Ubiquitination drives COPI priming and Golgi SNARE localization

  1. Swapneeta S Date
  2. Peng Xu
  3. Nathaniel L Hepowit
  4. Nicholas S Diab
  5. Jordan Best
  6. Boyang Xie
  7. Jiale Du
  8. Eric R Strieter
  9. Lauren P Jackson
  10. Jason A MacGurn
  11. Todd R Graham  Is a corresponding author
  1. Department of Biological Sciences, Vanderbilt University, United States
  2. Department of Cell and Developmental Biology, Vanderbilt University, United States
  3. Department of Chemistry, University of Massachusetts Amherst, United States

Abstract

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of β′-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI–SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE–Arf–COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

Editor's evaluation

This article will be of interest for cell biologists focused on understanding membrane biology, trafficking, and protein ubiquitination, as well as yeast geneticists. The main finding of this paper is that non-degradative ubiquitination is an important mechanism driving COPI-dependent SNARE trafficking and localization.

https://doi.org/10.7554/eLife.80911.sa0

Introduction

The sorting of proteins in the endomembrane system is a highly regulated, vesicle-mediated process important for many physiological events. Coat proteins drive the formation of vesicles by assembling onto the cytosolic surface of cellular membranes, where they select cargo proteins (Bonifacino and Glick, 2004; Brandizzi and Barlowe, 2013; Schmid, 1997; Spang, 2013). COPI-coated vesicles originate at the Golgi or endosomes, and mediate retrograde transport to early Golgi cisternae or back to the endoplasmic reticulum (ER) (Bonifacino and Glick, 2004; Letourneur et al., 1994; Tojima et al., 2019). COPI is a highly conserved heptameric protein complex (α, β, β', γ, δ, ε, and ζ subunits) that is recruited to Golgi membranes by the small GTP-binding protein Arf (Arf1 and Arf2 in budding yeast) (Hsu, 2011; Serafini et al., 1991; Thomas and Fromme, 2020; Waters et al., 1991). The N-terminal WD repeat (WDR) domains of α- and β′-COP recognize sorting signals on cargoes, such as dilysine motifs (KKxx and KxKxx) commonly found at the C-terminus of ER-resident membrane proteins (Eugster et al., 2004; Jackson et al., 2012; Ma and Goldberg, 2013). Mutations that impair the ability of both α- and β′-COP to bind dilysine motifs disrupt trafficking of well-studied dilysine cargoes, but these mutations do not affect cell viability (Jackson et al., 2012). However, COPI is essential in yeast and cells are unviable when both terminal propeller domains of α- and β′-COP are deleted (Eugster et al., 2004). These observations imply that important but poorly understood mechanisms exist for COPI recognition of cargoes lacking dilysine motifs. As Golgi cisternae mature from cis to trans in budding yeast, the retrograde movement of resident proteins, which primarily lack canonical dilysine signals, becomes critical in order to maintain a functional Golgi. Resident Golgi proteins, thus, are also important COPI cargo (Banfield, 2011; Glick et al., 1997; Ishii et al., 2016; Kurokawa et al., 2019).

SNAREs are another critical cargo of COPI vesicles because they are essential for vesicle fusion with the target membrane and are proposed to prime, or nucleate, coat formation (Chen and Scheller, 2001; Rein et al., 2002; Rothman and Warren, 1994; Springer et al., 1999). In addition to incorporating v-SNAREs into newly forming vesicle membranes, COPI must also mediate retrograde transport of early Golgi t-SNAREs moving through the Golgi by cisternal maturation to maintain Golgi organization, but how COPI mediates sorting of SNAREs is poorly understood. Because of the tail-anchored topology of SNAREs, none of these proteins contain a C-terminal dilysine motif on the cytosolic side of the membrane where it is accessible to COPI. Few sorting signals have been identified in SNARE proteins and how they are incorporated into COPI vesicles is incompletely understood (Black and Pelham, 2000; Daste et al., 2013; Fukasawa et al., 2004; Gao and Banfield, 2020; Jackson et al., 2010; Mancias and Goldberg, 2007; Martinez-Arca et al., 2003; Miller et al., 2007). The ArfGAP protein Glo3 may contribute to SNARE localization because it is known to interact with COPI, Arf-GTP, and SNAREs and is proposed to be part of the priming complex (Rein et al., 2002). In addition, Glo3 stimulates conversion of Arf-GTP to Arf-GDP, which destabilizes the COPI coat and is crucial for vesicle uncoating (Tanigawa et al., 1993). How these Glo3 interactions are regulated to keep the Arf-GAP activity of Glo3 in check to allow Arf-GTP-mediated COPI assembly during vesicle formation is unclear.

We recently found that COPI plays a role in the recycling of a budding yeast v-SNARE, Snc1, from the endocytic pathway to the trans-Golgi network (TGN) through recognition of a polyubiquitin (polyUb) signal (Xu et al., 2017). The cargo-binding WDR domains of α-COP and β′-COP bind specifically to polyUb (Xu et al., 2017). Deletion of the β′-COP N-terminal WDR domain (β′-COP ∆2–304) disrupts Snc1 recycling while the replacement of this domain with unrelated ubiquitin-binding domains (UBDs) restores Snc1 recycling. In addition, the β′-COP ∆2–304 mutant displays a slow growth phenotype, which is fully corrected by addition of the unrelated UBD. Because the β′-COP–UBD fusion proteins are incapable of binding dilysine motifs, these observations suggest a direct role for COPI–Ub interaction in Snc1 recycling (Xu et al., 2017). Thus, β′-COP plays a critical role in this ubiquitin-dependent trafficking route, but it was unclear if the COPI–ubiquitin interaction is important for trafficking of any other cargoes. The only other cargo known to be missorted in the β′-COP ∆2–304 mutant is Emp47, which bears a variant dilysine motif (KxKxx) that is uniquely recognized by the β′ propeller domain and the trafficking of Emp47 does not require ubiquitin interaction (Eugster et al., 2004; Xu et al., 2017). Another COPI cargo analyzed, Rer1-GFP, is sorted normally by β′-COP ∆2–304 (Xu et al., 2017).

In the current study, we seek to determine if the COPI–ubiquitin interaction is a general principle of SNARE trafficking and to define the molecular interplay between ubiquitination, COPI, and COPI vesicle components including Arf, ArfGAP, and COPI cargo. We show that the normal localization of several SNAREs functioning at the ER–Golgi interface or within the Golgi, including Bet1, Gos1, Snc2, Bos1, and Sec22, requires COPI–ubiquitin interactions. In addition, several Golgi SNAREs, COPI subunits, and the ArfGAP Glo3 are ubiquitinated under physiological conditions in Saccharomyces cerevisiae. Importantly, we show that ubiquitination of these components strengthens the interaction between Golgi SNAREs and COPI while apparently excluding Glo3, providing critical new mechanistic insight into priming mechanisms for COPI vesicle formation. These studies highlight the finely orchestrated role of ubiquitination in driving COPI priming and sorting of a specific set of Golgi SNAREs crucial to the functional organization of Golgi.

Results

A subset of SNAREs is mislocalized in the β′-COP ∆2–304 mutant

To determine the dependence of SNARE localization on COPI–ubiquitin interactions, we individually tagged 17 yeast SNAREs with mNeonGreen (mNG) and expressed them in S. cerevisiae wild-type (WT) cells or in a strain where the ubiquitin-binding N-terminal WDR of β′-COP had been deleted (β′-COP Δ2–304) (Figure 1a and Supplementary file 1, Table 1; Xu et al., 2017). This β′-COP mutation does not completely eliminate COPI polyUb binding because α-COP can also bind polyUb (Xu et al., 2017) therefore, the SNAREs were overexpressed from a strong ADH promoter so the screen would be more sensitive for detecting changes in localization. Many of the mNG-SNARE fluorescent patterns were indistinguishable between WT and β′-COP ∆2–304 cells (Figure 1—figure supplement 1a). However, for Bet1, Gos1, Snc1, and Snc2, a significant accumulation of individual SNAREs to elongated tube- and ring-like structures was observed in β′-COP ∆2–304 (Figure 1b, c). Bet1 and Snc2 were found in ring- and tube-shaped structures while Gos1 was rarely observed in the rings. As previously shown for Snc1 (Xu et al., 2017), Snc2 plasma membrane localization was also reduced in the COPI mutant. Tlg1 also localized to enlarged tubular structures and Snc1 colocalizes to these enlarged structures in the β′-COP ∆2–304 cells (Figure 1—figure supplement 1a; Xu et al., 2017). In addition, Sec22 and Bos1 were partially mislocalized to vacuolar structures in β′-COP ∆2–304 cells (Figure 1—figure supplement 1b). GFP is typically cleaved from protein chimeras upon arrival in the vacuole. Consistently, immunoblotting of cell lysates with anti-GFP indicated that 40% of the GFP-Sec22 chimera was cleaved in β′-COP ∆2–304 cells to release free GFP, in contrast to WT cells where less than 5% was cleaved (Figure 1—figure supplement 1c, d).

Figure 1 with 2 supplements see all
SNARE localization to morphologically aberrant structures in COPI mutants.

(a) Schematics of the experimental setup wherein SNAREs are individually tagged with mNeonGreen and expressed under constitutive ADH promoter in Saccharomyces cerevisiae wild-type (WT) cells or in cells with a deleted N-terminal WDR of β′-COP (Δ2–304). (b, c) Live-cell imaging shows significant differences in the appearance of SNAREs Bet1, Gos1, Snc1, and Snc2 where elongated tube- and ring-like structures (orange arrowheads) are seen in β′-COP Δ2–304 cells compared to control cells with full-length β′-COP (WT). Tubes were defined by having a long axis (length) at least twice as long as the short axis. The rings were defined as spherical structures having a larger diameter than a normal puncta and with a dimly fluorescent center. Data in (c) are the percentage of structures per cell optical section that are rings and tubules. The remainder of the fluorescent structures are punctae. Statistical differences were determined using a one-way analysis of variance (ANOVA) on the means of the three biological replicates (***p < 0.001). Scale bars represent 5 µm in the full panels and 2 µm for the insets. Full panel images are all scaled equally.

To test whether the observed morphological changes were caused by SNARE overexpression or loss of the β′-COP WDR domain, we expressed the Bet1, Snc1, and Snc2 mNG constructs using the weaker, inducible CUP1 promoter with a short (1 hr) induction time. Short induction times using the CUP1 promoter have been shown to approximate physiological protein abundance for SNAREs Bet1 and Snc1 (Best et al., 2020). Comparable morphological changes were observed with these SNAREs localizing to elongated tubular- and ring-like structures in β′-COP ∆2–304 relative to WT cells (Figure 1—figure supplement 2a, b). Thus, Bet1, Snc1, and Snc2 were localized to aberrant structures whether they were expressed using a strong, constitutive ADH promoter or the weaker, inducible CUP1 promoter. All subsequent imaging studies used the CUP1 promoter to approximate physiological SNARE expression. Together, these data suggest a dependence of a subset of SNAREs on COPI–ubiquitin interactions for their proper localization.

To further characterize the morphological changes observed for SNAREs, we performed colocalization analysis of mNG-Gos1 and mNG-Bet with four Golgi markers (early Golgi markers, Anp1 and Mnn9 and late Golgi markers Sec7 and Chs5) (Zhu et al., 2019). mNG-Gos1 colocalized substantially with Chs5, Sec7, and Mnn9 in WT cells but showed a significant decrease in colocalization with these markers in in β′-COP ∆2–304 cells. Gos1 weakly colocalized with Anp1 in WT cells and this overlap was also diminished in β′-COP ∆2–304 (Figure 2a, b). mNG-Bet1 also showed a significant reduction in colocalization with all four Golgi markers in β′-COP ∆2–304 (Figure 2—figure supplement 1a, b). We also observed colocalization of Bet1 with the medial-Golgi marker Aur1 in WT cells and the degree of this colocalization was also reduced in β′-COP ∆2–304 (Figure 2—figure supplement 2a, b). Where colocalization was observed between mNG-SNAREs and Golgi markers in β′-COP ∆2–304 cells, the Golgi marker only overlapped with a small region of the tube-like structures labeled with the mNG-SNARE (Figure 2a, Figure 2—figure supplement 1). We suspect these incidents of colocalization are adjacent structures, but it is possible they are single compartments where the two proteins are mostly segregated into separate domains. Thus, there was loss of mNG-Gos1 and mNG-Bet1 from the Golgi compartments where they normally localize when the terminal UBD of β′-COP was deleted. In addition, there was no noticeable difference in Anp1, Mnn9, Chs5, or Sec7 localization or the morphology of the Golgi compartments these proteins marked. Similarly, no morphological difference for early- and medial-Golgi markers, Sed5 and Aur1, respectively, was observed for β′-COP ∆2–304 cells compared to WT cells (Figure 2—figure supplement 3).

Figure 2 with 4 supplements see all
mNG-Gos1 accumulates in tube-like sequestration compartments lacking Golgi markers in β′-COP Δ2–304 cells.

(a, b) Colocalization analysis of mNG-Gos1 with two late Golgi markers Chs5-mKate, Sec7-mKate and two early Golgi markers, Mnn9-mKate and Anp1-mKate indicates a general loss of mNG-Gos1 from early and late Golgi in β′-COP Δ2–304 cells compared to wild-type (WT) cells. Gos1 accumulates in tube-like structures (orange arrowheads) in the COPI mutant. Scale bars represent 5µm. Statistical analysis was done on means using three biological replicates, t-test (****p ≤ 0.0001,*p < 0.05, Ns p > 0.05). (c) The abundance of mNG-tagged Gos1 and Bet1 tube- or ring-like structures is increased β′-COP Δ2–304 cells compared to WT cells, but the total number of fluorescent structures is unchanged. The number and appearance of mKate-tagged Chs5, Sec7, Mnn9, and Anp1 structures are not altered in β′-COP Δ2–304 cells compared to WT cells. (d) The average size and size distribution of intracellular structures for mNG-Gos1 are significantly increased in β′-COP Δ2–304 cells compared to WT control but the average size and size distribution of mKate-tagged Chs5, Sec7, Mnn9, and Anp1 structures are not altered in β′-COP Δ2–304 cells compared to WT control.

To further characterize the Gos1-labeled structures and Golgi morphology, we calculated the abundance of intracellular fluorescent structures grouped as either punctate structures (approximately 0.45 μm dotted structures), or tube-like structures (structures for which the longer axis is 2–3× longer than the shorter axis, length of the longer axis ranges from 0.6 to 1.8 μm) and ring-like structures (enlarged punctate-type structures but with a hole in the middle akin to a ring, approximate size 0.6–0.9 μm across both long and short axis) (Figure 2c). No difference between WT and β′-COP ∆2–304 was observed for the total number of mNG-Gos1 or mNG-Bet1 fluorescent structures per optical section. However, relative to WT cells, a greater number of these structures were rings and tubes in the β′-COP ∆2–304 cells with a concomitant reduction in punctae. For the Golgi markers, the number of punctae per cell optical section was the same in WT and mutant cells and the tube or ring structures were rarely observed. The average size of intracellular structures was not significantly different for the Golgi markers in WT or β′-COP ∆2–304 cells (Figure 2d). For mNG-Gos1, the average length of the structures was significantly increased in β′-COP ∆2–304. Thus, the COPI mutation used here does not cause whole organelle-level changes in Golgi morphology or the localization of markers used here (Figure 2, Figure 2—figure supplements 1 and 3). Bet1 and Gos1 are substantially lost from Golgi compartments and mislocalized to tube- or ring-like structures (Figures 1 and 2, Figure 2—figure supplements 1 and 2).

To test if Bet1 and Gos1 were mislocalized to the endosomal system, we stained the WT and COPI mutant cells with FM-64 for 3 min to mark endosomal compartments. The majority of FM4-64-stained endosomes had a normal morphology in the mutant cells (Figure 2—figure supplement 4). We did see a subset of the mNG-SNARE tubular structures stain weakly with FM4-64. However, the degree of colocalization between the SNAREs and FM4-64 as measured by the Mander’s colocalization coefficient was not significantly different between WT and COPI mutant cells. Therefore, it does not appear that Bet1 and Gos1 are being mislocalized from the Golgi to the endosomal system in the β′-COP ∆2–304 cells. We suspect that a failure to retrieve these SNAREs during maturation of the Golgi cisternae, combined with a normal retrieval of most resident Golgi proteins, results in Gos1 and Bet1 accumulating in dead-end compartments that we describe here as sequestration compartments.

The β′-COP-RKR mutant is incapable of binding dilysine motif cargoes through β′-COP. To determine whether Bet1 and Gos1 localization are dependent on the ability of β′-COP to bind dilysine cargoes, we compared the appearance and localization of mNG-Gos1 and Bet1 in WT or in β′-COP-RKR cells. No observable difference in the appearance or localization of Bet1 and Gos1 was observed. These observations suggest that the ability of β′-COP to bind ubiquitin but not the dilysine motifs is critical for proper localization of SNAREs Bet1 and Gos1 (Figure 2—figure supplement 3b).

β′-COP binding to ubiquitin is essential for proper SNARE localization

The N-terminal WDR domain of β′-COP binds ubiquitin, and the COPI–ubiquitin interaction is critical for Snc1 retrieval (Xu et al., 2017). To determine whether mislocalization of other SNAREs in β′-COP 2–304 is due to the inability of β′-COP to bind ubiquitin, we used a set of COPI constructs (Figure 3—figure supplement 1b–e) where the N-terminal WDR domain of β′-COP was replaced with (1) a general UBD of Doa1 (UBDDoa1), which is known to bind ubiquitin irrespective of the ubiquitin linkage type (Mullally et al., 2006) and (2) a UBD from Tab2 (NZFTab2) which specifically binds K63–ubiquitin linkages (Moritsugu et al., 2018). Compared to WT cells, Bet1, Gos1, and Snc1 were mislocalized to elongated tube structures in β′-COP Δ2–304 cells as seen previously (Figure 1b; Figure 3a–f). The replacement of the β′-COP 2–304 domain with the general UBD, UBDDoa1 restored SNARE localization to punctate structures comparable to WT cells (Figure 3a–f). β′-COP-UBDDoa1 does not restore the dilysine interaction (Xu et al., 2017); therefore, it is the ability of β′-COP to bind Ub that is critical for normal SNARE localization. Surprisingly, however, the K63-linkage restricted β′-COP NZFTab2 construct did not significantly correct the Bet1 or Gos1 localization pattern. We previously found that the β′-COP ∆2–304 Snc1 recycling defect was corrected by replacing the WDR domain with either the UBDDoa1 or NZFTab2 (Xu et al., 2017). Consistently, we found here that both the UBDDoa1 and NZFTab2 constructs significantly restored the WT pattern of intracellular structures labeled with mNG-Snc1 (Figure 3b, e). However, even though Snc2 is functionally and evolutionarily closely related to Snc1, we found that UBDDoa1 restored the mNG-Snc2 WT pattern, but the K63-restricted NZFTab2 domain did not (Figure 3—figure supplement 2). For GFP-Sec22, β′-COP-UBDDoa1 fully prevented vacuolar mislocalization while a partial rescue was conferred by β′-COP NZFTab2 (Figure 1—figure supplement 1c, d). Thus, Snc1 and Sec22 can use K63-linked polyUb chains for their trafficking, but Bet1, Gos1, and Snc2 appear to rely on COPI binding to some other ubiquitin linkage type.

Figure 3 with 2 supplements see all
β′-COP binding to ubiquitin is critical for proper SNARE localization.

Deletion of the N-terminal ubiquitin-binding WDR of β′-COP (∆2–304) leads to mislocalization of (a) mNG-Bet1, (b) mNG-Snc1, and (c) mNG-Gos1 into elongated tubular and ring-like structures (orange arrowheads). This phenotype is rescued by the replacement of the N-terminal ubiquitin-binding WDR of β′-COP by the general ubiquitin-binding domain Doa1 (β′-COP UBDDoa1) (Figure 3—figure supplement 1). The replacement of N-terminal UBD of β′-COP with K63-specific UBD, NZFTab2 (β′-COP NZFTab2) rescues the mislocalization phenotype for Snc1 but not for Bet1, Gos1, and Snc2 (Figure 3—figure supplement 2). The mislocalization of Bet1 and Snc1 observed in β′-COP Δ2–304 cells comparable to COPI inactivation phenotype observed for ret1-1 at nonpermissive temperatures. (d–f) Statistical differences were determined using a one-way analysis of variance (ANOVA) on the means of the three biological replicates (****p<0.0001, ***p < 0.001). (g) GST-β′-COP (1–604) binds linear and K63-linked triUb and to some extent to K6, K11, and K29 triUb relative to the GST-only control. 0.5 mM of GST and GST-tagged WDR proteins immobilized glutathione beads were incubated 250 nM Ub3 for corresponding linkages. (h) Quantitation of Ub3 polymers binding (GST-only background subtracted) relative to input. The values represent mean ± standard error of the mean (SEM) from three independent binding experiments. Scale bar represents 5 µm.

Next, we examined the localization of mNG-tagged Bet1, Snc1, and Snc2 in a temperature-sensitive COPI mutant (ret1-1) grown at the permissive temperature and shifted to the nonpermissive temperature of 37°C for 1 hr. The ret-1 mutation is within α-COP and substantially inactivates all known COPI functions (Gaynor et al., 1998; Ishii et al., 2016; Letourneur et al., 1994). Bet1, Snc1, and Snc2 were observed in tubular and ring-like structures in ret1-1 at the nonpermissive temperature (Figure 3a–f, Figure 3—figure supplement 2). Interestingly, the localization pattern seen for Bet1, Snc1, and Snc2 in β′-COP Δ2–304 cells was comparable to ret1-1 at the nonpermissive temperature (Figure 1b–c; Figure 3a–c). These data indicate that perturbations in the ability of β′-COP to bind ubiquitin in β′-COP Δ2–304 substantially disrupt COPI function with respect to Bet1, Snc1, and Snc2 localization.

β′-COP has been shown to bind K63-polyUb chains but not K48-polyUb or monoubiquitin (monoUb) (Xu et al., 2017). Since a general UBD rescued the localization for all 4 SNAREs, but not K63-specific UBD (Figure 3a–f), we reasoned that β′-COP might be able to bind other polyUb chains. To test this hypothesis, we assayed the ability of heterologously purified GST-tagged β′-COP to bind K6-, K11-, K29-, K33-, and linear (M1)-linked polyUb chains. K63-polyUb was used as a positive control, and GST-only was used to determine background levels of ubiquitin binding to GST (Figure 3g, h). β′-COP is capable of binding linear ubiquitin chains and more weakly to K6-, K11-, and K29-polyUb chains (Figure 3g, h).

Fusion of a deubiquitinase domain to COPI leads to SNARE mislocalization

To analyze the functional significance of ubiquitination within the COPI–SNARE system, we designed constructs where a deubiquitinase domain, UL36 (DUB) from Herpes Simplex Virus 1 (Kattenhorn et al., 2005), was fused to either α- or β′-COP. A catalytically dead version of UL36 (DUB*) wherein an active site Cys is mutated to Ser and thus cannot deubiquitinate substrates was engineered as a control. Strains expressing COPI-DUB constructs, irrespective of whether α- or β′-COP was fused to DUB, were enlarged in size (Figure 4a, b). Additionally, we observed mislocalization of Bet1 and Gos1 in COPI-DUB constructs wherein mNG-tagged SNAREs were observed in enlarged punctate structures, elongated tube structures, or ring-like structures (Figure 4a, c). COPI-DUB* constructs did not display significant phenotypic changes. The fusion of a DUB domain to COPI phenocopies the mislocalization pattern for Bet1 and Gos1 in the COPI (ret1-1) mutant at nonpermissive temperatures, supporting the importance of ubiquitination in COPI-mediated regulation of SNARE localization.

Deubiquitinase fusion to COPI subunits causes SNARE mislocalization.

mNG-tagged Bet1 and Gos1 were imaged in cells in which a deubiquitinase domain (DUB) was fused to the C-terminus of α- and β′-COP to generate αCOP-DUB and β′-COP-DUB, respectively, along with catalytically dead controls αCOP-DUB* and β′-COP-DUB*. (a, b) Cells carrying COPI-DUB fusion were larger in size compared to wild-type (WT) cells as well as catalytically dead controls . (a, c) Significant accumulation of Bet1 and Gos1 in the elongated tube- or ring-like or enlarged punctate structures (orange arrows) was observed in αCOP-DUB and β′-COP-DUB backgrounds compared to corresponding DUB* control or WT cells . Statistical differences were determined using a one-way analysis of variance (ANOVA) on the means of the three biological replicates (****p < 0.0001).

Ubiquitination is associated with Gos1, Ykt6, and Sed5 SNARE complexes

Global analyses of the budding yeast proteome have identified ubiquitinated lysines in Gos1, Snc1, and Snc2 but not Bet1 (Swaney et al., 2013). We set out to test if ubiquitination could be detected by immunoprecipitating the SNAREs and probing for ubiquitin on immunoprecipitated samples and by detecting the pooled ubiquitin released off of immunoprecipitated samples following a deubiquitinase (DUB) treatment. We individually tagged Bet1, Gos1, and Snc1 with 6xHIS-TEV-3xFLAG at their C-termini by chromosomal integration of the tag constructs. Following FLAG immunoprecipitation, the samples were treated with mock buffer (no DUB) or deubiquitinases (DUB) (Figure 5a) and probed with FLAG (Figure 5b) or ubiquitin antibodies (Figure 5c). Art1, a ubiquitinated protein from S. cerevisiae, was used as a positive control, and untagged cells (Ctrl) were used as a negative control. The FLAG antibody recognizes a nonspecific band at approximately 20 kDa (Figure 5b, Ctrl Lane) that unfortunately comigrates with Bet1-FLAG and Snc1-FLAG as indicated by the increased band intensity at 20 kDa in those samples relative to the untagged control (Ctrl) sample. In addition, Bet1-FLAG exhibited a significant smear extending to greater than 40 kDa (Figure 5b). However, this smeared pattern for Bet1-FLAG was not collapsed by DUB treatment, nor was this smear recognized by the antiubiquitin antibody. Moreover, the amount of monoUb released from Bet1-FLAG by DUB treatment was not significantly different from the control sample (Figure 5c, d).

Figure 5 with 2 supplements see all
Multiple Golgi SNARE complexes are modified with ubiquitin.

(a) Schematic of the experimental setup wherein SNAREs were individually tagged with FLAG and immunoprecipitated using anti-FLAG beads. Half the samples were mock treated, and the other half was treated with deubiquitinase (DUB). Western blots of samples are probed with FLAG (b) or ubiquitin antibody (c). Blue arrows in (b) indicate the position of FLAG-tagged protein and the asterisk indicates the position of a background band. (d) Quantitation of the amount of monoubiquitin released from the samples by deubiquitinases. (e) SILAC mass spectrometric analysis of Gos1-FLAG pulldown samples indicates enrichment of SNAREs Sft1, Ykt6, and Sed5 (red dots) and COPI subunits (green dots) with Gos1 . (f–i) Western blot analysis showing HA-tagged Ykt6, and Sed5 probed for ubiquitination following HA immunoprecipitation and deubiquitinase treatment. (g, i) Quantitation of monoubiquitin. Statistical differences were determined using a one-way analysis of variance (ANOVA) with multiple comparison test on three biological replicates (****p ≤ 0.0001, *p < 0.05, Ns p > 0.05).

For Gos1-FLAG immunoprecipitations probed with antiubiquitin antibody, a smeared pattern was observed in the 50–80 kDa molecular weight region (Figure 5b) when probed with anti-Ub, which collapsed, releasing a significant amount of monoUb following DUB treatment (Figure 5c, d, Gos1 lanes). A similar smeared pattern is seen for Art1 in mock-treated samples around 75–130 kDa molecular weight region, which was converted to monoUb by DUB treatment (Figure 5c, d, Art1 lanes). Although the smeared pattern for Snc1 was not apparent in these samples, DUB treatment released more monoUb than control samples (Figure 5d, Snc1). We initially focused our attention on Gos1 because it appeared to be ubiquitinated and evidence for the importance of Snc1 ubiquitination has already been reported (Chen et al., 2011; Xu et al., 2017; Figure 5c, d).

To identify other proteins specifically associated with Gos1 when purified under conditions that preserved ubiquitination, we employed a Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) mass spectrometry (MS) approach. A strain expressing Gos1-FLAG was grown in a light isotope medium and untagged control cells used to determine the nonspecific background proteins in the FLAG IP, were grown in a heavy isotope medium. Importantly, the samples were processed in the presence of DUB inhibitors to preserve ubiquitination on Gos1 and other proteins in the samples. Gos1 is reported to form a functional t-SNARE complex with Ykt6 and Sed5 that mediates fusion with intra-Golgi retrograde vesicles bearing Sft1 (Parlati et al., 2002). We observed significant enrichment of peptides from these partner SNAREs with Gos1-FLAG and known SNARE regulators like Sec17 and Sly1 (Demircioglu et al., 2014; Song et al., 2021; Figure 5e). Importantly, we also found several COPI subunit peptides that were enriched to comparable levels as Ykt6, Sft1, and Sed5 in the Gos1 pulldown samples (Figure 5e).

To probe the ubiquitination status of Gos1-binding SNARE partners, we individually tagged Ykt6 and Sed5 with 3xHA tag on the N-terminus (attempts at C-terminally tagging Ykt6 and Sed5 were unsuccessful potentially owing to structurally/functionally important modifications at the C-terminus, such as Ykt6 palmitoylation). HA-tagged Ykt6 and Sed5 were immunoprecipitated using anti-HA and probed for their ubiquitination status. A smeared pattern associated with ubiquitination was observed for both Ykt6 (Figure 5f) and Sed5 (Figure 5h) in mock-treated samples, which was collapsed by DUB treatment to monoUb (Figure 5f–i). These data support previously published high-throughput results indicating that Gos1, Ykt6, and Sed5 are ubiquitinated (Swaney et al., 2013). The differences in the size distributions of polyUb smear in each SNARE immunoprecipitate suggest that this assay is primarily detecting direct modification of Gos1, Ykt6, and Sed5 as opposed to the aggregate polyUb associated with the entire SNARE complex.

Nondegradative ubiquitination is associated with Gos1, COPI, and Glo3 complexes

We observed significant enrichment of COPI subunits in the Gos1 pulldown samples analyzed with SILAC MS (Figure 5e). Therefore, we probed the ubiquitination status of FLAG-tagged COPI (α- and β′-COP subunits) and Glo3, as this ArfGAP is reported to bind COPI and SNAREs (Rein et al., 2002). The FLAG IPs probed with antiubiquitin antibody show a substantial amount of monoUb released from COPI and Glo3 immunoprecipitates following the DUB treatment (Figure 5—figure supplement 1a–e). K48-linked polyUb chains are known to target proteins for proteasomal degradation. To address whether Gos1, COPI, and Glo3 complexes are modified with K48-linked polyUb, we treated the samples with a K48-specific DUB. No significant change in the smeared electrophoretic pattern or the release of monoUb in the samples was observed with or without K48-specific DUB treatment suggesting that the ubiquitination associated with COPI and Glo3 is not a degradation signal (Figure 5—figure supplement 1a-e).

We also probed Gos1, Ykt6, Sed5, COPI, and Glo3 FLAG-immunoprecipitated samples with K63-specific deubiquitinase (K63-DUB [Sato et al., 2008]), linear ubiquitin-specific deubiquitinase (M1-DUB [Keusekotten et al., 2013]), or a general deubiquitinase (DUB [Baek et al., 1997]) as a control (Figure 5—figure supplement 2). No significant release of ubiquitin was observed following K63- or M1-DUB treatment compared to the untagged control (Figure 5—figure supplement 2). A detectable amount of ubiquitin was released from Gos1 following K63-DUB treatment, but the signals were not significantly above the background levels (Figure 5—figure supplement 2). A significant level of released ubiquitin was detected for these samples when treated with the general deubiquitinase. The lack of K63 linkages on these components is also consistent with live-cell imaging data (Figure 3a–f), showing that β′-COP with a K63-specific binding domain failed to support the trafficking of Bet1, Gos1, and Snc2. Thus, the ubiquitination associated with Gos1, COPI, and Glo3 complexes appears to be nondegradative (non-K48 or non-K63) in nature and may modulate protein interactions in the COPI-dependent retrieval of SNAREs within the Golgi.

Ubiquitination stabilizes Golgi SNARE–COPI complexes

To explore the possibility that ubiquitination is an important regulator of protein–protein interactions in the COPI–SNARE system, we used comparative pulldown studies using FLAG-tagged SNAREs under conditions that preserved endogenous ubiquitination (w Ub) or catalyzed removal of ubiquitin (w/o Ub) (Figure 6a). An equal amount of Gos1-FLAG was pulled down in both w Ub and w/o-Ub conditions (Figure 6c). Probing samples with a ubiquitin antibody showed a ubiquitin smear associated with Gos1 immunoprecipitated using ‘w Ub’ conditions, most of which was stripped off under ‘w/o-Ub’ conditions (Figure 6b). We next probed these samples with COPI and Arf antibodies. Significant enrichment of COPI subunits and Arf was observed with Gos1 when ubiquitination was preserved, compared to ‘w/o-ub’ conditions (Figure 6e–g). The ubiquitination, thus, appears to play a role in the assembly and/or stability of COPI coatomer complex with Gos1.

Figure 6 with 1 supplement see all
Ubiquitin modification stabilizes a priming complex between COPI, Arf, and SNAREs.

(a) Schematic of the experimental setup wherein FLAG-tagged SNAREs are divided into two equal portions, and one is processed under ‘w Ub’ conditions (DUB inhibitors used during the lysis step and no deubiquitinases [mock] treatment) and the other portion is processed under ‘w/o Ub’ condition (no deubiquitinase inhibitors used during lysis and immunoprecipitated samples are treated with deubiquitinases). (b–g) Western blot data showing comparative pulldowns of Gos1-FLAG (b–f) and Bet1-FLAG (Figure 6—figure supplement 1) processed under ‘ubiquitin-preserved’ (w Ub) and ‘no-ubiquitin’ (w/o Ub) condition, and probed for Ub (b), FLAG (c), COPI (e), and Arf (f). Untagged cells processed under ‘w UB’ condition to determine background binding were used as a control (Cntr) and abundant background bands are marked with an asterisk. Quantitation of Gos1-FLAG (d) and Arf (g) in the pulldown samples. Band intensities are measured using ImageJ. Quantitation was done on three biological replicates using a t-test (**p < 0.01, Ns p > 0.05).

Our assays and previous reports indicate that Gos1 is ubiquitinated (Swaney et al., 2013), but ubiquitination has not been detected on Bet1 (Figure 5b–d). Loss of the UBD of COPI in β′-COP ∆2–304 led to mislocalization of both Bet1 and Gos1 to the elongated tube- and ring-like structures (Figures 1b and 3a, c). Therefore, we tested whether ubiquitination affects the interaction of Bet1 with COPI coat complex components. The smeared pattern associated with Bet1 in the blot probed with anti-FLAG antibody is similar under ‘w Ub’ and w/o Ub’ conditions (Figure 6—figure supplement 1b,c). Nonetheless, we see the enrichment of COPI subunits with Bet1 under ‘w Ub’ conditions compared to ‘w/o Ub’ (Figure 6—figure supplement 1d). Similarly, Arf is significantly enriched with Bet1 when ubiquitin was present on the complexes (Figure 6—figure supplement 1e, f). Control experiments indicated that the presence of DUB inhibitors during cell lysis was most critical to preserve the SNARE–COPI complex (Figure 6a, Figure 6—figure supplement 1g, h). Therefore, the role of ubiquitination in the assembly and stability of COPI coatomer complex with Bet1 appears to function independently of the Bet1 ubiquitination status. Altogether, the data reveal ubiquitin-mediated stabilization of COPI–Golgi SNARE complexes.

Glo3 is not enriched in ubiquitin-stabilized SNARE–COPI–Arf complexes and Gos1 localization is unaffected in glo3Δ cells

Glo3 is proposed to be part of a SNARE–Arf–COPI priming complex, but we failed to detect any Glo3 peptides in the Gos1 immunoprecipitates by MS (Figure 5e). To further test whether ArfGAP Glo3 is present in the ubiquitin-stabilized SNARE–COPI–Arf complex, we performed Gos1-FLAG pulldowns under w Ub and w/o-Ub conditions in cells expressing Glo3 C-terminally tagged with GST. We detected a small amount of Glo3-GST coimmunoprecipitating with FLAG-Gos1, but no significant difference was observed in the presence or absence of Ub (Figure 7a, c). In contrast, association of Arf with Gos1 was significantly enriched using w Ub conditions compared to w/o-Ub conditions (Figure 7a, d). Cell lysate controls probed for GST in cells expressing only Gos1-FLAG or both FLAG-Gos1 and Glo3-GST confirmed the identity of the Glo3-GST band (Figure 7b).

ArfGAP is not enriched in ubiquitin-stabilized SNARE–Coat complexes and is not required for Gos1 localization.

(a–e) Western blot data showing comparative pulldowns of Gos1-FLAG from cells expressing FLAG-tagged Gos1 and GST-tagged Glo3. Samples were processed under ‘ubiquitin-preserved’ (w Ub) and ‘no-ubiquitin’ (w/o Ub) condition, and probed for Glo3 (anti-GST), Gos1 (anti-FLAG), Arf, and Ub. Untagged cells processed under ‘w UB’ condition to determine background binding were used as a control (Ctrl). Cell lysates from cells expressing only FLAG-tagged Gos1 or both FLAG-tagged Gos1 and GST-tagged Glo3 probed with anti-GST antibody are included as controls to ensure expression of GST-tagged Glo3. Quantitation of (c) Glo3-GST, (d) Arf, and (e) Gos1-FLAG samples. Band intensities are measured using ImageJ. Quantitation was done on three biological replicates using a t-test (*p < 0.05, Ns p > 0.05). (f) SILAC mass spectrometric analysis of αCOP-FLAG pulldown samples indicate the enrichment of other COPI subunits (green dots), ArfGAP Glo3 (purple dots), and dilysine COPI cargo (red dots) but no SNAREs . (g) Live cell imaging of mNG-Gos1 in wild-type (WT), β′-COP Δ2–304 and glo3∆ cells. Quantification of % tube structures for each strain was from three biological replicates with 60 or more cells analyzed for each sample . Statistical differences were determined using a one-way analysis of variance (ANOVA) on the means of the three biological replicates (****p < 0.0001). Scale bar represents 5 µm. (h) Model showing ubiquitination-mediated priming of a SNARE–Arf–COPI complex. Glo3 is recruited at later stages after vesicle budding leading to hydrolysis of Arf-GTP and disassociation of COPI complex.

To determine whether we can detect Glo3 interaction with COPI, we performed SILAC coimmunoprecipitation analysis using C-terminally FLAG-tagged α-COP under ubiquitin preserved conditions. As expected, Glo3 was enriched with COPI, indicating eventual recruitment of Glo3 onto the COPI coat (Figure 7f). The COPI sample was also enriched for several ER residence membrane proteins bearing C-terminal dilysine motifs (Figure 7f, red datapoints). However, no SNAREs were coimmunoprecipitated with COPI, indicating that the COPI complexes recovered here were significantly different from SNARE–COPI complexes that contained Gos1.

In addition, we analyzed mNG-Gos1 localization in glo3Δ cells with WT and β′-COP Δ2–304 cells as control. Compared to WT cells, no significant changes in the size or distribution of mNG-Gos1 punctae were observed in glo3Δ (Figure 7g). As reported earlier, Gos1 is mislocalized to elongated tube-like structures in β′-COP Δ2–304 cells. Together, these data suggest that Glo3 can weakly bind Gos1 but unlike COPI, this interaction is not stabilized by the presence of Ub. In addition, Glo3 does not appear to be required for COPI-dependent Gos1 localization.

Altogether, these results indicate that ubiquitin plays critical role in stabilizing a complex between SNAREs, COPI, and Arf that is important for COPI function in retrieving a subset of SNAREs. Glo3 appears to be present at low levels in the Gos1–Arf–COPI complex, but this interaction is not modulated by ubiquitination.

Discussion

We previously discovered that COPI binds specifically to polyUb chains, and that this interaction is crucial for recycling Snc1, an exocytic v-SNARE, back to the TGN. In this study, we broadly probed the role of COPI–ubiquitin interactions on the localization of 16 additional budding yeast SNAREs to determine whether ubiquitination of coat components is a general mechanism for SNARE sorting. While localization of most mNG-tagged SNAREs was unaffected by deletion of the ubiquitin-binding N-terminal WDR domain of β′-COP, we found a significant change in the localization pattern for Bet1, Gos1, Snc1, Snc2, and partially for Bos1, Tlg1, and Sec22 in β′-COP ∆2–304 (Figures 1b, c, 3a–f, Figure 1—figure supplement 1). Normal SNARE localization is restored by the replacement of β′-COP N-terminal WDR domain (β′-COP-UBD) with an unrelated UBD (Figure 3, Figure 3—figure supplement 2). Moreover, we found nondegradative (non-K48 and -K63) ubiquitin associated with multiple Golgi SNAREs (Gos1, Ykt6, and Sed5) and the COPI machinery (Figure 5b–d, f–i, Figure 5—figure supplements 1 and 2), and that these ubiquitin modifications were essential for the stabilization of COPI, Arf, and Golgi SNARE complexes (Figure 6). For Gos1, the ubiquitin-stabilized SNARE–COPI–Arf complex is not enriched for the ArfGAP Glo3 (Figure 7). These studies highlight the important role of ubiquitination in COPI-mediated trafficking, specifically in the regulation of Golgi SNARE localization.

SNAREs are essential components of the endomembrane system where they play a major role in determining the directionality and specificity of vesicular transport. Our data provide exciting insights into regulation of SNARE localization required to maintain a functional endomembrane system. In the case of Sec22, the failure of COPI-mediated retrieval of this SNARE causes mislocalization to the vacuole. Bet1 and Gos1, however, display an unusual mislocalization phenotype from normal punctate Golgi in WT cells to abnormal tubular and ring-shaped structures that lack Golgi markers in β′-COP ∆2–304 cells. Although the degree of colocalization with Golgi markers is substantially reduced in this COPI mutant, neither Bet1 or Gos1 significantly accumulate at downstream compartments such as the plasma membrane, endosomes, or vacuoles. We propose that Bet1 and Gos1 accumulate in dead-end sequestration structures in β′-COP ∆2–304 that are remnants of the Golgi cisternal maturation process. None of the Golgi marker proteins assessed (Sed5, Anp1, Mnn9, Rer1, Aur1, Chs5, or Sec7) localized to the same abnormal structures containing Bet1 or Gos1 or showed any evidence of being mislocalized (this study and Xu et al., 2017). These data indicate that COPI is still functioning well to recycle most proteins from late Golgi compartments back to early compartments or the ER in β′-COP ∆2–304 cells. A failure of COPI to retrieve Gos1 and Bet1, and perhaps an inability of these SNAREs to enter vesicles destined for the plasma membrane or endocytic system, could lead to the accumulation of compartments that house only a few mislocalized proteins. It is also possible that Gos1 and Bet1 are transported to endosomes in β′-COP ∆2–304 cells but are very efficiently retrieved by retromer or sorting nexins. In this case, the abnormal Gos1 or Bet1 structures could represent functional intermediates that have lost most of their Golgi identity and only weakly stain with the endocytic tracer FM4-64. The appearance of abnormal Gos1 and Bet1 structures is almost identical in a COPI temperature-sensitive mutant that displays a broader defect in protein trafficking. This suggests that the β′-COP ∆2–304 cells have substantially lost COPI function in sorting Bet1 and Gos1, which may underpin the slow growth phenotype of this mutant. In contrast, β′-COP-UBDDoa1 and β′-COP-RKR cells sort Gos1 and Bet1 normally, even though these β′-COP variants cannot bind to dilysine cargoes, and both variants support WT growth of yeast. Thus, the Gos1 and Bet1 phenotypes observed in β′-COP ∆2–304 are directly caused by a deficiency in β′-COP binding to polyUb and are not a secondary effect of disrupting the interaction with dilysine motifs.

The type of ubiquitin linkages required for intra-Golgi SNARE interaction with COPI appears to be different from the ubiquitin linkages required to sort Snc1. β′-COP binds preferentially to K63-linked polyUb chains and does not bind monoUb, diUb, or K48-linked polyUb chains (Xu et al., 2017). The replacement of the N-terminal WDR domain of β′-COP with the NZF domain from Tab2, which binds specifically to K63-linked polyUb, substantially restores Snc1 trafficking to the plasma membrane (Xu et al., 2017; Figure 3b). Surprisingly, the β′-COP-Tab2NZF fusion fails to support the trafficking of Bet1 or Gos1 and only partially supports the trafficking of Sec22 (Figure 3a–f, Figure 1—figure supplement 1). We further explored the binding specificity of β′-COP and found that it is also capable of binding linear polyUb chains and to K6-, K11-, and K29- polyUb chains (Figure 3g, h). Moreover, the polyUb chains detected in the SNARE or COPI pulldowns are resistant to M1-, K63-, or K48-specific DUBs (Figure 5—figure supplement 2). Thus, the ubiquitin modifications present are unlikely to be targeting COPI to the proteasome or the SNAREs to the vacuole for degradation. Our data provide compelling evidence that the nonproteolytic ubiquitin code regulates the COPI-dependent trafficking patterns for Golgi SNAREs.

PolyUb chains on SNAREs could form a sorting signal that COPI recognizes in order to recycle them from downstream compartments, as previously proposed for Snc1 (Xu et al., 2017). However, several observations in the current study suggest broader roles of ubiquitination in regulating SNARE trafficking. For example, the Golgi localization of Bet1 relies on COPI’s ability to bind ubiquitin, but we could not detect ubiquitinated forms of this SNARE (Figure 5b–d). However, ubiquitination was associated with other SNAREs, including Gos1, Ykt6, and Sed5, multiple COPI subunits, and ArfGAP Glo3 (Figure 5b–d, f–i, Figure 5—figure supplements 1 and 2). It is possible that Bet1 associates with another cargo protein that is ubiquitinated, and the ubiquitin serves as the COPI-dependent sorting signal for both proteins. It is also possible that ubiquitination induces conformational changes in COPI driven by β′-COP interaction with ubiquitin attached to itself or to other COPI subunits. Such a COPI conformational change could produce a high-affinity binding site for Bet1. The role of ubiquitin in mediating the stability of the COPI–SNARE complex is further supported by the observation that Bet1 and Gos1 are mislocalized when ubiquitin is stripped from COPI–SNARE system by fusing a deubiquitinase domain to COPI components (Figure 4).

We were surprised to find that COPI was co-enriched with Gos1-FLAG in the SILAC-based MS data (Figure 5e) because cargo–coat interactions are typically low affinity. Arf1 was also present in this dataset, although not as highly enriched as the COPI subunits. We considered the possibility that the conditions used to pulldown Gos1-FLAG that preserve ubiquitination may have stabilized the COPI–Gos1 interaction. Indeed, performing these Gos1-FLAG pulldowns in the presence of active DUBs to remove ubiquitin dramatically reduces the amount of COPI and Arf recovered with Gos1-FLAG relative to samples prepared with DUB inhibitors present (Figure 6e–g). The interaction between Gos1 and COP1/Arf is nearly undetectable if ubiquitination of the components is not preserved. Bet1 interaction with Arf/COPI is also enhanced substantially under conditions that preserve ubiquitination (Figure 6—figure supplement 1). Therefore, ubiquitination appears to regulate the assembly and/or stability of the COPI–cargo complex independent of the ubiquitination status of cargo. Not all ubiquitinated SNAREs relied on COPI–ubiquitin interaction for their sorting, For example, Sed5 is ubiquitinated but its localization is not affected by the alterations in the ability of COPI to recognize and bind ubiquitin (Figure 5h, i, Figure 1—figure supplement 1, Sed5), and Sed5 appears to be independent of COPI for its Golgi localization (Gao and Banfield, 2020). A subset of Golgi SNAREs is dependent on the ability of COPI to bind ubiquitin (Figure 6—figure supplement 1), and it is likely that other ubiquitin-independent interactions contribute to cargo selection.

One of the long-standing questions about COPI-mediated vesicular trafficking has been the essential roles of α and β′COP WDR domains needed to sustain yeast viability. α and β′COP WDR domains are required for the sorting of dilysine motif COPI cargoes, but cells are viable when all dilysine-binding sites are mutated (Jackson et al., 2012). These studies implicated additional roles for the COPI WDR domains in cells. Our data suggest that ubiquitin binding by these COPI WDR domains contributes to the essential function of COPI because yeast harboring β′COP-UBDDoa1 grow much better than strains harboring β′COP-∆2–304 (Xu et al., 2017). We have previously shown that the human β′COP-β-propeller also binds polyUb and can function in yeast to support the normal localization of mNG-Snc1, suggesting that the COPI–ubiquitin interaction is functionally conserved. Another key element of vesicle-mediated trafficking is the ability of the coat to bind cargo during vesicle formation, followed by dissociation after the vesicle forms. SNAREs are thought to prime coat assembly through interactions with Arf, ArfGAPs, and COPI as a mechanism to ensure vesicles form with an adequate load of v-SNAREs (Rein et al., 2002; Spang et al., 2010). The ArfGAP Glo3 contains a BoCCS motif that mediates binding to both COPI and to several different SNAREs, suggesting that Glo3 is a key determinant of the priming complex (Schindler and Spang, 2007). However, it is unclear how Glo3 could facilitate coat assembly when its enzymatic function is to inactivate Arf. We have identified a ubiquitin-stabilized complex between Gos1, Arf, and COPI that appears to lack endogenous Glo3 (Figure 5). We were able to detect a tagged form of Glo3 in Gos1 immunoprecipitates that lack ubiquitin; however, preserving ubiquitin in these Gos1 pulldowns had no influence on Glo3 recovery even though substantially more Arf and COPI were recovered. The presence of ubiquitin modifications on COPI and Glo3 does not prevent their interaction because we observed enrichment of Glo3 in COPI pulldowns under the same ubiquitin-preserved conditions. Therefore, we suggest that the SNARE/Arf-GTP/COPI priming complex is stabilized by ubiquitination of the components and is devoid of ArfGAP. Arf-GDP and COPI likely dissociate rapidly from the complex as the ArfGAP binds (Figure 7h).

Ubiquitin-dependent enrichment of Arf and COPI with SNAREs suggests that cycles of ubiquitination and deubiquitination could control the switch from Arf-GTP/SNARE-mediated assembly of COPI during budding and ArfGAP-mediated disassembly and uncoating of vesicles prior to fusion. Ubiquitination–deubiquitination cycles for key components within the COPI-SNARE system thus may alter the coatomer assembly–disassembly dynamics regulating COPI function.

Materials and methods

Reagents

ANTI-FLAG M2 Magnetic Beads (M8823), EZview Red ANTI-FLAG M2 Affinity Gel (F2426), 3xFLAG Peptide (F4799), N-ethylmaleimide (E3876), iodoacetamide (GERPN6302), 1,10-phenanthroline (131377), N-ethylmaleimide (E3876), deubiquitinase inhibitor PR-619 (SML0430), protease inhibitor tablets (04693159001), and phosphatase inhibitors tablets (PHOSS-RO) were purchased from MilliporeSigma (St Louis, MO). Coomassie Brilliant Blue R-250 Dye (20278) and FM4-64 dye (T-3166) were purchased from Thermo Fisher Scientific (San Jose, CA). ECL Prime Western Blotting Chemiluminescent Substrate (34580) and Pierce Anti-HA Agarose (26181) were purchased from Thermo Scientific (Rockford, IL). Deubiquitinases (DUBs) Usp2 (E-504), MINDY2 (E-620), MINDY3 (E-621), OTULIN (E-558), AMSH (E-548B), K6-ubiquitin trimer (Ub3) chains (UC-20-025), K11-Ub3 chains (UC-50-025), K29- Ub3 chains (UC-85-025), and K33-Ub3 (UC-105-025) were from BostonBiochem – R&D Systems, Inc (MN, USA).

Antibodies

ANTI-FLAG antibody produced in mouse (clone M2, F3165, 1:3500) and Anti-HA antibody produced in rabbit (H6908, 1:1000) were purchased from MilliporeSigma (St. Louis, MO). VU101: Anti-ubiquitin Antibody (VU-0101, 1:1000) was purchased from LifeSensors (PA, USA). Anti-mouse HRP conjugate (W4021, 1:10,000) and Anti-Rabbit HRP conjugate (W4011, 1:10,000) were purchased from Promega (Madison, WI). Anti-COPI antibody was a gift from Charles Barlowe (Dartmouth Univ, 1:3000). Anti-Arf antibody (1:3000) used was reported previously (Liu et al., 2008). Anti-GST antibody (1:1000) was purchased from Vanderbilt Antibody Product Store (VAPR, Nashville, TN).

Strains and plasmids

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Standard media and techniques for growing and transforming yeast were used. Epitope tagging of yeast genes was performed using a PCR toolbox (Janke et al., 2004; Longtine et al., 1998). For the construction of mKate-tagged Golgi markers in β′COP-∆2–304 background, the PXY51 strain was used followed by shuffling of p416-SEC27 with p315-SEC27Δ2–304. The list of yeast strains used in this study is included as a table file (Supplementary file 1). Plasmid constructions were performed using standard molecular manipulation. Mutations were introduced using Gibson Assembly Master Mix. The list of plasmids used in this study is included as a table file (Supplementary file 2).

Imaging and image analysis

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To visualize mNG- or mScarlet-tagged proteins, cells were grown to early-to-mid-logarithmic phase, harvested, and resuspended in imaging buffer (10 mM Na2PHO4, 156 mM NaCl, 2 mM KH2PO4, and 2% glucose). Cells were then mounted on glass slides and observed immediately at room temperature. Images were acquired using a DeltaVision Elite Imaging system equipped with a ×63 objective lens followed by deconvolution using SoftWoRx software (GE Healthcare Life Science). Overlay images were created using the merge channels function of ImageJ software (National Institutes of Health). To quantify SNAREs colocalization, a Pearson’s correlation coefficient for the two markers in each cell (n = 3, over 20 cells each) was calculated using the ImageJ plugin Just Another Colocalization Plugin with Costes Automatic Thresholding (Bolte and Cordelières, 2006).

Identification and quantitation of fluorescence-based morphological patterns were performed as below: the punctate pattern indicates small, dotted structures, typically around 0.45 µm, the ring-like structures indicate larger, round structures with a hole in the middle, akin to a ring or a donut, roughly 0.6–0.9 µm along the longer and short axis, and elongated tube-like structures indicate tube-like structures whose longer axis is approximately two to five times in length of the shorter axis, approximately 0.6–1.8 µm along the long axis. Each fluorescent structure in the cell was categorized as puncta, ring, or tubule and the number of tubules + ring divided by total fluorescent structures was used to quantify the % tubular and ring structures. Measurements were done in minimum of 50 cells (n ≥ 50) for three biological replicates. Fluorescence pattern identification and quantitation were repeated in a blinded fashion and/or by a second observer to avoid bias.

Synthesis of K63 and linear ubiquitin chains

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To synthesize K63-linked Ub chains, 2 mM Ub, 300 nM E1, 3 μM UBE2N/UBE2V2 were mixed in the reaction buffer (50 mM Tris–HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2, 20 mM ATP, and 2 mM Dithiothreitol (DTT)) overnight at 37°C. Reactions were quenched by lowering the pH to 4.5 with addition of 5 M ammonium acetate pH 4.4. K63 tri-Ub was isolated and further purified using size-exclusion chromatography (Hiload 26/600 Superdex 75 pg, GE Healthcare) in gel filtration buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM DTT). Purified chains were buffer exchanged into H2O and lyophilized. Recombinant M1-linked tri-Ub-FLAG-6XHis was expressed and purified as previously described (Hepowit et al., 2020). Briefly, E. coli C41 (DE3) cells at OD600 of 0.6 were induced with 1 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG), lysed by sonication in ice-cold Tris buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM imidazole, 2 mM (2-Mercaptoethanol) βME, complete protease inhibitors (Roche, Basel, Switzerland), 1 μg/ml DNase, 1 μg/ml lysozyme, and 1 mM phenylmethylsulfonyl fluoride (PMSF)), and clarified by centrifugation (50,000 × g for 30 min at 4°C) and filtration (0.45 μM filter). M1 tri-Ub was purified to homogeneity by Ni2+-NTA affinity column (Thermo Scientific, Rockford, IL) chromatophraphy, HiPrep Q FF anion exchange column (GE Healthcare Life Sciences, Marlborough, MA) chromatography, and HiLoad Superdex size-exclusion column (GE Healthcare Life Sciences, Marlborough, MA) chromatography.

Construction FLAG-, HA-, and GST-tagged constructs

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Multiple strains of S. cerevisiae were generated in a manner where one of the components is tagged with an epitope tag. Bet1, Gos1, and Snc1 were C-terminally tagged with 6xHis-TEV-3xFLAG by integration of a PCR product amplified from pJAM617 into the BET1, SNC1, and SNC2 locus, respectively (Janke et al., 2004). Due to the low recombination rate, a GOS1 PCR product with longer 5′ and 3′ regions of homology (over 200 bp) was generated from pJAM617 and gene synthesized DNA fragments and integrated into the GOS1 locus (two-step PCR and integration method). Properly integrated clones were confirmed by genotyping PCR as well as by immunoblot using anti-FLAG antibody. Similarly, COP1, Sec27, and ArfGAP Glo3 were C-terminally tagged with 6xHis-TEV-3xFLAG using two-step PCR and integration method. Additionally, Glo3 was C-terminally tagged with GST (using pFA6a-GST-HisMX6 as a template) in WT S. cerevisiae as well as in cells harboring 6xHis-TEV-3xFLAG-tagged Gos1. Efforts to C-terminally tag Ykt6 and Sed5 were unsuccessful; consequently, Ykt6 and Sed5 were N-terminally tagged with 6xHA tag by integration of a PCR product amplified from pYM-N20 cassette (Euroscarf #P30294).

Purification of FLAG- or HA-tagged proteins

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Affinity isolation of FLAG- or HA-tagged proteins was performed with anti-FLAG magnetic beads or Anti-HA Agarose, respectively. 800 OD600 of untagged WT cells (BY4742) and cells with C- or N-terminally tagged protein of interest were grown in YPD and harvested by centrifugation when the OD600 reached ~0.8. After washing with cold water, the pellets were resuspended in 3 ml lysis buffer (100 mM Tris pH 7.4, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10% glycerol, 1% Triton X-100, 100 μM PR619, 5 mM 1,10-phenanthroline, 50 mM N-ethylmaleimide, phosphatase inhibitors, and complete protease inhibitor tablet). Cells were broken using a Disruptor Genie (Scientific Industries) at 4°C for 10 min at 3000 setting with 0.5 mm diameter glass beads. The lysates were centrifuged at 13,000 rpm for 15 min at 4°C and the supernatant was incubated with 50 µl FLAG or HA beads overnight at 4°C. The next morning the beads were washed 3× with washing buffer (100 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.5% Triton X-100) and eluted in sodium dodecyl sulfate (SDS) running buffer.

Heterologous expression and purification of GST-β′COP and ubiquitin-binding assays were performed as reported previously (Xu et al., 2017). Briefly, 0.5 mM of GST and GST-tagged β′COP (604) proteins immobilized glutathione beads were incubated 250 nM ubiquitin trimer (Ub3) for corresponding linkages, washed 3× and eluted using reduced glutathione.

DUB treatments

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The DUB treatments were performed as described (Hospenthal et al., 2015). Briefly, the beads with target proteins were equally split into two parts. One part was subjected to mock treatment as a control, and the other part was incubated with deubiquitinases in the following reaction: 5 μl of 10xDUB reaction buffer (1 M Tris pH 7.4, 1.5 M NaCl, 10 mM DTT), 0.5 μl of deubiquitinase enzyme, and water for a 50 μl reaction volume. The samples were incubated at 37°C for 45 min and reactions were stopped with 2× Laemmli sample buffer by incubating at 95°C for 5 min. Supernatants were collected and used for electrophoresis followed by Western transfer. Deubiquitinases were used following manufacturer recommended concentrations as follows: DUB: the general deubiquitinase Ups2 (1–5 nM); K48-DUB: K48 linkage-specific deubiquitinase MINDY2 and MINDY3 (10–30 nM); K63-DUB: K63 linkage-specific deubiquitinase AMSH (100–500 nM); M1-DUB: OTULIN (0.05­ 1 μM). Data were generated from independent experiments from three biological replicates and quantified as described later.

For comparative pulldown samples processed under conditions that preserved endogenous ubiquitination (w Ub) or catalyzed removal of ubiquitin (w/o Ub), a similar immunoprecipitation and DUB protocol were used with the following modifications. The cell pellets (800 OD600) were divided into two equal portions. For samples processed under ‘w Ub’ condition, lysis buffer with deubiquitinase inhibitors (100 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% glycerol, 0.2% NP40, 100 μM PR619, 5 mM 1,10-phenanthroline, 50 mM N-ethylmaleimide, phosphatase inhibitors, and complete protease inhibitor tablet) was used. Immunoprecipitated samples were washed 2×. As a mock treatment for immunoprecipitated samples under ‘w Ub’ condition, the deubiquitinase buffer did not have any deubiquitinases. For the samples processed under conditions that catalyzed removal of ubiquitin (w/o Ub), the lysis buffer did not have the deubiquitinase inhibitors 100 μM PR619, 5 mM 1,10-phenanthroline, or 50 mM N-ethylmaleimide, and furthermore the immunoprecipitated samples were processed using DUB buffer containing 1 µl of each deubiquitinase Usp2, AMSH, and OTULIN. Data were generated from independent experiments from three biological replicates and quantified as described later.

For systematic screening of comparative enrichment of Arf with Gos1 under various ubiquitin-preserved/-removed conditions in combination with phosphorylation preserved/phosphorylation removed conditions the samples were processed as described above with the following modifications to the procedure. Cells were lysed in the presence of (1) deubiquitinase inhibitors (PR619, O-PA, and NEM), (2) phosphatase inhibitors (PhosSTOP), (3) both deubiquitinase and phosphatase inhibitors, or (4) no additional inhibitors other than the protease inhibitors. Cell lysis in the presence of deubiquitinase and phosphatase inhibitors is expected to preserve ubiquitin- and phosphorylation-mediated complexes. Cell lysis in the presence of just deubiquitinase or phosphatase inhibitors is expected to preserve only ubiquitin- or phosphorylation-mediated complexes. Cell lysis in the absence of both deubiquitinase and phosphatase inhibitors in expected to not preserve ubiquitin- or phosphorylation-mediated complexes. Following immunoprecipitation using anti-FLAG resin, the samples were treated with (1) deubiquitinases (USP2, AMSH, and Otulin), (2) phosphatases (Lambda phosphatase), (3) both deubiquitinases and phosphatases, and (4) no post-IP treatment. Post-IP deubiquitination (with USP2, AMSH, and Otulin) and/or dephosphorylation (with Lambda phosphatase) is expected to strip off any preserved or remaining ubiquitination and phosphorylation, respectively, from the immunoprecipitated samples.

Immunoblotting with ECL

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Protein samples were separated by 4–20% gradient SDS–polyacrylamide gel electrophoresis followed by immunoblotting. For anti-ubiquitin antibody the membranes were treated with glutaraldehyde solution (supplied with the antibody) as per the manufacturer’s protocol and washed with PBS. The membranes were blocked in 5% nonfat milk for 1 hr, incubated with primary antibodies for 3 hr at room temperature, washed five times with Tris-buffered saline (TBS) with 0.1% Tween, incubated with appropriate secondary antibody for 1 hr at room temperature, washed five times and imaged using manufacturer recommended chemiluminescence protocol. The membranes were imaged with AI600 Chemiluminescent Imager (GE Life Sciences). Quantitative analysis of Western blot images was performed using ImageJ software.

SILAC MS

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SILAC-based mass spectrometric analysis of Gos1-FLAG with untagged control was performed using a similar protocol as described previously (Hepowit et al., 2020). Briefly, an equal amount of cells (labeled with either light or heavy Arg and Lys) expressing endogenous FLAG-tagged Gos1 or untagged cells were harvested from the mid-log phase and disrupted by bead beating using ice-cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.2% NP-40, 10 mM iodoacetamide, 1 mM 1,10-phenanthroline, 1× EDTA free protease inhibitor cocktail [Roche], 1 mM phenylmethylsulfonyl fluoride, 20 µM MG132, 1× PhosStop [Roche], 10 mM NaF, 20 mM N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide, dihydrochloride (BGP), and 2 mM Na3VO4). Lysate was clarified by centrifugation at 21,000 × g for 10 min at 4°C and supernatant was transferred into a new tube and diluted with three-fold volume of ice-cold TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl). Samples were incubated with 50 µl of EZview anti-FLAG M2 resin slurry (Sigma) for 2 hr at 4°C with rotation. The resin was washed three times with cold TBS and incubated with 90 µl elution buffer (100 mM Tris–HCl, pH 8.0, 1% SDS) at 98°C for 5 min. The collected eluate was reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide, and precipitated with 300 µl precipitation solution (50% acetone, 49.9% ethanol, and 0.1% acetic acid). Light and heavy protein pellets were dissolved with Urea-Tris solution (8 M urea, 50 mM Tris–HCl, pH 8.0). Heavy and light samples were combined, diluted fourfold with water, and digested with 1 µg MS-grade trypsin (Gold, Promega) by overnight incubation at 37°C. Phosphopeptides were enriched by immobilized metal affinity chromatography using Fe(III)-nitrilotriacetic acid resin as previously described (MacGurn et al., 2011) and dissolved in 0.1% trifluoroacetic acid and analyzed by liquid chromatography (LC–MS)/MS using an Orbitrap XL mass spectrometer. Data collected were searched using MaxQuant (ver. 1.6.5.0) and chromatograms were visualized using Skyline (ver. 20.1.0.31, MacCoss Lab). Coimmunoprecipitation followed by SILAC-based mass spectrometric analysis of α-COP-FLAG was performed as described above.

Statistical analysis

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Statistical differences between two groups for SNARE morphology were determined using a Fisher’s exact test. For multiple group comparison, one-way analysis of variance on the means using GraphPad Prism (GraphPad Software Inc). Probability values of less than 0.05, 0.01, and 0.001 were used to show statistically significant differences and are represented with *, **, or ***, respectively. To quantify western blot data, at least three independent replicates were used, and intestines were calculated using ImageJ software and statistical analyses, as indicated, were performed using GraphPad Prism.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1 and 3-7.

References

Decision letter

  1. Felix Campelo
    Reviewing Editor; Institute of Photonic Sciences, Spain
  2. Suzanne R Pfeffer
    Senior Editor; Stanford University School of Medicine, United States
  3. Felix Campelo
    Reviewer; Institute of Photonic Sciences, Spain

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Ubiquitination drives COPI priming and Golgi SNARE localization" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Felix Campelo as the Reviewing Editor and Reviewer #1 and the evaluation has been overseen by a Senior Editor.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this manuscript in its current form will not be considered further, but we would welcome a new submission if it can address, in full, the reviewers' concerns.

Specifically, all the reviewers valued the strong interest of the scientific question you tackled here. However, after consultation among the three reviewers, we agreed that there are a number of important points that would need to be clarified (requiring a number of additional experimental work), before this manuscript could be further considered. Given that the amount of extra work is relatively considerable (although, we believe, doable in a 3-6 month time period), we decided to reject this manuscript at the present moment, so you can decide whether you want to work on the requested revisions (see below) and – depending on the results found – submit again to eLife (as a new submission), or otherwise maybe temper the claims of the paper for a prompt submission elsewhere.

In our opinion, the main points we would like to see so we are convinced about the claims of this paper are:

1) Provide more convincing evidence for a trafficking defect, not just a morphology change. As an example of what can be done (see detailed report below for more details) is to use co-localization as a readout of trafficking. For example, use Mnn9-mCherry or a number of other established Golgi proteins that can be successfully tagged with RFP.

2) Test if the COPI b' mutant (∆2-304) has a (more) general effect on Golgi trafficking and morphology as compared to what they report here. In particular, comparisons to the KK-binding site β'-COP mutant will be an important control to include for most of the experiments, as this is the canonical COPI binding site and is lacking in the β'-COP (∆2-304) mutant.

3) Improve on the image quantitation to better understand the nature of the aberrant compartment where SNAREs relocalize.

There were also other important questions raised by the reviewers (see below) that would require some clarification/explanation (e.g. difference between o/expression end. expression levels; better Bet1 localization together with other Golgi/TGN markers, etc.).

Reviewer #1 (Recommendations for the authors):

Date et al. present a thorough study of the effects and roles of protein ubiquitination in the association of different COPI subunits and COPI-associated factors with SNARE proteins in the yeast S. cerevisiae. In a previous work from this lab (Xu et al. eLife 2017), it was shown that the SNARE Snc1 is mislocalized when COPI is not able to associate with the ubiquitination in Snc1. Here, Date et al. expand on those observations and describe that (i) COPI components, Glo3 (ArfGAP), and some but not all tested Golgi SNARES are ubiquitinated; (ii) the localization of some of these SNARES is dependent on the binding between COPI and the ubiquitinated cargoes (SNAREs) ; and (iii) they propose a model in which non-degradative ubiquitination of SNAREs enhances and/or stabilizes the COPI/Arf/SNARE complex.

I think this is an interesting and timely topic and that the authors have generally provided sufficient experimental support for their claims. I particularly value the clarity of the SILAC data as well as the different ubiquitination/deubiquitination treatments/conditions. However, I think some of the claims could be better supported by having additional experimental data and/or analyses. In particular, the quantification of the fluorescence microscopy images could be improved to better characterize the observed phenotypes. It is not 100% clear to this reviewer whether the observed changes in the morphology of the SNARE-positive structures are a result of a trafficking defect only or that morphological changes of the Golgi cisternae can explain the morphological changes herein reported.

That being said, I think this is a nice piece of work that can potentially be an important contribution to understanding a still obscure cellular process as is the regulation of SNARE incorporation into COPI carriers.

1) Suppl. Data Figure 4: The Aur1 images are of a relatively poor quality (also in S3c). Is there a specific technical reason for that? These data are part of the main ones to support the idea that it is not a change in Golgi morphology but a trafficking defect of the SNAREs that the tubes and rings represent, but I think it will be important to strongly clarify this. Related to this, I have not understood the meaning of the last sentence of this paragraph (line 124-125): what is the evidence that supports the statement that "Bet1 is mislocalizing to a downstream (trans-Golgi) comportment in the COPI mutants tested"? It might be a correct statement, but I missed the evidence to support it, so I would appreciate it if the authors could clarify it.

2) Regarding the quantification of the microscopy images (% tubes and rings), I do appreciate the fact that the authors do it in an unbiased way as possible as detailed in the methods. However, I think this is fundamental for many of the claims in the paper, and it would need to be somehow improved. For instance, the authors present it as a fraction, but also total cell-by-cell distributions of dots, tubes and rings could be informative. Also, I miss details on how the classification was actually done (lines 524-532): how specifically are the different structures identified (software, thresholding, signal to background ratio, etc.), and differentiated (visually? or were the lengths, aspect ratios actually measured?).

3) Line 520: Is PCC the best way to quantify colocalization of these kinds of structures (see e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074624/)?

4) Again, I might have missed it, but e.g. in Figure 4f, how do you know that the released mono-ubiquitin (IB:Ub + condition) comes from Ykt6 totally and not also from other ubiquitinated co-IPed proteins? I understand that the blots on the left show indeed that Ykt is ubiquitinated (loss of 100 kDa band), but is there anything else co-IPed there?

Reviewer #2 (Recommendations for the authors):

In this manuscript the authors investigate the role of ubiquitination in the trafficking of SNARE proteins. They previously reported that trafficking of the SNARE Snc1 requires ubiquitination. In this work, the authors determine that multiple Golgi-localized SNAREs are also polyubiquitinated. They find that the ubiquitin-binding domain of the COPI β' subunit is required for proper morphology of compartments containing these GFP-SNAREs. They conclude that ubiquitination of these SNAREs is important for their trafficking. Additional co-IP experiments indicate that COPI coat components interact with at least one of these SNAREs in a ubiquitin-dependent manner.

Some of the claims and conclusions may be premature and would benefit from additional experiments, as detailed below:

1. The fluorescence morphology assay used to assess whether SNAREs are being properly trafficked appears problematic. There are at least two issues with this approach: First, the assay cannot distinguish between whether the GFP-SNARE has been trafficked to another compartment or whether the compartment itself has changed morphology. This seems problematic as the model is that ubiquitination is important for SNARE trafficking, yet the assay does not directly measure trafficking. Second, as described in the methods, the authors are scoring the fluorescent structures as either "punctate", "tubules", or "rings". Not only does this appear to involve some judgement in assignments to these categories, but more problematically it results in a loss of information regarding potentially important aspects of the morphology, including numbers of structures per cell, average size or intensity of the structures, etc. Therefore, two genotypes could both be declared to have "wt morphology" based on the % structures that are judged to be punctate, even if in one genotype the cells have many more puncta compared to wt, or if some other parameter is quite different but not measured in the quantitation.

2. My interpretation of the imaging data presented in the manuscript is that the COPI β' mutant (delta2-304) appears to be affecting Golgi trafficking and morphology more generally, rather than specific trafficking of Gos1 and Bet1 Golgi SNAREs. The domain removed from the β' mutant harbors the canonical KKXX COPI cargo binding motif which is known to be important for trafficking of KKXX and KXKXX containing cargos. It is therefore not surprising (and perhaps expected) that Golgi trafficking and morphology would be perturbed by this mutant.

The authors provide some control experiments meant to rule out this possibility, but due to concerns detailed in Point 1 above, and examination of the imaging data examples provided, I do not think the authors have ruled out the possibility that the β' mutant is affecting Golgi morphology rather than specific trafficking of Gos1 and Bet1.

3. The use of overexpression and inducible promoters rather than endogenous expression is potentially problematic. The authors see no difference in results when comparing the CUP1 promoter to the ADH1 promoter, but both promoters could result in non-physiological results if the protein levels are significantly higher than endogenous levels.

4. To summarize this point, there are two main issues with this visual assay: The first is that morphology is simply not a good readout of trafficking. The second is that, as implemented, the quantitation method used does not appear to capture all aspects of morphology, because genotypes that look different are yielding similar quantitative measurements. I will explain here a bit more my observations and reasoning:

The phenotype observed is a morphological change of the compartment that the SNARE localizes to. The authors assert that this means the SNARE now localizes to a different compartment because it cannot engage with COPI to be sorted into COPI vesicles, but an alternative explanation is that the COPI β' mutant, which lacks the ability to bind canonical KKXX cargos, changes the morphology of the compartment that the SNARE normally localizes to. This latter possibility seems supported by the data, as the GFP-SNAREs shown in Figure 1b appears to localize to swollen Golgi compartments in the β' mutant. These swollen compartments also appear to be visible (although I assume to a lesser extent) for the other Golgi SNAREs Sed5 and Tlg1, judging by the images shown in Figure 1-supplement 1a.

The authors try to address this possibility by showing data such as Figure 1-supplement 4 in which they claim that Golgi morphology (as assessed by imaging Sed5, Aur1, and Sec7 fusions) is unaffected by the β' mutant but the images in this figure are not clear and the Sed5 morphology in the wild-type cells appears already a bit unusual in this figure.

Examination of related data in the 2017 eLife paper (Figure 4) shows a similar morphological effect in which both the dilysine-binding site mutant and the delta2-304 mutant both result in enlarged Tlg1 compartments.

My concern about the visual phenotype extends to figure 2, because in Figure 2a, GFP-Bet1 appears quite perturbed in the cells with the UBD-Doa1 construct, with an appearance that is not normal for wt cells. In addition, in the 2017 eLife paper Figure 4 appears to suggest that the same UBD-Doa1 construct does not rescue the Tlg1 morphological phenotype, which again argues for a more general perturbation of Golgi trafficking. This suggests that the author's use of "% tubular+ring structures" for quantitation may not be the best way to assess whether these SNAREs are properly trafficked.

Therefore, I do not think the authors have ruled out a simple morphological change resulting from aberrant COPI trafficking.

If the authors think the GFP-SNAREs are trafficked to a different compartment in the β' mutant, one alternative would be to demonstrate this via a clear-cut gain and/or loss of colocalization with established compartment markers. The data shown in Figure 1-supplement 3c,d is not convincing.

If they continue using the morphological assay, and are able to significantly improve the quantitation, then additional Golgi membrane proteins that are not SNARE proteins should also be tested in the various mutant conditions, along with the controls already used, in order to better validate the use of this assay to monitor SNARE trafficking.

5. Assuming the authors can improve their methodology and quantitation for assessing whether SNAREs are mis-trafficked, there are still other issues that need to be addressed, mainly centered around the fact that KKXX COPI cargos are also mis-sorted in the β' delta2-304 mutant:

There appears to be some confusion over whether the β' delta2-304 mutant effects KKXX cargos: On lines 175-177, the authors state: "We previously showed that β'-COP Δ2-304 does not perturb Golgi to ER trafficking of cargoes bearing the KKXX or HDEL motifs (Xu et al., 2017). Thus, it is the ability of the β'-COP N-terminal WDR domain to bind ubiquitin, not dilysine motifs, that is critical for SNARE localization." But I think this statement is incorrect, unless I am confused, because in the 2017 Xu et al. eLife paper, the authors find that the β' mutant does indeed mislocalize the KXKXX-motif containing cargo Emp47, and they state in that paper: "The β'-COP N-terminal di-lysine binding site has a specific role in sorting Emp47 within the Golgi. As previously reported, β'-COP (∆2-304) and the RKR mutant mislocalizes Emp47 to the vacuole where it is degraded (Eugster et al., 2004). Replacement of the N-terminal propeller of β'-COP with the NZFTab1 or UBDDoa1 domains predictably failed to stabilize Myc-Emp47 because these domains lack the di-lysine binding site (Figure 5C)."

Therefore, I suggest these experiments:

Ideally the authors would have a mutant in β' that blocks ubiquitin binding while preserving the KKXX binding site. In the absence of such a mutant, more experiments and controls are needed to convince me that the model is correct. For example:

Does the KK-binding site mutant of β' result in the same trafficking effects on Gos1 and Bet1?

The DUB fusion experiment shown in Figure 3 should be repeated to include additional controls to examine whether other membrane proteins that localize to the same compartment(s) as Bet1 and Gos1 are similarly affected.

Does a mutant of Gos1 that cannot be ubiquitinated (or is fused to a DUB, as was previously done with Snc1 in the 2017 eLife paper) result in the same trafficking defects? [This point arises because the authors state the phenotypes they observe are incomplete because the COPI α subunit also binds Ub. Therefore, a mutant of Gos1 that cannot be ubiquitinated (or is fused to a DUB) would be expected to have stronger sorting phenotypes.]

6. Is there a strong rationale for using the CUP1 promoter instead of native promoters? Even though expression driven by CUP1 is lower than ADH1, it may still be significantly higher than endogenous levels. The authors see no difference in results comparing CUP1 to ADH1 promoters, but both promoters could result in non-physiological results if CUP1 expression is significantly higher than the endogenous levels. Perhaps expression under CUP1 promoter could be compared by western blot to expression by endogenous promoter for the key proteins (Gos1 and Bet1). There also appears to be some cell-to-cell variation arising from varying plasmid copy numbers (and therefore varying expression levels) in cells.

7. Although this may be a relatively minor point, I think it is puzzling that the authors think that Bet1 is localized to the medial/late-Golgi, when it is an established ER-Golgi SNARE (it is the v-SNARE of the Sec22/Bos1/Sed5/Bet1 SNAREpin). There are two potential issues with the experiment they used to determine this (Figure 1-supplement 3a): the first issue is how do the authors know that Aur1 is medial/late? The second issue is that the appearance of Bet1 in the middle row (when co-expressed with Aur1) is quite different from its appearance when co-expressed with Sed5. Furthermore, the bottom row shows a mixture of cells in which some cells express a low level of Bet1 and some cells express a higher level of Bet1. There appears to be some colocalization with Sec7 when Bet1 is expressed at a higher level but not when Bet1 is expressed at a lower level. This highlights potential problems with analyzing proteins expressed on plasmids, as some cells harbor more copies of the plasmid. If the authors wish to convincingly demonstrate the localization of Bet1, the gold-standard is to do time-lapse analysis and use endogenous expression. Although this doesn't seem too relevant to the overall point of the paper, it is nonetheless a poorly supported conclusion.

Reviewer #3 (Recommendations for the authors):

In this manuscript Date and colleagues expand on their previous publication (Xu et al., 2017) and show that a sub-set of Golgi SNAREs are mis-localised in a yeast mutant lacking the Β'-COP N-terminal WDR domain. Fusion of a deubiquitinase (DUB) domain to COPI leads to the same mis-localisation phenotype, strengthening their hypothesis. By immunoprecipitating FLAG-tagged SNAREs followed by DUB treatment they are able to show that, while Bet1 does not appear to be ubiquitinated, Gos1 and Snc1 are. SILAC analysis of the Gos1 IP sample shows a stable complex with its partner SNAREs Ykt6 and Sed5 which are also ubiquitinated. By performing IP assays under conditions in which endogenous ubiquitination is preserved or ubiquitin removal is catalyzed, the authors show strong enrichment of Arf and COP1 under ubiquitination conditions. Interestingly this is true also when performing the same experiments with Bet1 which is not itself ubiquitinated. The Arf GAP Glo3 is however not enriched in these SNARE-COPI-Arf complexes and SNAREs localize correctly in a Glo3 mutant. Based on their data, the authors propose a model in which ubiquitination stabilizes an Arf-SNARE-COP complex which does not contain a GAP.

The experiments are generally well executed, however what in my opinion is lacking is a characterization of the aberrant compartments in which the SNAREs are mis-localized in the BetaCOP mutant. This would greatly help to understand the mechanism. If SNAREs are not recycled back via COPI do they leak to downstream compartments? The authors say that Bet1 is mis-localizing to a downstream (trans-Golgi) compartment (lines 126-127), however, Figure 1 figure supplement 3c-d show a decreased co-localisation with the late Golgi marker Aur1. Is it possible Bet1 is in the TGN? Co-localization with Sec7 is not tested. Snc1 is an exocytic SNARE however it mis-localises to the same aberrant compartment as the other Golgi SNAREs?

https://doi.org/10.7554/eLife.80911.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

In our opinion, the main points we would like to see so we are convinced about the claims of this paper are:

1) Provide more convincing evidence for a trafficking defect, not just a morphology change. As an example of what can be done (see detailed report below for more details) is to use co-localization as a readout of trafficking. For example, use Mnn9-mCherry or a number of other established Golgi proteins that can be successfully tagged with RFP.

Our experiments show that a subset of SNAREs localize to tube-like and ring-like structures when the ubiquitin-binding domain in β’COP is deleted (β’COP Δ2-304 cells). To further characterize these aberrant structures, we performed colocalization analysis of mNG-Gos1 with mKate tagged early Golgi markers Anp1 and Mnn9, late Golgi markers Sec7 and Chs5 as well as with FM4-64 to mark endosomes. Our extensive colocalization experiments show significant loss of colocalization of mNG-tagged Gos1 with both early-Golgi as well as both late-Golgi markers in β’COP Δ2-304 cells compared to wild-type (WT) control (Figure 2, Figure 2 figure supplements 1). We showed in the original submission a reduction in Bet1 co-localization with the Golgi marker Aur1. None of the Golgi markers tested displayed the same morphological phenotype observed with Bet1, Gos1 or Snc2. Altogether these data indicate a significant loss of SNAREs from Golgi compartments in β’COP Δ2-304. We further tested if Gos1 was being mislocalized to endosomes by labeling cells with FM4-64 for a short period of time to stain endosomes (Figure 2, Figure 2 figure supplements 4). While some of the tubular structures stained weakly with FM4-64, no significant increase in colocalization of mNG-Gos1 or mNG-Bet1 with FM4-64 was observed in β’COP Δ2-304 relative to WT cells; therefore, these Golgi SNAREs are not being mislocalized to the endosomal system. Our conclusion for these studies as stated on page 8 is that “We suspect that a failure to retrieve these SNAREs during maturation of the Golgi cisternae, combined with a normal retrieval of most resident Golgi proteins, results in Gos1 and Bet1 accumulating in dead-end compartments that we describe here as sequestration compartments.” Additional figures with these colocalization studies are now in a new Figure 2 and Figure 2 —figure supplement 1 and 4 and described on page 7 and 8.

2) Test if the COPI b' mutant (∆2-304) has a (more) general effect on Golgi trafficking and morphology as compared to what they report here. In particular, comparisons to the KK-binding site β'-COP mutant will be an important control to include for most of the experiments, as this is the canonical COPI binding site and is lacking in the β'-COP (∆2-304) mutant.

To further address this comment, we compared the localization of mNG-Gos1 and mNG-Bet1 in the β’COP-RKR mutant (which is not capable of binding dilysine-motif cargo) with WT cells and with β’COP Δ2-304 cells. No observable difference in the appearance and localization of mNG-Bet1 and mNG-Gos1 was observed in β’COP-RKR mutant compared to WT cells. This result is now shown in (Figure 2 figure supplement 3b).

Data from our previous work and current efforts indicate that there is no general effect on Golgi morphology or overall vesicle-trafficking events in β'-COP (∆2-304) as supported by the following observations:

2.1: Localization of Gos1, Bet1, Snc1 and Snc2 is restored to the WT pattern when the terminal βpropeller domain of β’COP is replaced with ubiquitin-binding domain of Doa1 (Figure 3a-3f). While Doa1UBD can bind ubiquitin, it is not capable of binding dilysine cargo (Xu et al. 2017). If the mislocalization of SNARE was the result of impaired ability of β’COP to bind dilysine cargo, restoration to WT phenotype in Doa1-UBD fusion constructs would not have been possible. We previously demonstrated that the Doa1-UBD fusion failed to rescue the Emp47 trafficking defect exhibited by ∆2-304 (Xu et al. 2017). Thus, di-lysine binding and ubiquitin-binding are separable functions of the COPI β’ subunit. Emp47 is the only cargo we are aware of that has been shown to be mislocalized in the β’COP RKR mutant. Most dilysine cargoes are handled efficiently by alphaCOP.

2.2: We now show that the localization and appearance of several Golgi markers including Anp1, Mnn9, Aur1, Sec7, Chs5 and Sed5 are not different in β’COP Δ2-304 cells compared to WT cells (Figure 2 and its supplements). We previously showed that Rer1 trafficking, a sensitive indicator for overall loss of COPI function, was unaffected in β’COP Δ2-304 cells. These data indicate that there is no general defect in Golgi morphology or COPI-dependent trafficking.

2.3: The localization and appearance of several ER, Golgi, and endosomal SNAREs are not affected in β’COP Δ2-304 cells compared to WT cells (Figure 1 figure supplement 1), indicating this phenomenon is specific to a subset of SNAREs and not a generalized trafficking defect.

2.4: In data not presented here, we initially looked at the trafficking of several different GFP-tagged proteins (Mup1, Tat2, Pma1, Ena1, Can1, Ina1, Pdr5) known or suspected to recycle between the plasma membrane, endosomes, Golgi and back to the plasma membrane because we hypothesized that the COPI-Ub interaction was specifically driving this pathway (similarly to Snc1). However, none of these proteins were mislocalized in β’COP Δ2-304 cells.

3) Improve on the image quantitation to better understand the nature of the aberrant compartment where SNAREs relocalize.

We have now included additional figures with colocalization analysis of mNG-SNAREs with Golgi markers and endosomes. We have use Mander’s co-efficients to quantify the degree of co-localization for the new co-localization data. We have also provided detailed scoring and quantitative analyses for (a) the number of fluorescent mNG-Gos1 and mNG-Bet1 structures in WT and in β’COP Δ2-304 cells and (b) the average size of these structures as requested by reviewer 1 (Figure 2 and its supplements).

There were also other important questions raised by the reviewers (see below) that would require some clarification/explanation (e.g. difference between o/expression end. expression levels; better Bet1 localization together with other Golgi/TGN markers, etc.).

Please see point-by-point responses below.

Reviewer #1 (Recommendations for the authors):

Date et al. present a thorough study of the effects and roles of protein ubiquitination in the association of different COPI subunits and COPI-associated factors with SNARE proteins in the yeast S. cerevisiae. In a previous work from this lab (Xu et al. eLife 2017), it was shown that the SNARE Snc1 is mislocalized when COPI is not able to associate with the ubiquitination in Snc1. Here, Date et al. expand on those observations and describe that (i) COPI components, Glo3 (ArfGAP), and some but not all tested Golgi SNARES are ubiquitinated; (ii) the localization of some of these SNARES is dependent on the binding between COPI and the ubiquitinated cargoes (SNAREs) ; and (iii) they propose a model in which non-degradative ubiquitination of SNAREs enhances and/or stabilizes the COPI/Arf/SNARE complex.

I think this is an interesting and timely topic and that the authors have generally provided sufficient experimental support for their claims. I particularly value the clarity of the SILAC data as well as the different ubiquitination/deubiquitination treatments/conditions. However, I think some of the claims could be better supported by having additional experimental data and/or analyses. In particular, the quantification of the fluorescence microscopy images could be improved to better characterize the observed phenotypes. It is not 100% clear to this reviewer whether the observed changes in the morphology of the SNARE-positive structures are a result of a trafficking defect only or that morphological changes of the Golgi cisternae can explain the morphological changes herein reported.

That being said, I think this is a nice piece of work that can potentially be an important contribution to understanding a still obscure cellular process as is the regulation of SNARE incorporation into COPI carriers.

We thank this reviewer for the supportive comments and we have worked to better address the trafficking and morphological phenotypes as describe above.

1) Suppl. Data Figure 4: The Aur1 images are of a relatively poor quality (also in S3c). Is there a specific technical reason for that? These data are part of the main ones to support the idea that it is not a change in Golgi morphology but a trafficking defect of the SNAREs that the tubes and rings represent, but I think it will be important to strongly clarify this. Related to this, I have not understood the meaning of the last sentence of this paragraph (line 124-125): what is the evidence that supports the statement that "Bet1 is mislocalizing to a downstream (trans-Golgi) comportment in the COPI mutants tested"? It might be a correct statement, but I missed the evidence to support it, so I would appreciate it if the authors could clarify it.

We have now performed SNARE colocalization analysis with several additional Golgi markers (two early Golgi and two late Golgi markers) that are mKate-tagged and give a better observable fluorescence pattern. We have also analyzed cells pulsed with FM4-64 to label the endosomes and find that Gos1 or Bet1 are not substantially mislocalized to endosomes. The new analyses indicate that in β’COP Δ2-304 cells there is a significant loss of SNAREs from early, medial, and late Golgi compartments to what we are now describing as sequestration compartments (Figure 2 and its supplements). We, at this point, have not identified another non-SNARE marker protein that co-labels this compartment. However, the colocalization analyses, complementation analyses with general ubiquitin-binding domains, and comparisons of Golgi morphology in WT vs β’COP Δ2-304 show that SNAREs are lost from Golgi compartments without affecting Golgi morphology or general trafficking. The text has been revised to clarify the nature of the compartment we are observing as described above for the editor’s summary.

2) Regarding the quantification of the microscopy images (% tubes and rings), I do appreciate the fact that the authors do it in an unbiased way as possible as detailed in the methods. However, I think this is fundamental for many of the claims in the paper, and it would need to be somehow improved. For instance, the authors present it as a fraction, but also total cell-by-cell distributions of dots, tubes and rings could be informative. Also, I miss details on how the classification was actually done (lines 524-532): how specifically are the different structures identified (software, thresholding, signal to background ratio, etc.), and differentiated (visually? or were the lengths, aspect ratios actually measured?).

As outlined above, colocalization studies with 4 mKate-tagged Golgi markers and FM4-64 marked early endosomes were performed to provide unbiased, software-based readouts for changes in colocalization using Mander’s coefficients on thresholded images. The rings and tubes were visually scored and binned in a blinded fashion for most of the analyses. We have now included detailed information about scoring of these structures and additional plots as requested in the new Figure 2 and its supplementary figures. We find no significant change in the total number of fluorescent structures for Bet1 and Gos1, but do observe a significant change in the distribution of rings/tubes versus punctae between WT vs β’COP Δ2-304.

3) Line 520: Is PCC the best way to quantify colocalization of these kinds of structures (see e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074624/)?

This is a good point and we have quantified all of the new data using Mander’s coefficients.

4) Again, I might have missed it, but e.g. in Figure 4f, how do you know that the released mono-ubiquitin (IB:Ub + condition) comes from Ykt6 totally and not also from other ubiquitinated co-IPed proteins? I understand that the blots on the left show indeed that Ykt is ubiquitinated (loss of 100 kDa band), but is there anything else co-IPed there?

We agree with the reviewer’s suggestion that there is a possibility that the ubiquitin we are detecting may also come from other proteins co-IPed with Ykt6. We have carefully used the language that ubiquitination is associated with SNAREs and/or IPed complexes to try and avoid the possible misinterpretation that ubiquitin is solely linked with the protein being IPed. With that being noted, the smeared patterns detected on the western blots (or average mass) associated with ubiquitination is different when different SNAREs and COPI components are IPed and probed for ubiquitin. If we were observing the same complex over and over again with different components being pulled down, we anticipate the smeared pattern would be identical. These differences in the ubiquitin-associated migration pattern and different extent of ubiquitin being released suggests that the bait protein is the primary contributor for the associated ubiquitination. In support of this contention, COPI co-IPs with Bet1 and yet we cannot detect the presence of ubiquitin associated with this complex above background, even though we know many COPI subunits are ubiquitinated.

Reviewer #2 (Recommendations for the authors):

In this manuscript the authors investigate the role of ubiquitination in the trafficking of SNARE proteins. They previously reported that trafficking of the SNARE Snc1 requires ubiquitination. In this work, the authors determine that multiple Golgi-localized SNAREs are also polyubiquitinated. They find that the ubiquitin-binding domain of the COPI β' subunit is required for proper morphology of compartments containing these GFP-SNAREs. They conclude that ubiquitination of these SNAREs is important for their trafficking. Additional co-IP experiments indicate that COPI coat components interact with at least one of these SNAREs in a ubiquitin-dependent manner.

Some of the claims and conclusions may be premature and would benefit from additional experiments, as detailed below:

1. The fluorescence morphology assay used to assess whether SNAREs are being properly trafficked appears problematic. There are at least two issues with this approach: First, the assay cannot distinguish between whether the GFP-SNARE has been trafficked to another compartment or whether the compartment itself has changed morphology. This seems problematic as the model is that ubiquitination is important for SNARE trafficking, yet the assay does not directly measure trafficking. Second, as described in the methods, the authors are scoring the fluorescent structures as either "punctate", "tubules", or "rings". Not only does this appear to involve some judgement in assignments to these categories, but more problematically it results in a loss of information regarding potentially important aspects of the morphology, including numbers of structures per cell, average size or intensity of the structures, etc. Therefore, two genotypes could both be declared to have "wt morphology" based on the % structures that are judged to be punctate, even if in one genotype the cells have many more puncta compared to wt, or if some other parameter is quite different but not measured in the quantitation.

Please see responses above. We have now addressed these concerns with additional experiments presented as the new Figure 2 and associated supplemental figures.

2. My interpretation of the imaging data presented in the manuscript is that the COPI β' mutant (delta2-304) appears to be affecting Golgi trafficking and morphology more generally, rather than specific trafficking of Gos1 and Bet1 Golgi SNAREs. The domain removed from the β' mutant harbors the canonical KKXX COPI cargo binding motif which is known to be important for trafficking of KKXX and KXKXX containing cargos. It is therefore not surprising (and perhaps expected) that Golgi trafficking and morphology would be perturbed by this mutant.

Please see the response to the editor. We have revised the manuscript in several places to better explain how published data and our current results make it highly unlikely that our results are a secondary effect of perturbing dilysine trafficking.

The authors provide some control experiments meant to rule out this possibility, but due to concerns detailed in Point 1 above, and examination of the imaging data examples provided, I do not think the authors have ruled out the possibility that the β' mutant is affecting Golgi morphology rather than specific trafficking of Gos1 and Bet1.

Please see responses above (comment 1 and 2). We have now imaged a large number of Golgi markers and quantified the number and size of the Golgi compartment marked by Anp1, Mnn9, Chs5 and Sec7 (new Figure 2). Gos1 and Bet1 quantifiably colocalize with Golgi markers in WT cells but this colocalization is significantly lost in the COPI mutant. We hoped to find a Golgi or endosomal marker that labeled the abnormal structures but this is not what we have found. However, we feel that the improved analysis and quantitation makes a strong case that Bet1 and Gos1 are mislocalized from the compartments where they normally reside.

3. The use of overexpression and inducible promoters rather than endogenous expression is potentially problematic. The authors see no difference in results when comparing the CUP1 promoter to the ADH1 promoter, but both promoters could result in non-physiological results if the protein levels are significantly higher than endogenous levels.

Please see response above to reviewer 1.

4. To summarize this point, there are two main issues with this visual assay: The first is that morphology is simply not a good readout of trafficking. The second is that, as implemented, the quantitation method used does not appear to capture all aspects of morphology, because genotypes that look different are yielding similar quantitative measurements. I will explain here a bit more my observations and reasoning:

The phenotype observed is a morphological change of the compartment that the SNARE localizes to. The authors assert that this means the SNARE now localizes to a different compartment because it cannot engage with COPI to be sorted into COPI vesicles, but an alternative explanation is that the COPI β' mutant, which lacks the ability to bind canonical KKXX cargos, changes the morphology of the compartment that the SNARE normally localizes to. This latter possibility seems supported by the data, as the GFP-SNAREs shown in Figure 1b appears to localize to swollen Golgi compartments in the β' mutant. These swollen compartments also appear to be visible (although I assume to a lesser extent) for the other Golgi SNAREs Sed5 and Tlg1, judging by the images shown in Figure 1-supplement 1a.

We’ve looked through several Sed5 images, including the Figure 1 supplement 1 images and do not see any difference between WT and mutant. We thank the reviewer for correctly pointing out that Tlg1 is displaying tubular structures and we inadvertently failed to mention this in the initial submission. We assumed these were the same structures that also label with Snc1 based on our earlier studies and decided to focus on other Golgi SNAREs for this paper. We have revised the text to indicate we also observe abnormal structures with Tlg1.

The authors try to address this possibility by showing data such as Figure 1-supplement 4 in which they claim that Golgi morphology (as assessed by imaging Sed5, Aur1, and Sec7 fusions) is unaffected by the β' mutant but the images in this figure are not clear and the Sed5 morphology in the wild-type cells appears already a bit unusual in this figure.

See comments above and the new Figure 2. We haven’t performed a lot of imaging experiments with Sed5 but this mScarlet-Sed5 morphology did not strike us as being unusual. The mNG-Sed5 images in Figure 1 supplement 1 showed a larger number of spots per cell than mScarlet-Sed5, which we assume may be because of the brighter fluorescence of mNG.

Author response image 1

Examination of related data in the 2017 eLife paper (Figure 4) shows a similar morphological effect in which both the dilysine-binding site mutant and the delta2-304 mutant both result in enlarged Tlg1 compartments.

This is a good point and we have revised the manuscript accordingly.

My concern about the visual phenotype extends to figure 2, because in Figure 2a, GFP-Bet1 appears quite perturbed in the cells with the UBD-Doa1 construct, with an appearance that is not normal for wt cells. In addition, in the 2017 eLife paper Figure 4 appears to suggest that the same UBD-Doa1 construct does not rescue the Tlg1 morphological phenotype, which again argues for a more general perturbation of Golgi trafficking. This suggests that the author's use of "% tubular+ring structures" for quantitation may not be the best way to assess whether these SNAREs are properly trafficked.

The morphological influence of the UBD-Doa1 construct on Tlg1 morphology has not been quantified in the 2017 eLife paper or the current work. Extensive quantitation across multiple biological replicates would be needed to draw conclusions about Tlg1 and we feel that initiating a careful study of Tlg1 is beyond the scope of the current work. Please see above for the detailed discussion regarding Golgi morphology.

Therefore, I do not think the authors have ruled out a simple morphological change resulting from aberrant COPI trafficking.

If the authors think the GFP-SNAREs are trafficked to a different compartment in the β' mutant, one alternative would be to demonstrate this via a clear-cut gain and/or loss of colocalization with established compartment markers. The data shown in Figure 1-supplement 3c,d is not convincing.

We have now provided a clear-cut loss of colocalization with 4 Golgi markers.

If they continue using the morphological assay, and are able to significantly improve the quantitation, then additional Golgi membrane proteins that are not SNARE proteins should also be tested in the various mutant conditions, along with the controls already used, in order to better validate the use of this assay to monitor SNARE trafficking.

This has now been done using Golgi glycosyltransferases and Sec7 as markers.

5. Assuming the authors can improve their methodology and quantitation for assessing whether SNAREs are mis-trafficked, there are still other issues that need to be addressed, mainly centered around the fact that KKXX COPI cargos are also mis-sorted in the β' delta2-304 mutant:

Based on our careful analysis of literature and our own experiments, we have not identified any dilysine cargo other than Emp47 that is mislocalized by any mutations to β’ COP that renders it incapable of binding dilysine cargo. If we have missed any published work, please provide a reference. There seems to be a common view in the trafficking field that sorting of dilysine cargos is the essential function of COPI and this emphasizes why the current study is important. Here is a quote from Jackson et al. 2012 (PMID 23177648).

“Finally, we generated a yeast strain in which both the α- and β'-COP N-terminal WD-repeat domains had lost the ability to bind the carboxy terminus of dilysine motifs (sec27::URA3 R15A K17A R59A ret1::TRP1 R13A K15S R57S). Although viable at 37°C, the ability of this mutant to traffic both KKxx and KxKxx reporter constructs was severely impaired (Figure S4E). The inability to support retrograde dilysine-based transport causes a slight growth defect at 37°C, resulting in smaller colonies as compared to wild-type, but this mutant is not lethal.”

There appears to be some confusion over whether the β' delta2-304 mutant effects KKXX cargos: On lines 175-177, the authors state: "We previously showed that β'-COP Δ2-304 does not perturb Golgi to ER trafficking of cargoes bearing the KKXX or HDEL motifs (Xu et al., 2017). Thus, it is the ability of the β'-COP N-terminal WDR domain to bind ubiquitin, not dilysine motifs, that is critical for SNARE localization." But I think this statement is incorrect, unless I am confused, because in the 2017 Xu et al. eLife paper, the authors find that the β' mutant does indeed mislocalize the KXKXX-motif containing cargo Emp47, and they state in that paper: "The β'-COP N-terminal di-lysine binding site has a specific role in sorting Emp47 within the Golgi. As previously reported, β'-COP (∆2-304) and the RKR mutant mislocalizes Emp47 to the vacuole where it is degraded (Eugster et al., 2004). Replacement of the N-terminal propeller of β'-COP with the NZFTab1 or UBDDoa1 domains predictably failed to stabilize Myc-Emp47 because these domains lack the di-lysine binding site (Figure 5C)."

The reviewer is correct that the second sentence should not have started with “Thus” because that conclusion is based on more data than the preceding sentence spells out. Emp47 has a variant dilysine motif that depends on its interaction with β'-COP and is the only dilysine cargo we are aware of that exclusively relies on β’COP. Emp47 is mislocalized to vacuole in β'-COP ∆2-304 as well as in the β'-COP RKR mutant. In case of SNAREs, they do not have a dilysine binding motif and SNARE mislocalization is restored when terminal propeller of β'-COP is replaced with an unrelated ubiquitin-binding domain UBDDoa1 (which can bind ubiquitin but not dilysine cargo). This supports our observation that SNARE interaction with COPI is not dilysine motif dependent but it is dependent on the ability of β'-COP to bind ubiquitin. We have modified this sentence to improve accuracy.

Therefore, I suggest these experiments:

Ideally the authors would have a mutant in β' that blocks ubiquitin binding while preserving the KKXX binding site. In the absence of such a mutant, more experiments and controls are needed to convince me that the model is correct. For example:

Does the KK-binding site mutant of β' result in the same trafficking effects on Gos1 and Bet1?

Additional experiments were done to analyze the appearance and localization of mNG-Bet1 and mNGGos1 in β'-COP RKR mutant (see above). No mislocalization or change of morphology for mNG-Gos1 or mNG-Bet1 was observed in β'-COP RKR mutant.

The DUB fusion experiment shown in Figure 3 should be repeated to include additional controls to examine whether other membrane proteins that localize to the same compartment(s) as Bet1 and Gos1 are similarly affected.

The original COPI-DUB fusions have a relatively weak phenotype. We now have a better collection of DUB fusions to other COPI subunits that are providing fascinating new data (we believe we can now interfere with α-COPI binding to ubiquitin). We are performing several studies with multiple cargoes and controls to provide a full characterization of these new COPI-DUB fusion constructs. We hope the reviewer will give us the latitude to report these additional experiments in the next manuscript on this topic. We feel these COPI-DUB fusion studies are moving beyond the scope of the current work and that we have adequately addressed the primary concerns related to compartment morphologies and marker localizations with the new data we have included (please see above).

Does a mutant of Gos1 that cannot be ubiquitinated (or is fused to a DUB, as was previously done with Snc1 in the 2017 eLife paper) result in the same trafficking defects? [This point arises because the authors state the phenotypes they observe are incomplete because the COPI α subunit also binds Ub. Therefore, a mutant of Gos1 that cannot be ubiquitinated (or is fused to a DUB) would be expected to have stronger sorting phenotypes.]

In many cases, ubiquitination is promiscuous, meaning if the primary site of ubiquitination is mutated another Lys within the same substrate is often ubiquitinated. Additionally, there are several lysines within Gos1 and mutating all of these lysines could lead to non-ubiquitin-dependent but Lys-dependent phenotypes. Further, mNG has several lysine residues, which could potentially be ubiquitinated in the context of the fusion. Due to these reasons the Lys mutagenesis studies have not been employed. Two people in our lab tried to fuse DUB to mNG-Gos1, but the construct seems to be toxic to E. coli because all of the clones isolated carried deletions or mutations.

6. Is there a strong rationale for using the CUP1 promoter instead of native promoters? Even though expression driven by CUP1 is lower than ADH1, it may still be significantly higher than endogenous levels. The authors see no difference in results comparing CUP1 to ADH1 promoters, but both promoters could result in non-physiological results if CUP1 expression is significantly higher than the endogenous levels. Perhaps expression under CUP1 promoter could be compared by western blot to expression by endogenous promoter for the key proteins (Gos1 and Bet1). There also appears to be some cell-to-cell variation arising from varying plasmid copy numbers (and therefore varying expression levels) in cells.

We compared expression of Snc1 and Bet1 from the CUP1 and ADH promoters by western blot and correlated this to the endogenous expression of ADH and yeast SNAREs (from the SGD page – average values from several studies). We see more than a 100-fold reduction in SNARE expression from the CUP1 promoter under the conditions we use to induce expression, which puts this in the range of normal expression for the yeast SNAREs. These data were published as supplemental figure 3 in Best et al. (2020) and this was done concurrently with many of the imaging studies for the submitted manuscript. This was described in the submitted manuscript but we have added another sentence in the Results section to better communicate this result.

Author response image 2

7. Although this may be a relatively minor point, I think it is puzzling that the authors think that Bet1 is localized to the medial/late-Golgi, when it is an established ER-Golgi SNARE (it is the v-SNARE of the Sec22/Bos1/Sed5/Bet1 SNAREpin). There are two potential issues with the experiment they used to determine this (Figure 1-supplement 3a): the first issue is how do the authors know that Aur1 is medial/late? The second issue is that the appearance of Bet1 in the middle row (when co-expressed with Aur1) is quite different from its appearance when co-expressed with Sed5. Furthermore, the bottom row shows a mixture of cells in which some cells express a low level of Bet1 and some cells express a higher level of Bet1. There appears to be some colocalization with Sec7 when Bet1 is expressed at a higher level but not when Bet1 is expressed at a lower level. This highlights potential problems with analyzing proteins expressed on plasmids, as some cells harbor more copies of the plasmid. If the authors wish to convincingly demonstrate the localization of Bet1, the gold-standard is to do time-lapse analysis and use endogenous expression. Although this doesn't seem too relevant to the overall point of the paper, it is nonetheless a poorly supported conclusion.

It is true that Bet1 is an established ER/Golgi SNAREpin and it is puzzling that Bet1 localizes to the Golgi and does not appear to recycle back to the ER. Dieter Schmitt’s lab explored the steady-state distribution of Bet1 using a C-terminal tag and found that it localized to the Golgi by immunofluorescence and subcellular fractionation (Ossipov et al. 1999). The tag contained an alphafactor fragment that was cleaved rapidly by Kex2 after the fusion protein was synthesized, indicating that Bet1 moves rapidly to the TGN. They also saw that Bet1 remained in the Golgi in a sec23 (COPII) ts mutant at the nonpermissive temperature. We thought this was a surprising result but we repeated their experiments using the mNG-N-terminal tag and obtained the same results. In addition to Aur1, we have now co-localized mNG-Bet1 with several more Golgi markers and find it more enriched with lateGolgi markers than early Golgi markers in WT cells. To account for potential cell-cell variability in the plasmid-driven expression, we have ensured to include a large number of cells across biological replicates in our analyses. The same modest variation in expression is observed in WT and mutant cells and so this shouldn’t impact the overall conclusion. The primary point of these studies is not to define the precise Golgi compartment where Bet1 localizes or its dynamic retrieval in WT cells and we feel the time-lapse studies are beyond the scope of the current study. In addition, we deleted a paragraph in the Discussion on the implications of Bet1 trafficking on its SNARE function at the ER/Golgi interface. We will incorporate these ideas into a review article where they can be developed more completely.

Reviewer #3 (Recommendations for the authors):

In this manuscript Date and colleagues expand on their previous publication (Xu et al., 2017) and show that a sub-set of Golgi SNAREs are mis-localised in a yeast mutant lacking the Β'-COP N-terminal WDR domain. Fusion of a deubiquitinase (DUB) domain to COPI leads to the same mis-localisation phenotype, strengthening their hypothesis. By immunoprecipitating FLAG-tagged SNAREs followed by DUB treatment they are able to show that, while Bet1 does not appear to be ubiquitinated, Gos1 and Snc1 are. SILAC analysis of the Gos1 IP sample shows a stable complex with its partner SNAREs Ykt6 and Sed5 which are also ubiquitinated. By performing IP assays under conditions in which endogenous ubiquitination is preserved or ubiquitin removal is catalyzed, the authors show strong enrichment of Arf and COP1 under ubiquitination conditions. Interestingly this is true also when performing the same experiments with Bet1 which is not itself ubiquitinated. The Arf GAP Glo3 is however not enriched in these SNARE-COPI-Arf complexes and SNAREs localize correctly in a Glo3 mutant. Based on their data, the authors propose a model in which ubiquitination stabilizes an Arf-SNARE-COP complex which does not contain a GAP.

The experiments are generally well executed, however what in my opinion is lacking is a characterization of the aberrant compartments in which the SNAREs are mis-localized in the BetaCOP mutant. This would greatly help to understand the mechanism. If SNAREs are not recycled back via COPI do they leak to downstream compartments? The authors say that Bet1 is mis-localizing to a downstream (trans-Golgi) compartment (lines 126-127), however, Figure 1 figure supplement 3c-d show a decreased co-localisation with the late Golgi marker Aur1. Is it possible Bet1 is in the TGN? Co-localization with Sec7 is not tested. Snc1 is an exocytic SNARE however it mis-localises to the same aberrant compartment as the other Golgi SNAREs?

Please see the comments above and the new Figure 2 along with its supplemental figures.

https://doi.org/10.7554/eLife.80911.sa2

Article and author information

Author details

  1. Swapneeta S Date

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4086-110X
  2. Peng Xu

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7103-3692
  3. Nathaniel L Hepowit

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Contribution
    Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Nicholas S Diab

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Jordan Best

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Boyang Xie

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2829-9254
  7. Jiale Du

    Department of Chemistry, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Eric R Strieter

    Department of Chemistry, University of Massachusetts Amherst, Amherst, United States
    Contribution
    Supervision, Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3447-3669
  9. Lauren P Jackson

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Resources, Supervision, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3705-6126
  10. Jason A MacGurn

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Resources, Supervision, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5063-259X
  11. Todd R Graham

    Department of Biological Sciences, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    tr.graham@vanderbilt.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3256-2126

Funding

National Institutes of Health (R35GM144123-01)

  • Todd R Graham

National Institutes of Health (1R35GM119525)

  • Lauren P Jackson

National Institutes of Health (R35GM144112)

  • Jason A MacGurn

Pew Charitable Trusts

  • Lauren P Jackson

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Scott Emr (Cornell University) and Dr. Aki Nakano (Riken Institute) for plasmids and strains. We thank Charles Barlow for the anti-COPI antibody. We thank Kristie Lindsey Rose (Vanderbilt University, Proteomics Core Laboratory) for help with processing mass spectrometry samples. Funding sources: These studies were supported by NIH Grants R35GM144123-01 (to TRG), 1R35GM119525 (to LPJ), and R35GM144112 (to JAM). Lauren P Jackson is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trusts.

Senior Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Reviewing Editor

  1. Felix Campelo, Institute of Photonic Sciences, Spain

Reviewer

  1. Felix Campelo, Institute of Photonic Sciences, Spain

Publication history

  1. Preprint posted: November 29, 2021 (view preprint)
  2. Received: June 9, 2022
  3. Accepted: July 1, 2022
  4. Accepted Manuscript published: July 29, 2022 (version 1)
  5. Version of Record published: August 12, 2022 (version 2)

Copyright

© 2022, Date et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Swapneeta S Date
  2. Peng Xu
  3. Nathaniel L Hepowit
  4. Nicholas S Diab
  5. Jordan Best
  6. Boyang Xie
  7. Jiale Du
  8. Eric R Strieter
  9. Lauren P Jackson
  10. Jason A MacGurn
  11. Todd R Graham
(2022)
Ubiquitination drives COPI priming and Golgi SNARE localization
eLife 11:e80911.
https://doi.org/10.7554/eLife.80911
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    Jie Li, Jiayi Wu ... Eunhee Choi
    Research Article

    The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.