Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity*

Background: Proteolytic activation of fungal SREBP requires the five-subunit Golgi Dsc E3 ligase. Results: Dsc1 is an active E3 ligase; loss of Dsc E3 ligase activity leads to ER localization of the Dsc complex. Conclusion: ER exit of the Dsc E3 ligase requires E3 ligase activity. Significance: This is the first example of enzyme activity-dependent protein sorting in the secretory pathway. Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1–Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

growth, exponentially grown cells were pelleted, transferred to an Invivo 2 400 work station (Biotrace), and resuspended in deoxygenated medium. Anaerobic conditions were maintained by 10% hydrogen gas with balanced nitrogen in the presence of palladium catalyst. Media were deoxygenated by incubation in an Invivo 2 400 work station for at least 24 h.
S. pombe uba1 (SPBC1604.21c) and ubc4 (SPBC119.02) were PCR-amplified from a cDNA library (11) and cloned into XhoI and NheI sites of pET28a (Novagen) as N-terminal His 6 fusion proteins. The plasmid harboring full-length uba1 was transformed into E. coli Rosetta DE3 competent cells (EMD Millipore) and induced overnight with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside at 22°C in the presence of 2% ethyl alcohol. The plasmid containing full-length ubc4 was transformed into E. coli BL21 Codon Plus (DE3)-RIPL (Stratagene) and induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside for 4 h. In both cases, E. coli cell pellets were lysed by sonication in buffer A (50 mM Na 2 HPO 4 pH 8.0, 300 mM NaCl), and cell debris was removed by centrifugation at 20,000 ϫ g for 20 min. The supernatant was allowed by bind to nickel-nitrilotriacetic acid-agarose beads (Qiagen) and washed with buffer A containing 20 mM imidazole. Bound protein was eluted with buffer A containing 250 mM imidazole. Protein purity was checked by SDS-PAGE and GelCode Blue. Antibodies-Polyclonal antibodies against Sre1, Dsc1, Dsc2, Dsc3, Dsc4, and Dsc5 used in this study were described previously (3,5). Dsc1 antiserum was affinity-purified by passing through a column containing Dsc1 N terminus (aa 1-300) coupled to agarose beads (AminoLink Plus immobilization kit, Thermo Scientific) according to the manufacturer's protocol. HRP-conjugated Dsc antibodies were prepared using the EZlink Plus activated peroxidase kit (Thermo Scientific) following the manufacturer's instructions. Anti-GST antibody was from Covance, and anti-ubiquitin (P4D1) and anti-FLAG (M2) were from Sigma.
Sre1 and Sre2 Cleavage Assays-Sre1 and Sre2 cleavage assays were performed as described previously (3,9). Briefly, for Sre1 cleavage, cells were grown to exponential phase under normoxic conditions and then shifted to anaerobic chamber (Invivo 2 400 work station) for the indicated times. Cells were collected and washed, and lysates were prepared under denaturing conditions as described (3). Processing of Sre1 was detected by Western blot analysis using anti-Sre1 antibody. Anti-Dsc5 antibody was used as a loading control. For quantification purposes, blots were developed using IRDye secondary antibodies and an Odyssey CLx imager from LI-COR. Signals for Sre1 were normalized to Dsc5, and data were quantified from three independent experiments using Image Studio software from LI-COR.
For Sre2 cleavage assays, strains expressing 3ϫFLAG-Sre2 (aa 423-793) from the cauliflower mosaic virus promoter (9) were grown at 30°C to exponential phase, and whole cell lysates were assayed by immunoblotting using anti-FLAG antibody. Immunoblots were developed using chemiluminescence except where noted in the figure legends.
Deglycosylation Assay-Cells (5 ϫ 10 8 ) were grown to exponential phase and collected by centrifugation at 500 ϫ g for 5 min. Cell pellets were washed once and resuspended in B88 buffer (20 mM HEPES, pH 7.2, 150 mM KOAc, 5 mM Mg(OAc) 2 , 250 mM sorbitol) and supplemented with 1ϫ protease inhibitors and with an additional 1ϫ Complete, EDTA-free protease inhibitor (Roche Diagnostics). Cell lysis was performed by vortexing with glass beads (Sigma, 400 -600 m) for 10 min, and debris was removed by centrifugation at 500 ϫ g. The resulting supernatant was subjected to 20,000 ϫ g centrifugation for 20 min. The membrane pellet was resuspended in B88 buffer containing 1% Nonidet P-40 and nutated at 4°C for 1 h followed by centrifugation at 20,000 ϫ g for 20 min. Supernatant was collected and used as Nonidet P-40solubilized membrane. Solubilized membrane proteins (30 g) were denatured by adding SDS and 2-mercaptoethanol to final concentrations of 0.5 and 1%, respectively, in 15 l and heating at 37°C for 30 min. Peptide-N-glycosidase F (500 units) (New England Biolabs) was added to the denatured membrane protein and incubated at 37°C for 1 h. The reaction was stopped by the addition of 5ϫ SDS-PAGE loading dye (150 mM Tris-HCl, pH 6.8, 15% SDS, 25% glycerol, 0.02% bromphenol blue, and 12.5% 2-mercaptoethanol) and heating at 37°C for an additional 30 min. Dsc1 mobility was assayed by immunoblotting using affinity-purified Dsc1 antiserum.
Cycloheximide Treatment-Temperature-sensitive sar1-1 or ubc4-P61S strains were grown overnight at permissive 25°C to exponential phase. Cycloheximide (100 g/ml) was added to the cells and shifted to non-permissive 36°C for the indicated times before harvesting. Cell membranes were processed as described above or treated cells were directly used for live cell microscopy.
Microscopy-S. pombe cells expressing fluorescently tagged proteins were immobilized on 2% agarose as described previously (5) and imaged using a Marianas/Yokogawa CSU22 spinning disk confocal microscope (3i) equipped with Axio Observer (Zeiss), Photometrics Cascade II EM-CCD camera (Roper Scientific), and environmental chamber (Tokai HIT). Cells were imaged at room temperature, 25°C, or 36°C as indicated in the figure legends. Images were captured using 100ϫ oil objective with 1.0 NA. We used SlideBook 5.0 for data acquisition. Fifteen Z-images (0.34-m step size) were collected, and three-dimensional reconstitution of the confocal slices was performed by ImageJ (National Institutes of Health). All the images described in this study were acquired and processed in an identical manner.
Co-immunoprecipitation-Co-immunoprecipitation assays were performed as described previously (8). Briefly, cells (6 ϫ 10 8 ) from exponentially growing wild-type or mutant strains were collected and lysed using glass beads in digitonin lysis buffer (50 mM HEPES, pH 6.8, 1% (w/v) digitonin, 50 mM KOAc, 2 mM Mg(OAc) 2 , 1 mM CaCl 2 , 200 mM sorbitol, 1 mM NaF, 0.3 mM Na 3 VO 2 , supplemented with 1ϫ protease inhibitors). Cellular debris was removed by centrifugation at 100,000 ϫ g for 10 min, and 0.75 mg of lysate supernatant was incubated with 5 l of Dsc2 antiserum for 15 min at 4°C followed by the addition of 40 l of protein A-agarose beads. Binding reaction was rotated overnight at 4°C, and unbound proteins were removed by three washes with digitonin lysis buffer. The bound fraction was eluted by boiling 5 min in SDS-lysis buffer (10 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1% SDS and 1 mM EDTA) and analyzed by Western blotting using HRP-conjugated Dsc antibodies.

Results
The fission yeast Golgi Dsc E3 ligase complex contains five integral membrane proteins, Dsc1-Dsc5. Bioinformatic analysis identified S. pombe Dsc1 as a homolog of the S. cerevisiae Golgi Tul1 E3 ligase (6). Although Dsc1 and Tul1 have low sequence similarity (less than 30%), both are predicted to contain a large luminal N-terminal domain (about 300 -400 aa long), followed by seven transmembrane segments, and a C-terminal, cytosolic RING domain. The RING domain is a well characterized zinc finger in which a total of eight cysteine and histidine residues coordinate two zinc atoms (13). Dsc1 contains an H2-type RING domain (C3H2C3) that differs from the classical RING domain (C3HC4) by having two histidines in the fourth and fifth positions (Fig. 1B) (14). RING domains interact directly with E2 ubiquitin-conjugating enzymes to ubiquitinate protein substrates (13,15,16). Although the Dsc1 RING domain contains residues required for E2 binding (Fig. 1B), not all predicted RING domains possess E3 ligase activity (13). To test whether the Dsc1 RING domain is a functional E3 ligase, we performed an in vitro ubiquitination assay. RING domains will auto-ubiquitinate, allowing assessment of E3 ligase activity in the absence of substrate (17,18). Purified Dsc1 RING domain (aa 608 -695) fused to GST was incubated with ubiquitin-activating enzyme E1 (Uba1) and cognate ubiquitinconjugating enzyme E2 (Ubc4) (Fig. 1C). GST-Dsc1 RING was efficiently ubiquitinated in a reaction that required ATP, E1 and E2 enzymes, and Dsc1 RING domain (Fig. 1D). The Dsc1 RING domain lacks lysine residues, indicating that ubiquitination occurred on lysine(s) in GST or on non-lysine residues in Dsc1. The E3 ligase activity of Dsc1 RING required both zinc-coordinating residues (C634A and H668A) and E2-interacting residues (I636D or L675D) (Fig. 1D, lanes 6 -9). These results demonstrate that Dsc1 is a functional RING E3 ligase.

ER Exit of Dsc E3 Ligase Requires Enzyme Activity
further tested whether SREBP activation required a RING domain surface residue essential for E2 binding (Dsc1-L675D) and E3 ligase activity (Fig. 1D) (15). In addition, we tested a second zinccoordinating residue (Dsc1-C634A). We assayed proteolytic cleavage of both fission yeast SREBPs, Sre1 and Sre2. Consistent with previous results (5), mutation of either residue blocked SREBP activation (Fig. 2, A and B), further demonstrating that SREBP activation requires Dsc1 E3 ligase activity. Quantification of Sre1 cleavage showed an ϳ20-fold increase in Sre1N in wild-type cells shifted to low oxygen ( Fig. 2A). Inter-estingly, we observed an ϳ4-fold decrease in Sre1 precursor in dsc1⌬ cells under normoxic conditions. This decrease likely results from reduced levels of the Sre1-binding protein Scp1 in dsc1⌬ cells (19). Scp1 stabilizes Sre1 precursor, and in the absence of Scp1, Sre1 precursor is degraded via ER-associated degradation (20). In addition, Sre1 precursor decreased under low oxygen in dsc mutant cells. Low oxygen stimulates ER-to-Golgi transport of the Sre1-Scp1 complex for Sre1 cleavage in the Golgi. In dsc mutants, Sre1N is not made (Fig. 2A, lanes  3-12), the Sre1 precursor is likely degraded, and the supply of  , wild-type, dsc1⌬, dsc1-1, dsc1-C634A, dsc1-L675D, and sre1⌬ cells were shifted to low oxygen for 4 h to induce Sre1 activation. Sre1 cleavage was assayed by immunoblotting whole cell lysates with anti-Sre1 antibody. P and N denote precursor and cleaved nuclear form, respectively. Sre1 levels were normalized to the Dsc5 loading control, and quantification was performed with a LI-COR Odyssey CLx using three biological replicates. Error bars indicate S.E. AU, arbitrary units. B, strains carrying empty vector (Ϫ) or a plasmid expressing 3ϫFLAG-Sre2 (423-793) (ϩ) were cultured at 30°C. Sre2 cleavage occurs in the presence of oxygen and was assayed by immunoblotting cell lysates with anti-FLAG antibody. CaMV, cauliflower mosaic virus. . Dsc1 glycosylation requires RING activity. A, detergent-solubilized membranes from wild-type, dsc2⌬, dsc1 RING mutants, and dsc1⌬ were treated without or with the glycosidase peptide-N-glycosidase F (PNGase F) and then immunoblotted using Dsc1 antiserum. B, wild-type, dsc1⌬, and temperaturesensitive ubc4-P61S cells were grown at 25°C to exponential phase and then shifted to 36°C. Cells were harvested at the indicated times, and detergentsolubilized membranes were immunoblotted using anti-Dsc1 antibody. C, wild-type, temperature-sensitive sar1-1, and dsc1⌬ cells were grown at 25°C to exponential phase and then shifted to 36°C in the absence or presence of cycloheximide (CHX, 100 g/ml) for 1 h. Cells were harvested at the indicated times, and detergent-solubilized membranes were treated without or with peptide-N-glycosidase F and immunoblotted using anti-Dsc1 antibody. D, wild-type, Sre1 precursor is not replenished by positive feedback activation of the sre1 promoter (21). Sre2 does not bind Scp1, and Sre2 cleavage is Scp1-independent (3,5).
The N-terminal luminal domain of Dsc1 contains five potential N-glycosylation sites and glycosylation of three sites (Asn-52, Asn-115, and Asn-220) has been confirmed by mass spectrometry (22). Interestingly, complete glycosylation of Dsc1 requires Dsc2, Dsc3, and Dsc4 (8). In the absence of Dsc2, Dsc3, or Dsc4, Dsc1 migrated with an intermediate mobility as compared with the mature and deglycosylated forms (Fig. 3A, lane  2) (8). Unexpectedly, we observed that Dsc1 mutants lacking E3 ligase activity also displayed incomplete glycosylation (Fig. 3A,  lanes 3 and 4). Changes in mobility were due to differential N-linked glycosylation because Dsc1 mutants showed the same mobility as wild type after treatment with peptide-N-glycosidase F (Fig. 3A, lanes 6 -9). These data indicate that Dsc1 E3 ligase activity is required for complete Dsc1 glycosylation. Dsc1 E3 ligase activity requires the E2 enzyme Ubc4 in vitro (Fig. 1D), and SREBP cleavage activation requires ubc4, demonstrating that Ubc4 is the cognate E2 enzyme for Dsc1 (5). ubc4 is an essential gene in fission yeast (23); thus we used a temperature-sensitive mutant (ubc4-P61S) as an independent test of whether Dsc1 E3 ligase activity is required for Dsc1 glycosylation. The intermediate Dsc1 band was absent at permissive temperature, but appeared after 30 min at non-permissive temperature and accumulated with time (Fig. 3B, lanes 1-5). Wild-type cells showed no change in Dsc1 mobility upon temperature shift (Fig. 3B, lanes 7 and 8). To this point, these results indicate that Dsc1 ubiquitin E3 ligase activity is required for complete Dsc1 glycosylation.
In fungi, secretory proteins receive mannose-rich core glycosylation in the ER (24). Core glycosylated proteins move to the Golgi via COPII vesicles, and in the Golgi, enzymes sequentially add and modify sugar residues to create the mature glycosylated protein (24). The Sar1 GTPase initiates COPII coat assembly and is required for ER-to-Golgi transport (25,26). To test whether complete Dsc1 glycosylation requires Golgi enzymes, we used a temperature-sensitive sar1-1 strain to block ER-to-Golgi transport of newly synthesized Dsc1 (27). Upon shifting to the non-permissive temperature, we observed accumulation of the intermediate form of Dsc1 in sar1-1, but not wild-type cells (Fig. 3C, lanes 4 and 5). Treatment with cycloheximide blocked the appearance of the Dsc1 intermediate form (Fig. 3C, lane 6), indicating that this species represents newly synthesized, incompletely glycosylated Dsc1. Treating membrane extracts with peptide-N-glycosidase F confirmed that the observed mobility differences were due to altered N-linked glycosylation. Importantly, the mobility of Dsc1 from sar1-1 cells grown at non-permissive temperature was identical to that seen in dsc1-L675D cells defective for Dsc1 E3 ligase activity (Fig. 3D). Together, these data indicate that complete Dsc1 glycosylation requires Dsc1 E3 ligase activity and ER-to-Golgi transport for modification by Golgi enzymes.
We speculated previously that the Dsc1 glycosylation defect in cells lacking Dsc subunits was due to either improper complex assembly or Dsc1 mislocalization (8). To assess Dsc E3 ligase complex assembly in strains lacking E3 ligase activity, we performed co-immunoprecipitation studies with digitonin-solubilized membranes from wild-type and RING domain mutant cells. When Dsc2 was immunopurified, all Dsc components (Dsc1, Dsc3, Dsc5, and Dsc5) co-purified in wild-type cells and in Dsc1 RING mutants that are truncated (Q673X), fail to bind E2 ubiquitin-conjugating enzymes (L675D), or do not coordinate zinc (C634A) (Fig. 4A, lanes 7-12), indicating that complex assembly is unaffected by RING domain mutations. We further investigated Dsc E3 ligase complex assembly in ubc4-P61S cells defective for the cognate E2 ubiquitin-conjugating enzyme Ubc4. We observed above that after shifting to nonpermissive temperature, Dsc1 accumulates as a faster migrating, intermediate form in ubc4-P61S cells (Fig. 3B). When purified with Dsc2, the Dsc1 intermediate form co-purified with other Dsc subunits as in wild-type cells (Fig. 4B, lanes 7-12). These data demonstrate that the incompletely glycosylated Dsc1 assembles into the Dsc E3 ligase complex and that E3 ligase activity is not required for complex assembly.  , dsc1⌬, dsc1-1, dsc1-L675D, and dsc1-C634A cells using Dsc2 antiserum. Binding of Dsc proteins was assayed by immunoblotting. B, digitonin-solubilized membranes from wild-type, ubc4-P61S, and dsc2⌬ cells either grown at 25°C or grown after shifting to 36°C for 2 h were subjected to immunoprecipitation using Dsc2 antiserum. Dsc proteins in bound fraction were assayed by immunoblotting.
Alternatively, incomplete Dsc1 glycosylation could result from a failure of Dsc1 to interact with Golgi glycosylation enzymes. The Dsc E3 ligase complex has a hierarchical organization where Dsc2-Dsc3-Dsc4 define the core, and Dsc1 and Dsc5 are peripheral subunits (Fig. 1A) (8). To assay Dsc E3 ligase localization, we determined the localization of the core subunit Dsc2 in mutant strains using live cell fluorescence confocal microscopy. As reported previously in wild-type cells, Dsc2 localized to Golgi puncta, distinct from the ER glycosyltransferase Ost1 (Fig. 5, top panels) (5). Deletion of dsc1, dsc3, or dsc4, but not dsc5, caused Dsc2 to localize to the ER (Fig. 5). These results correlated with the presence of incompletely glycosylated Dsc1 observed previously in dsc3⌬ and dsc4⌬ cells, but not dsc5⌬ cells (8). In addition, Dsc1 is incompletely glycosylated in dsc2⌬ cells (Fig. 3A). Thus, assembly of subunits Dsc1-Dsc4 is required for proper Golgi localization of the Dsc E3 ligase, and Dsc5 is not required for ER exit. Often, failure of a multi-protein complex to assemble or exit the ER results in subunit degradation by quality control pathways like ER-associated degradation or autophagy (28). Notably, Dsc complex subunits were stable when retained in the ER, for example, in dsc1⌬ cells (Fig. 4A, lane 2) (8).
Interestingly, Dsc2 also accumulated in the ER in dsc1-1 cells that assemble the Dsc complex normally (Fig. 4A), but lack Dsc E3 ligase activity due to a RING domain truncation (Fig. 4)  the non-permissive temperature when E3 ligase activity is inhibited (Fig. 3B). As expected, incompletely glycosylated Dsc1 accumulated in ubc4-P61S cells (Fig. 6A, lanes 1-3). Cycloheximide treatment specifically blocked production of the Dsc1 intermediate form upon Ubc4 inactivation, indicating that Dsc1 fails to receive Golgi-dependent carbohydrate modifications in the absence of Ubc4 activity (Fig. 6A). Next, we examined Dsc E3 ligase localization in ubc4-P61S cells. At permissive temperature, the majority of cells showed Dsc2 localization only in Golgi puncta (88%) (Fig. 6B, left panel, magenta  arrowhead), and a small number of cells showed both ER and Golgi localization (green arrowhead). Upon incubation at non-permissive temperature for 2 h, Dsc2 localization changed with 82% of cells showing mixed ER and Golgi localization and 18% displaying only Golgi localization (Fig. 6B, middle panel). Treatment with cycloheximide when shifting to the non-permissive temperature prevented ER accumulation of Dsc2 (88% Golgi only, 12% ER and Golgi) (Fig. 6B, right panel). Dsc2 localized to the Golgi in wildtype cells under each of the three conditions, showing that ER localization required Ubc4 inactivation (Fig. 6C). Taken together, the data in Fig. 6 demonstrate that the Dsc E3 ligase complex fails to exit the ER in the absence of Ubc4 activity. Given that inactive Dsc E3 ligase complex assembles normally when retained by ER in both dsc1-1 and ubc4 cells, we conclude that failure to exit the ER is due to the absence of E3 ligase activity and not structural alterations in the Dsc E3 ligase.
To test whether Dsc E3 ligase activity is required specifically for Dsc complex ER exit, we assayed secretory pathway function in dsc mutant strains. ER-to-Golgi protein trafficking is essential for cell viability (29). We hypothesized that growth at elevated temperature will stress cells and may reveal dsc-dependent defects in ER-to-Golgi transport. We . Cell growth and Golgi morphology does not require Dsc E3 ligase activity. A, overnight grown cells from wild-type, dsc1⌬, dsc2⌬, and dsc1-L675D strains were diluted in rich medium and incubated at 37°C. Cell growth was monitored by measuring OD 600 . Error bars indicate standard error. B, localization of the Golgi mannosyltransferase subunit Anp1-GFP was determined in indicated strains by imaging cells at room temperature using a 3i spinning disk confocal microscope. C, localization of Dsc2-6ϫGFP and Anp1-mCherry in dsc1⌬ cells as in B. Scale bar ϭ 10 m.
compared growth of wild-type and dsc mutant cells at 37°C and observed no growth difference in exponentially growing cells (Fig. 7A). Next, we looked at the localization of cis-Golgi resident, mannosyltransferase subunit Anp1. Inhibition of COPII-dependent vesicle formation results in Anp1 mislocalization to the ER (5). Anp1-GFP localized to Golgi puncta in wild-type, dsc1⌬, and dsc2⌬ cells (Fig. 7B), indicating that COPII vesicle transport does not require Dsc E3 ligase activity. In an independent experiment, we co-localized Dsc2 and Anp1 in dsc1⌬ cells, confirming that Golgi-resident Anp1 localizes normally under conditions in which the Dsc E3 ligase is mislocalized to the ER (Fig. 7C).

Discussion
Multiple lines of evidence demonstrate that Dsc1 E3 ligase activity is required for ER exit of the Dsc complex, thus ensuring that only fully functional Dsc E3 ligase moves to the Golgi where it acts in SREBP activation. Although E3 ubiquitin ligases are known to regulate their abundance through auto-ubiquitination (30 -32), in this instance, enzyme activity controls protein localization. Multi-subunit membrane complexes such as MHC class I and Kir potassium channels require proper assembly prior to ER exit (33)(34)(35). However, even when the Dsc E3 ligase complex assembles properly, ER exit requires E3 ligase activity. To our knowledge, this represents the first example of enzyme activity-dependent protein sorting in the secretory pathway.
How ligase activity controls ER exit is unknown, but potential mechanisms exist. First, the Dsc E3 ligase complex may assemble with an ER retention protein whose removal and possibly degradation require Dsc1-dependent ubiquitination. Alternatively, the Dsc1 E3 ligase may ubiquitinate one or more COPII components whose modification is required for Dsc E3 ligase ER exit. Indeed, packaging of pro-collagen requires CUL3-KLHL12-mediated mono-ubiquitination of the COPII component Sec31 (36), and mono-ubiquitination of Sec23 has been detected in budding yeast (37). Interestingly, Dsc1 E3 ligase utilizes the E2 Ubc4 that preferentially adds mono-ubiquitin to substrates (38). Finally, a Dsc subunit could be a direct substrate, and the addition of ubiquitin may alter Dsc complex conformation or recruit an effector protein to facilitate ER exit. Although the mechanistic details are under investigation, these findings add to the growing complexity of ER cargo sorting and open up an opportunity to investigate the role of ubiquitination in this process.
Lastly, these findings have important implications for the current model of SREBP activation in fungi in which we proposed that SREBP activation requires its ubiquitination. The fact that the Dsc E3 ligase mislocalizes to the ER when inactive necessitates additional studies of the role for SREBP ubiquitination in its proteolytic activation.