been corrected.\nLine 444 – Cdc48/p97 is not a Ub-adaptor protein. The AAA unfoldase cannot bind Ub on its\nown but requires adaptor proteins (UFD1/NPL4) to engage with ubiquitinated substrates.\nCdc48/p97 works with a host of adaptors, some of which are the SHP box adaptors.\nPlease clarify in the text.\n> Yes, DOI: 10.1038/35087056 notwithstanding, Cdc48 uses a host of Ub-binding partners\nto reach its substrates. We no longer refer to Cdc48 itself as a Ub-adaptor protein.\nPlease highlight the vacuole/delimiting membrane, ER, and other cellular organelles where\nappropriate in fluorescence microscopy images for people not familiar with imaging\nstudies in yeast.\n> Localizations of fluorescent proteins are now pointed out in Fig. 1, 2, and 8.\nThe comprehensive literature review in the Supplement is a great resource. Possibly\nconsider presenting in Table format in the main text.\n> Thank you. We are not sure it fits physically there but will work with the editors\naccordingly.\nFielding, L. (2007). NMR methods for the determination of protein–ligand dissociation\nconstants. Progress in Nuclear Magnetic Resonance Spectroscopy, 51(4), 219-242.\nhttps://doi.org/https://doi.org/10.1016/j.pnmrs.2007.04.001\nSaerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., Wyns, L.,\nMuyldermans, S., & Conrath, K. (2005). Identification of a universal VHH framework to\ngraft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol,\n352(3), 597-607. https://doi.org/10.1016/j.jmb.2005.07.038\nresponse to referees: NCOMMS-25-30550B\nWe have made the following changes in response to Reviewer 1, who pointed out some clumsy grammar.\nThanks for the opportunity to correct this.\nOld: The resulting ΔI values were fit to a single-site binding model described in equation ..\nNew: The resulting ΔI values were used to fit the single-site binding model described in equation..\nOld: Ub L71 was selected because it lies within the binding interface and exhibits both large chemical shift perturbations and\npeak intensity changes.\nNew: Ub L71 was selected because it lies within the binding interface and exhibits both larger chemical shift perturbations\nand peak intensity changes."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Minard and co-workers submitted a revised version of their manuscript entitled “K48-ubiquitin-dependent proteases cut-up\npost-ER proteins”.\nGenerally, they have addressed the points raised in my previous review. However, I recommend to fix the following items\nbefore publication (numbering used here refers to the numbering used in the rebuttal letter):\n2. The data presented in Figure 3D, E (left) aim to report on the interaction between 15N isotopically enriched ubiquitin and\nHDD-RVB domains from Sc (D) and human (E). The manuscript does not comprise the correct information about the\nanalytical function the authors have used to fit (continuous line in the plots) the experimental data. The statement “... fitted to\na standard quadratic equation ...” is not only superficial but also wrong.", "reviewer_2": "In this revised version of their article, the authors have adequately addressed most of my concerns. The article has been\nsignificantly improved, and I have no further questions or comments.", "reviewer_3": "All questions raised in my previous review were thoroughly addressed and I was excited to see the new additional data.\nThank you for pointing me to the publication I missed, describing the direct interaction of VCP with ubiquitin.\nWhile reading the revised version I noticed one typo in the description of the catalytic dead Rbd2 in the text.\nI congratulate the authors to this very exciting and excellent manuscript!"}, "author_response": "We have made the following changes in response to Reviewer 1, who pointed out some clumsy grammar.\nThanks for the opportunity to correct this.\nOld: The resulting ΔI values were fit to a single-site binding model described in equation ..\nNew: The resulting ΔI values were used to fit the single-site binding model described in equation..\nOld: Ub L71 was selected because it lies within the binding interface and exhibits both large chemical shift perturbations and\npeak intensity changes.\nNew: Ub L71 was selected because it lies within the binding interface and exhibits both larger chemical shift perturbations\nand peak intensity changes."}]

Decision letter