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Golgi membrane protein Erd1 Is essential for recycling a subset of Golgi glycosyltransferases

  1. Richa Sardana  Is a corresponding author
  2. Carolyn M Highland
  3. Beth E Straight
  4. Christopher F Chavez
  5. J Christopher Fromme
  6. Scott D Emr  Is a corresponding author
  1. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, United States
  2. Department of Molecular Medicine, Cornell University, United States
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Cite this article as: eLife 2021;10:e70774 doi: 10.7554/eLife.70774

Abstract

Protein glycosylation in the Golgi is a sequential process that requires proper distribution of transmembrane glycosyltransferase enzymes in the appropriate Golgi compartments. Some of the cytosolic machinery required for the steady-state localization of some Golgi enzymes are known but existing models do not explain how many of these enzymes are localized. Here, we uncover the role of an integral membrane protein in yeast, Erd1, as a key facilitator of Golgi glycosyltransferase recycling by directly interacting with both the Golgi enzymes and the cytosolic receptor, Vps74. Loss of Erd1 function results in mislocalization of Golgi enzymes to the vacuole/lysosome. We present evidence that Erd1 forms an integral part of the recycling machinery and ensures productive recycling of several early Golgi enzymes. Our work provides new insights on how the localization of Golgi glycosyltransferases is spatially and temporally regulated, and is finely tuned to the cues of Golgi maturation.

Editor's evaluation

The authors took the critiques seriously and responded with substantial changes and enhancements. While we are still not fully convinced by the interpretations, the authors have done a thorough, rigorous piece of work that makes a valuable contribution about a topic of broad interest.

https://doi.org/10.7554/eLife.70774.sa0

Introduction

Glycosylation is the most abundant and diverse post-translational protein modification and plays a critical role in modulating protein interactions, stability, and physiological functions Stanley, 2011; Schjoldager et al., 2020. The importance of proper glycosylation is highlighted by the multi-system failures, immune dysfunction, and many inherited disorders of glycosylation arising due to the loss of activity or localization of Golgi enzymes Reily et al., 2019 ; Ng and Freeze, 2018; Rodrigues et al., 2018. The complex structure of glycosylation modifications is generated by the sequential action of specific sets of membrane-embedded glycosyltransferases localized to distinct Golgi cisternae Schjoldager et al., 2020; Colley, 1997; Orlean, 2012. Maintenance of these enzymes in the appropriate Golgi compartments therefore allows accurate protein glycosylation Moremen et al., 2012.

All known Golgi glycosyltransferases are type II integral membrane proteins with three key features – a short amino-terminal cytosolic tail, a single transmembrane domain (TMD), and a lumenal region containing the catalytic domain Welch and Munro, 2019; Banfield, 2011; Tu and Banfield, 2010. Previous work from multiple groups have reported the individual contributions of cytosolic, transmembrane, and lumenal domains of glycosyltransferases in their localization Burke et al., 1994; Colley, 1997; Fenteany and Colley, 2005; Banfield, 2011; Schmitz et al., 2008; Tu et al., 2008; Welch and Munro, 2019; Nilsson et al., 1993; Tu and Banfield, 2010. However, how these domains contribute collectively to the steady-state localization of these Golgi enzymes is not clear.

Cisternal maturation is the most established model for Golgi organization and protein recycling, and proposes that Golgi resident proteins are specifically segregated away from anterograde and secretory cargos in the maturing cisternae and packaged into recycling vesicles Pantazopoulou and Glick, 2019; Glick and Nakano, 2009. Dynamic retrograde trafficking of the Golgi residents from maturing late Golgi to early cisternae is key to maintaining their localization Banfield, 2011; Welch and Munro, 2019. The recycling of several Golgi glycosylation and glycosphingolipid synthesis enzymes requires the activity of a phosphatidylinositol-4-phosphate [PI(4)P] binding protein, Vps74 (human GOLPH3), that binds to the COPI coat complex and to the cytosolic tails of its client Golgi enzymes Schmitz et al., 2008; Tu et al., 2012; Tu et al., 2008; Rizzo et al., 2021; Welch et al., 2021. The binding affinities between Vps74 and the cytosolic tails of glycosyltransferases are reported to be weak Schmitz et al., 2008, suggesting that successful retrieval in vivo may require more than binding to Vps74 alone. Considering the TMD and lumenal domains of glycosyltransferases have also been shown to affect recycling, an integral membrane protein capable of extended engagement with the glycosyltransferases, as well as of stabilizing the interactions with cytosolic recycling machinery can provide a key missing mechanistic link in this process.

erd1 and erd2 (ER Retention Defective) mutants were isolated three decades ago in a screen for mutants that secrete HDEL-invertase Pelham et al., 1988. Erd2 was subsequently characterized as the receptor that binds and retrieves ER proteins containing a C-terminal HDEL sequence, providing the underlying mechanism for its erd phenotype Semenza et al., 1990. Despite a strong defect in ER retention of HDEL-invertase and Kar2, as well as defects in Golgi-dependent modification of glycoproteins observed in the erd1 mutant, the function of Erd1 remains uncharacterized Hardwick et al., 1990; Copic et al., 2009. Here, we provide evidence that Erd1 chaperones early Golgi glycosylation enzymes throughout their trafficking in the Golgi, and facilitates stable interaction with the cytosolic receptor Vps74, thus acting as a key component of the glycosyltransferase recycling pathway. Membrane-embedded Erd1 and cytosolic Vps74 are together required for successful recycling of a subset of enzymes from late Golgi to early Golgi compartments via COPI vesicles.

Results and discussion

Golgi localization of glycosyltransferases requires Erd1

We examined the genome-wide genetic interactions networks for several glycosyltransferases Costanzo et al., 2016, and observed significant profile similarity with Erd1. To uncover functional information from genetic network analysis, we performed spatial analysis of functional enrichment (SAFE) and generated a profile similarity network (PSN) for Erd1 Usaj et al., 2017; Costanzo et al., 2016; Baryshnikova, 2016 (Figure 1A and B). SAFE analysis allows the identification of regions of the global similarity network that are significantly enriched for genes exhibiting negative or positive genetic interactions with a gene of interest. PSN analysis correlates the genetic interaction profiles and allows annotation of genes with related functions. Our analysis indicated that Erd1 is functionally related to genes known to mediate glycosylation and transport events at the ER and Golgi.

Figure 1 with 1 supplement see all
Erd1 is required for Golgi protein glycosylation.

(A) Spatial analysis of functional enrichment (SAFE) analysis based on the genetic interaction profile of erd1Δ mutant (stringent cut-off (p < 6e-11)). (B) Profile similarity network (PSN) of Erd1 showing genes with similar genetic interactions (similarity cut-off 0.25). (C) Growth of wild type and erd1Δ mutant in the presence of indicated concentrations of HygromycinB in YPD liquid cultures at 30° C after 24 hr. (D) Sensitivity of wild type and erd1Δ mutant to indicated concentrations of Tunicamycin in YPD liquid cultures at 30° C. (E) Violin plots for the ratio of co-localized Erd1-mNeonGreen fluorescence with early (Mnn9-mCherry), medial (Gea2-3xmMars), and late (Sec7-6xDsRed) Golgi markers (n = 250 puncta for each condition). The median is indicated with dashed lines. (F) Live-cell fluorescence imaging of Och1-GFP, Kre2-GFP, and vacuole membrane marker (Vph1-mCherry) in wild type and erd1Δ mutant. Red dashed lines indicate the cell boundaries based on DIC images. (G) Live-cell fluorescence imaging of Erd2-GFP, GFP-Rer1, Aur1-GFP and GFP-Neo1 in wild type and erd1Δ mutant. (H) Quantification of the percent vacuolar to total GFP fluorescence for reporters in (F) and (G). Scale bars: 2.5µ m.

erd1Δ mutant cells exhibited sensitivity to the aminoglycoside hygromycin, consistent with defects in glycosylation Ballou et al., 1991; Dean, 1995 (Figure 1C, Figure 1—figure supplement 1A), as well as tunicamycin, an inhibitor of N-linked glycosylation (Figure 1D). Additionally, as compared to WT control cells, we observed altered mobility of reporters of O-linked (Gas1) and N-linked glycosylation (CPY/Prc1, Pep4) in erd1Δ lysates, indicative of defects in glycosylation at the Golgi (Figure 1—figure supplement 1B). These observations are also in agreement with the reported defects in Golgi based CPY and invertase modifications in the erd1 mutant isolated from the HDEL-invertase screen Hardwick and Pelham, 1990. Endogenously tagged Erd1-mNeongreen predominantly co-localized with the early-Golgi marker, Mnn9, and to some extent with the medial-Golgi marker, Gea2, suggesting its function in early to medial Golgi compartments (Figure 1E, Figure 1—figure supplement 1C).

To assess the role of Erd1 in protein trafficking, we examined the localization of several representative ER, cis-, medial-, and trans-Golgi proteins tagged with a green fluorescent protein (GFP) in WT and erd1Δ cells. All the proteins tested exhibited the expected ER or punctate localization in WT cells; however, the Golgi glycosyltransferases, Och1 and Kre2, were significantly mislocalized to the vacuole lumen in the erd1Δ mutant (Figure 1F, G and H). A similar mislocalization of Och1, but not Mnn9, was previously observed in the erd1Δ mutant Okamoto et al., 2008. This suggested a specific sorting defect in erd1Δ mutant cells rather than a more general Golgi trafficking defect.

Erd1 is required for Vps74 function in intra-Golgi recycling

The defect in glycosyltransferase recycling is highly reminiscent of a similar defect previously reported for the vps74Δ mutant Schmitz et al., 2008; Tu et al., 2008. Vps74 is a peripheral membrane protein that acts as the cytosolic receptor for COPI recycling Schmitz et al., 2008; Tu et al., 2012; Tu et al., 2008; Welch et al., 2021; Rizzo et al., 2021. To assess the relationship between Vps74 and Erd1, we first directly compared the glycosylation defects in erd1Δ and vps74Δ mutants. The erd1Δ and vps74Δ mutants phenocopied each other in the Gas1 glycosylation defect, as well as in tunicamycin and hygromycin sensitivities (Figure 2A, B, Figure 2—figure supplement 1A). Furthermore, SAFE analysis on the global genetic interactions indicated a robust functional overlap between Erd1 and Vps74 (Figure 2—figure supplement 1B). Simultaneous deletion of Erd1 and Vps74 did not worsen the temperature sensitive slow growth of vps74Δ mutant at 40° C suggesting the two act in a common pathway (Figure 2C, Figure 2—figure supplement 1A). While the specific Gas1 glycosylation defect in erd1Δ and vps74Δ mutants was most similar to the loss of Kre2 (Figure 2—figure supplement 1C), their sensitivity to hygromycin resembled a defect similar to the loss of multiple glycosyltransferases (Figure 2—figure supplement 1D). To test this directly, we examined the localization of a panel of endogenously tagged glycosyltransferases and early-Golgi proteins in WT, erd1Δ, and vps74Δ mutants (Figure 2D). Indeed, loss of Erd1 and Vps74 manifested in a defect in recycling of the same subset of Golgi glycosyltransferases. All the tested glycosyltransferases that were Erd1-dependent also contained the cytosolic motif previously reported to require Vps74 binding (Figure 2—figure supplement 2E) Tu et al., 2008.

Figure 2 with 2 supplements see all
Erd1 is required for Vps74-COPI dependent recycling of specific Golgi glycosyltransferases.

(A) Immunoblot analysis on yeast cell lysates from wild type, erd1Δ, and vps74Δ mutant for glycosylation reporter, Gas1. (B) Growth of wild type, erd1Δ, and vps74Δ mutant in the presence of indicated concentrations of Tunicamycin in YPD liquid cultures at 30° C after 24 hr. (C) Growth of serial dilutions of wild type, erd1Δ, vps74Δ, and erd1Δvps74Δ mutants on synthetic media at 26 °C and 40° C after 2 days. (D) Quantification of percent vacuolar fluorescence to total fluorescence of the indicated GFP tagged early and medial Golgi proteins in wild type, erd1Δ, and vps74Δ mutant. (E) Live-cell fluorescence imaging of Erd1-GFP and vacuolar dye, CMAC in wild type and the vps74Δ mutant. (F) Live-cell fluorescence imaging of mNeonGreen-Vps74 and medial Golgi marker, Gea2-3xMars in wild type and the erd1Δ mutant. (G) Growth of serial dilutions of vps74Δ mutant transformed with empty vector (EV) or plasmids overexpressing Vps74 and Erd1 on YPD with 50 µg/ml hygromycinB at 26 °C or synthetic media lacking uracil at 26 °C or 40° C after 2–3 days. (H) Growth of serial dilutions of wild type and erd1Δ mutant transformed with the indicated Vps74 mutants at 26° C after 3 days. (I) Growth of serial dilutions of wild type, erd1Δ, sec21-1, and sec21-1 erd1Δ mutants at 26 °C after 3 days. Scale bars: 2.5µ m.

To investigate the mechanism of interdependence of Erd1 and Vps74 function, we first asked if they were required for each other’s correct localization. In wild-type cells, Erd1-GFP exhibited punctate distribution similar to cis-Golgi markers (Figure 1—figure supplement 1C). Strikingly, Erd1-GFP was missorted to the vacuole lumen in the vps74Δ mutant, as indicated by the co-localization with the vacuole lumen stained with the fluorescent dye, CMAC (Figure 2E). This suggests that in the absence of Vps74, Erd1 itself is not incorporated into COPI recycling vesicles and is instead delivered to the vacuole. On the other hand, endogenously tagged mNG-Vps74 co-localized predominantly with the medial-Golgi marker, Gea2, in both WT and erd1Δ mutant (Figure 2F, Figure 2—figure supplement 1F and 1G). Thus, whereas Erd1 is not required for recruitment of Vps74, it appears to be required for the function of Vps74 at the Golgi. Even though Vps74 is recruited to the Golgi via its interaction with PI4P Wood et al., 2009, it is unable to mediate recycling in the erd1ΔΔ mutant. These observations also indicate that the Vps74-dependent pathway recycles single pass type II membrane proteins such as glycosyltransferases as well as multi-pass transmembrane proteins such as Erd1.

If Erd1 and Vps74 cooperate to facilitate recycling, we asked if overexpression of Erd1 and Vps74 can compensate for each other in functional assays. Overexpression of Erd1 in the vps74Δ mutant partially suppressed the hygromycin and temperature sensitivity of the vps74Δ mutant (Figure 2G). In contrast, overexpression of Vps74 in the erd1Δ mutant, severely inhibited its growth fitness (Figure 2I). This toxicity was specifically due to maintenance of Vps74 expressing plasmid, as the growth was normal on rich media with no selection for the plasmid. Mutations in Vps74 that disrupt COPI binding (vps74 R6-8A) or oligomerization (vps74Δ201–204) alleviated the toxicity (Figure 2H, Figure 2—figure supplement 1E). One interpretation of these results is that overexpression of Vps74 in the erd1Δ mutant resulted in futile Vps74-COPI complexes that reduced the level of free COPI available for Golgi function. Consistent with this, the erd1Δsec21-1 mutant, defective in Erd1 and COPI function, exhibited a similar synthetic growth defect even at permissive temperature (Figure 2I). Collectively, these observations indicate significant interdependence between Erd1 and Vps74 in the COPI-mediated intra-Golgi recycling of specific Golgi cargos.

Erd1 has previously been proposed to play a role in Golgi ion homeostasis by transporting phosphate from the Golgi lumen to the cytosol Snyder et al., 2017. The erd1Δ mutant led to loss of phosphate (Pi) via exocytosis, and required 1.5-fold higher concentration of Pi in the media for 50% maximal growth as compared to wild type Snyder et al., 2017. To test if the effect of Erd1 on glycosyltransferase recycling is distinct from effects on ionic balance, we compared the erd1Δ phenotypes with that of pmr1Δ mutant. Pmr1 pumps Ca2+ and Mn2+ ions into the Golgi lumen Dürr et al., 1998. As expected, deletion of Pmr1 results in a similar Gas1 glycosylation defect as loss of Erd1 or Vps74 (Figure 2—figure supplement 2A). However, while supplementing the media with Ca2+ or Mn2+ rescued the glycosylation defect observed in pmr1Δ mutant (Figure 2—figure supplement 2C), supplementing the media with even 50-fold higher Pi did not rescue the glycosylation defect in the erd1Δ mutant. Importantly, despite the glycosylation defect, pmr1Δ mutant had no defect in the localization and recycling of Kre2 (Figure 2—figure supplement 2D). Therefore, while it is possible that Erd1 plays a role in phosphate homeostasis at the early Golgi, our data support a distinct role of Erd1 in also being required for glycosyltransferase recycling.

Erd1 directly interacts with glycosyltransferases and Vps74 to facilitate recycling

Based on our observations, we predicted that Erd1 directly interacts with Vps74 as well as glycosyltransferases. To monitor interactions between Erd1 and Vps74, we employed bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, and split-ubiquitin based yeast two-hybrid (Y2H) assays Iyer et al., 2005; Feng et al., 2017. We made fusions to the N-terminal beta strands 1–10 of mNeongreen, and the C-terminal beta strand 11 of mNeongreen, and since Vps74 has been shown to oligomerize, it served as a positive control in the BiFC assay (Figure 3A) Cai et al., 2014; Wood et al., 2012; Schmitz et al., 2008. We observed fluorescence complementation between Erd1-NG11, and NG1-10-Vps74 (Figure 3A). Fluorescence complementation was also detectable between tagged Erd1 and COPI binding defective vps74 R6-8A mutant, but not between tagged Erd1 and the oligomerization defective vps74 Δ201–204 mutant. These data suggest that Erd1 and Vps74 interaction is likely not bridged via COPI binding. Erd1 and Vps74 interaction was also confirmed using co-immunoprecipitation and Y2H assays (Figure 3B, Figure 3—figure supplement 1A). Erd1 also showed interaction with itself in Y2H and co-immunoprecipitation assays, suggesting Erd1 likely oligomerizes in vivo (Figure 3—figure supplement 1A and B).

Figure 3 with 1 supplement see all
Erd1 interacts with glycosyltransferases and Vps74.

(A) Bimolecular fluorescence complementation of mNeongreen tested in cells with plasmids expressing split-mNG fragments fusions to the indicated proteins, in a strain also expressing the early Golgi marker, Mnn9-mCherry. BiFC signal is shown in green, and Mnn9-mCherry is shown in magenta. (B) Coimmunporecipitation analysis to test the interaction between Erd1-GFP and FLAG-Vps74 (FLAG tag listed in all cases is 6 x His-TEV cleavage site-3xFLAG) from yeast cell lysates. (C) Coimmunporecipitation analysis to test the interaction between Erd1-GFP and Och1-FLAG, and GFP-Vps74 and Och1-FLAG from yeast cell lysates. (D) Coimmunporecipitation analysis to test the interaction between GFP-Vps74 and Och1-FLAG from wild type and erd1Δ yeast cell lysates. (E) Quantification of ratio of the signal in the IP to input normalized to the loading control for GFP-Vps74 and Och1-FLAG. (F) Growth of serial dilutions of erd1Δ mutant transformed with plasmids expressing Gyp1, Dcr2, Cog5, Cog7, Ers1, Neo1, Ypt7 identified in the dosage suppressor screen on YPD with 60 µg/ml hygromycin at 26 °C for 2 days.

To test interaction with glycosyltransferases, we immunoprecipitated GFP tagged Erd1 and Vps74 from yeast cell lysates and probed for Och1-FLAG in the pulldown by immunoblotting (Figure 3C). Och1 co-immunoprecipitated with both Erd1 and Vps74. Since tagging the cytosolic N-terminus of the glycosyltransferases resulted in retention in the ER (Figure 3—figure supplement 1D), we tagged the lumenal facing ends of Och1 and Erd1 to test interaction using BiFC. We observed robust fluorescence complementation signal between Och1-NG1-10 and NG11-Erd1, that co-localized with the cis-Golgi marker Mnn9 (Figure 3A). We next asked if the interaction between Erd1 and glycosyltransferases may be required for stable complex formation with Vps74. Although GFP-Vps74 efficiently coimmunoprecipitated Och1-FLAG or Kre2-FLAG from WT yeast cell lysates, the interaction was dramatically reduced in the erd1Δ mutant (Figure 3D,E, Figure 3—figure supplement 1E). Overall, these findings support a model where Erd1 chaperones the Golgi enzymes and allows stable interaction with the cytosolic recycling machinery.

To identify mechanisms that can compensate for the loss of Erd1 function, we employed a dosage suppressor screen to identify genes which when expressed at a higher than normal level can substitute for Erd1 function or bypass its requirement. We transformed the erd1Δ mutant with a multicopy yeast gDNA library Herman et al., 1991, isolated and confirmed suppressors of its hygromycin sensitivity (Figure 3F). Interestingly, we identified several genes with known roles in membrane protein trafficking (Gyp1, Cog5, Cog7, Ypt7) as well as those with previously reported genetic interactions with either Erd1 or Vps74 (Dcr2, Neo1, Ers1) Wang et al., 2020; Miyasaka et al., 2020; Hardwick and Pelham, 1990. The suppression was specific to erd1Δ mutant, since the introduction of these plasmids did not suppress the growth inhibition of wild type cells at high concentrations of hygromycin (Figure 3—figure supplement 1C). Gyp1 is a GTPase-activating protein for the Rab GTPase Ypt1 at the cis-Golgi, and it negatively regulates Ypt1 Du and Novick, 2001. Consistent with this, we found that the GDP-locked (inactive) mutant of Ypt1 (ypt1 S22N) but not WT Ypt1 or the GTP-locked (active) form (ypt1 Q67L) partially suppressed the hygromycin sensitivity of the erd1Δ mutant, suggesting that reduction in Ypt1 function is the basis of Gyp1 suppression (Figure 3—figure supplement 1F). Also intriguing is the suppression by subunits of the COG complex which is known to be required for the tethering of vesicles for intra-Golgi glycosyltransferase recycling Pokrovskaya et al., 2011; Ungar et al., 2006. Overall, the hits identified from the multicopy suppressor analysis support a role of Erd1 in Golgi protein recycling.

Erd1 and Vps74 sequentially engage with the glycosyltransferases

At steady state Erd1 and the glycosyltransferases localize predominantly at the early Golgi, while Vps74 localizes to the medial and late Golgi compartments. If Erd1 and Vps74 function cooperatively in mediating glycosyltransferase recycling, they would be expected to exhibit temporal overlap during Golgi maturation. To investigate this, we examined the in vivo spatiotemporal dynamics of Erd1 and Vps74 relative to each other, and other markers of Golgi cisternal maturation. Endogenously tagged constructs of Vps74, Erd1, and Och1 were confirmed to be functional (Figure 4—figure supplement 1D). Using time-lapse imaging to track individual Golgi puncta, we found that Erd1 arrived and departed in synchrony with the Golgi glycosyltransferases Mnn9 and Och1 (Figure 4A,B, Figure 4—figure supplement 1A,B). Erd1 signal peaked at the same time as Mnn9, and significantly before the medial (Gea2) and late Golgi (Sec7) markers (Figure 4C,D, Figure 4—figure supplement 1E). In contrast, Vps74 levels peaked after Gea2, and before Sec7 or the PI4P-specific probe, P4C Luo et al., 2015; Highland et al., 2021 (Figure 4E, Figure 4—figure supplement 1C,F). Interestingly, COPI signal peaked after Erd1, but before the arrival of Vps74 (Figure 4F). In agreement with these observations, simultaneous kinetic analysis of Erd1 and Vps74 also indicated temporal overlap between the two (Figure 4G).

Figure 4 with 1 supplement see all
Kinetic analysis of Erd1 and Vps74 in the context of Golgi cisternal maturation.

(A) Time-lapse imaging series (2 s intervals) of the indicated single Golgi compartment in cells expressing Erd1-mNeonGreen and Mnn9-mCherry. (B) Averaged normalized fluorescence traces with 95% CI of time-lapse imaging analysis of 10 puncta described in (A). Dashed line represents the time corresponding to the peak signal of Erd1 and Mnn9. (C) Schematic describing the estimation of peak-to-peak fluorescence times of two markers from time-lapse imaging analysis. (D) Quantification of peak-to-peak times for Erd1-mNeonGreen with respect to early (Mnn9-mCherry), medial (Gea2-3xMars), and late (Sec7-6xDsRed) Golgi markers (n > 15 puncta for each condition). Peak Erd1 is set at t0 and indicated with the dashed line. The bold line represents the median value for each condition. (E) Quantification of peak-to-peak times for mNeonGreen-Vps74 with respect to early (Mnn9-mCherry), medial (Gea2-3xMars), and late (Sec7-6xDsRed) Golgi markers. Peak Vps74 is set at t0 and indicated with the dashed line. The bold line represents the median value for each condition. (F) Quantification of peak-to-peak times for Erd1 and Vps74 with respect to COP1-mCherry. Peak COP1 is set at t0 and indicated with the dashed line. (G) Time-lapse imaging series (2 s intervals) of the indicated single Golgi compartment in cells expressing Erd1-mNeonGreen and mScarlet-Vps74 (left) and normalized fluorescence of time-lapse imaging (right). (H) Schematic depicting the dynamics of tagged Erd1, Vps74 and COP1 at maturing Golgi compartments marked by Mnn9 (early), Gea2 (medial), and Sec7 (late) based on A-F. (I) A simplified model for the proposed role of Erd1 in the recycling of Golgi glycosyltransferase at two steps - (1) at the step of commitment of the cargos for recycling by allowing stable complex formation with the cytosolic recycling machinery, and (2) at the final step of vesicle tethering and fusion at the early Golgi by facilitating interaction with the tethering machinery.

The time-lapse imaging revealed that Erd1 has the same kinetics as its recycling clients, and the subsequent arrival of Vps74 coincides with recycling. These observations are consistent with sequential association of Erd1 and Vps74 with their client cargos and packaging into COPI vesicles (Figure 4H). Our data support a model where engagement of Erd1 with the membrane bound cargos allows stable interaction with Vps74, and commits them for recycling (Figure 4I). By chaperoning the early Golgi enzymes throughout the recycling process, Erd1 allows productive coordination of Vps74 binding with the timing of cargo recycling in the spatio-temporal context of Golgi cisternal maturation. Erd1 itself is also a cargo of the Vps74 pathway, and it is possible that Erd1’s role in Golgi recycling could be more complex and it may be involved in distinct steps for Golgi to ER Vps74-independent and intra-Golgi Vps74-dependent recycling steps.

Overall, our work demonstrates a role of Erd1 in Golgi protein recycling, and provides a mechanistic basis for the observed glycosylation defects. This work also provides new insight on how cooperative, yet hierarchical engagement of transmembrane (Erd1) and cytosolic receptors (Vps74), likely with different domains of their transmembrane cargos, is important for productive recycling. By extended engagement with the cargos, and with the cytosolic recycling machinery, Erd1 might serve as an important quality assurance sensor at multiple steps in the pathway. Other such transmembrane adaptors are also likely to exist. Recent screens in mammalian cells have identified transmembrane proteins TMEM165 and TM9SF2 that may serve similar roles in bridging the Golgi Gb3 synthase A4GALT to cytosolic recycling machinery Yamaji et al., 2019; Tian et al., 2018; Tanaka et al., 2017. Similarly, transmembrane Erv proteins can serve as receptors for COPII dependent ER to Golgi protein trafficking Barlowe and Miller, 2013. A lot remains to be discovered to understand the role and the interplay of different components at multiple steps of Golgi enzyme recycling- vesicle tethering complexes such as the COG and GARP complexes, as well as Golgi matrix proteins such as GRASP55 are also required for recycling Golgi glycosylation enzymes Khakurel et al., 2021; Pokrovskaya et al., 2011; Pothukuchi et al., 2021. Suppression of the erd1Δ mutant by COG subunits also suggests a potential role for Erd1 at the final tethering step of the recycling process that would need further examination.

The role of Erd1 and other EXS domain proteins such as human XPR1 and plant PHO1 in phosphate homeostasis has been previously reported, although unlike Erd1, XPR1 and PHO1 both also contain a phosphate sensing SPX domain Secco et al., 2012; Wege et al., 2016; Giovannini et al., 2013; Snyder et al., 2017; Legati et al., 2015; Bondeson et al., 2020; Arpat et al., 2012. XPR1 functions in phosphate transport at the cell surface, but also localizes intracellularly Bondeson et al., 2020. PHO1 localizes to the Golgi and trans-Golgi network Arpat et al., 2012, and it is possible that the intracellular pools of PHO1 and XPR1 also play roles in protein trafficking. Since glycosylation reactions in the Golgi produce inorganic phosphate (Pi) and protons (H+) as byproducts, these proteins could also help maintain glycosylation homeostasis more broadly. Future analysis will examine how Erd1 engages with only a subset of client glycosyltransferases and with Vps74. A systematic comparison of Erd1 and Vps74 cargo repertoire will also be important to determine the extent of overlap between their clients. Finally, it remains to be determined whether transmembrane proteins in recycling vesicles, such as Erd1, play a role in decisions that allow some COPI retrograde vesicles to be recycled within the Golgi, while others are trafficked to the ER. These and other exciting questions remain to be addressed as we move forward.

Materials and methods

Yeast strains and plasmids

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All yeast deletion or chromosomal tagged strains were constructed in the strain background SEY6210 using iterative markerless CRISPR/Cas9 genome engineering, or using PCR-based amplification and homologous recombination as described Shaw et al., 2019; Longtine et al., 1998. All yeast strains used in this study are listed in Supplementary file 1. Cells were grown in yeast synthetic media [0.17 % (w/v) yeast nitrogen base, 0.5 % (w/v) ammonium sulfate, 2 % (w/v) glucose, and supplemented with appropriate nutrients] or YPD [1 % (w/v) yeast extract, 1 % (w/v) peptone, 2 % (w/v) glucose] at 26 °C, unless otherwise specified. All plasmids used in this study are listed in Supplementary file 1, and were generated using standard cloning procedures. Yeast transformations were performed following the standard lithium acetate protocol Schiestl et al., 1993.

Growth assays

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For growth assays, ten-fold serial dilutions of mid-log phase cultures normalized to OD 0.3 were spotted onto the indicated media and incubated for 2–5 days. Y2H assay was performed in yeast strain L40 by co-transforming the indicated bait and prey vectors. Interaction was scored as growth on synthetic media lacking Leu, Trp and His and containing 1 mM 3-Amino-1,2,4-triazole (3-AT). Dosage suppressor screen was performed by transforming a 2 micron URA3 gDNA libray into erd1Δ mutant and suppressors were selected on YPD plates containing 75 µg/ml HygromycinB. The suppressing plasmids were extracted, amplified in bacteria, re-transformed into erd1Δ yeast cells and the suppressing genes were identified by sequencing.

Genetic interaction analysis

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Analysis of genetic interactions was performed on the genome-wide genetic interaction dataset available on the CellMap repository Usaj et al., 2017. SAFE analysis was performed as described Baryshnikova, 2016 using negative genetic interactions and stringent cut-off settings (p < 6e-11). Erd1 PSN was generated using a threshold cutoff off 0.25 and visualized as a Chord plot.

Fluorescence microscopy

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Cells were grown in synthetic dropout media to mid-log phase at 26° C and imaged at room temperature on glass coverslips or in glass-bottomed dishes. Images unless mentioned otherwise were captured using a DeltaVision RT system (Applied Precision, Issaquah, WA), equipped with a DV Elite CMOS camera, a 100 x objective, and a DV Light SSI 7 Color illumination system with Live Cell Speed Option (FITC for GFP and TRITC for mCherry/RFP). Image acquisition and deconvolution were performed using the provided DeltaVision software (softWoRx 6.5.2; Applied Precision, Issaquah, WA). Masks for vacuoles and the total cell were created based on DIC images in ImageJ. Mean vacuolar to total fluorescence ratios were calculated from three independent experiments with ∼25 cells in each set.

All time-lapse images were captured with a CSU-X spinning-disk confocal microscope system (Intelligent Imaging Innovations) using a DMI6000 microscope (Leica Microsystems) outfitted with a CSU-X1 spinning-disk confocal unit (Yokogawa Electric Corporation) with a QuantEM 512SC (Photometrics). The objective was a 100 × 1.46 NA Plan Apochromat oil immersion lens (Leica Microsystems). Images were captured every 2 s for 2 min using Slidebook six software (Intelligent Imaging Innovations). Images were processed in ImageJ and assembled in Adobe Illustrator CS6. Quantification of Golgi puncta was perfomed in Python. Briefly, images were corrected for hot pixels by using a median filter, and uneven illumination by applying a gaussian background subtraction. Puncta in both channels were defined by thresholding using the Otsu method. Object based co-localization was used to calculate the ratio of overlap between the two channels for ∼250 puncta for each set. The quantification data of each puncta was plotted, and the median and confidence intervals were annotated in violin plots. For generation of time lapse imaging traces, individual Golgi compartments that remained in the same focal plane throughout their lifetimes were used for analysis. Fluorescence intensities for the identified puncta were measured over time and intensity values were normalized to values between 0 and 1 for each channel. Peak-to-peak time values denote the time between maximum intensities of green and red fluorescence signals at individual maturing Golgi puncta. Averaged traces with confidence intervals were generated from ∼10 puncta for each channel.

Protein extraction and immunoprecipitation

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Yeast lysates for immunoblotting were prepared from 5 OD600 equivalents of cells. Briefly, cells were harvested and incubated in 10 % trichloroacetic acid (TCA) on ice for 1 hr. Cells were pelleted, washed with cold acetone, lysed by beating with glass beads in 80 µl of 2 x urea buffer (50 mM Tris-HCl pH 7.5, 8 M urea, 2 % SDS, 1 mM ethylenediaminetetraacetic acid [EDTA]) for 5 min at room temperature, followed by incubation at 42 °C for 10 min. 80 µl of 2 x sample buffer (150 mM Tris HCl, pH 6.8, 8 M urea, 8 % SDS, 24 % glycerol, bromophenol blue) supplemented with 100 mM dithiothreitol (DTT) was added, and samples were vortexed for 5 min followed by incubation at 42 °C for 10 min. The samples were centrifuged and 0.2 OD equivalents were resolved on eight or 10% SDS-Polyacrylamide gels, followed by transfer to nitrocellulose membranes (0.45 µm, GE healthcare) at 4 °C via wet transfer in transfer buffer (25 mM Tris, 192 mM Glycine, 10% v/v methanol, 0.006 % SDS) at 100 V for 90 min before immunoblotting. For glycosylation rescue experiments, cells were grown to mid-log phase in YPD at 30 °C, followed by growth in YPD supplemented with 50 mM CaCl2, 600 µM MnCl2, or 50 mM phosphate buffer pH 6.2 for 4 hr at 30 °C.

For immunoprecipitations, cells were grown to OD600 0.5–1.0 in 100 ml of synthetic growth media and harvested on ice. Cells were washed and resuspended in Lysis Buffer (50 mM Tris pH 7.5, 2 mM EDTA pH 8.0, 150 mM NaCl, 10 % glycerol, 1 mM PMSF, 1 X Roche cOmplete Protease Inhibitor Tablet/50 ml). Cell extracts were prepared by glass bead beating for three cycles of 1 min vortexing with 1 min breaks on ice. Membranes were solubilized by nutating for 30 min at 4 °C after addition of Saponin to 1 % final concentration. Crude extracts were clarified and the lysates was incubated with 25 µl of GFP-nanobody resin at 4 °C for 3 hr. The resin was washed five times with Lysis buffer and bound proteins were eluted by addition of 100 µl 2 x sample buffer (100 mM Tris-HCl pH 8.0, 1 % SDS, 10 mM DTT), followed by incubation at 65 °C for 10 min. 1% input and 10% immunoprecipitates were resolved on a 10% SDS-PAGE gel and subjected to immunoblotting.

The following antibodies and dilutions were used for western blotting: rabbit polyclonal anti-Gas1 (1:20000), rabbit polyclonal anti-CPY (1:5000), rabbit polyclonal anti-Pep4 (1:2000), rabbit polyclonal anti-GFP (1:5000) (TP401; Torrey Pines Biolabs, Secaucus, NJ), mouse monoclonal anti-PGK (1:5000) (22C5D8; Molecular Probes Inc), mouse monoclonal anti-FLAG (1:3000) (M2; Sigma), 800CW goat anti-rabbit (1:10,000) (926–32211; LI-COR Biosciences, Lincoln, NE) and 680LT goat anti-mouse (1:10,000) (926–68021; LI-COR Biosciences).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, Fig 2-S2, Fig 3-S1.

The following previously published data sets were used
    1. Usaj M
    2. Tan Y
    3. Wang W
    4. VanderSluis B
    5. Zou A
    6. Myers CL
    7. Costanzo M
    8. Andrews B
    9. Boone C
    (2017) thecellmap
    TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network.
    https://doi.org/10.1534/g3.117.040220

References

    1. Pelham HR
    2. Hardwick KG
    3. Lewis MJ
    (1988)
    Sorting of soluble ER proteins in yeast
    The EMBO Journal 7:1757–1762.
    1. Stanley P
    (2011) Golgi glycosylation
    Cold Spring Harbor Perspectives in Biology 3:a005199.
    https://doi.org/10.1101/cshperspect.a005199

Decision letter

  1. Benjamin S Glick
    Reviewing Editor; The University of Chicago, United States
  2. Vivek Malhotra
    Senior Editor; The Barcelona Institute of Science and Technology, Spain
  3. Benjamin S Glick
    Reviewer; The University of Chicago, United States
  4. Alberto Luini
    Reviewer; National Research Council, Italy

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

Decision letter after peer review:

Thank you for submitting your article "Golgi membrane protein Erd1 is essential for recycling of Golgi glycosyltransferases" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Benjamin S Glick as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Alberto Luini (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reason for these revisions is that the evidence about the role of Erd1 could be consistent with mechanisms different from the one presented in the manuscript. In the spirit of constructive feedback, the authors are encouraged to dig a bit deeper and potentially reconsider their conclusions.

1) Perform a simultaneous kinetic analysis of Erd1 and Vps74 to determine the extent of temporal overlap between these two proteins during maturation. This result will help to clarify the functional relationship between Erd1 and Vps74, and may point to a more complex model along the lines of the suggestion from Reviewer #2.

A related issue is that Erd1 seems to resemble glycosyltransferases in being a cargo of Vps74-mediated recycling. This point should be kept in mind when considering whether the Erd1-Vps74 interaction has a richer meaning.

2) Provide evidence for direct interactions between Erd1 and glycosyltransferases. Such data are important to support a model in which Erd1 plays a receptor-like role in recycling.

3) Carefully evaluate the earlier work implicating Erd1 in fungal phosphate transport, especially in light of the statement by Reviewer #3 that the entire Erd1 protein is an EXS domain. A key paper here is reference 46 in the manuscript.

Proteins known to maintain the luminal ionic environment of the Golgi, such as Pmr1, are also required for normal glycosylation. It will be informative if the authors can present arguments that Erd1 is in a different category.

4) Cite and discuss the earlier paper by Okamoto et al.

Reviewer #1:

Glycosylation in the Golgi is carried out by resident enzymes that recycle within the organelle while secretory proteins move forward in the maturing cisternae. The mechanism of Golgi enzyme recycling is incompletely understood, but it seems to involve retrograde COPI coated vesicles. In yeast, a subset of Golgi enzymes interact with the peripheral membrane protein Vps74, which is thought to serve as an adaptor for capturing Golgi enzymes in COPI vesicles. The current manuscript identifies the integral membrane protein Erd1 as a facilitator of Golgi enzyme recycling. Evidence is presented that Erd1 interacts with Golgi enzymes as well as Vps74, and that Erd1 is needed for normal Golgi enzyme localization. Therefore, Erd1 is an important new player in the maintenance of Golgi organization.

This study is of high quality. The data are generally strong, the experiments are logical, and multiple complementary approaches are used to answer the questions. A persuasive case is made that Erd1 plays a role in recycling Golgi enzymes.

Less persuasive is the interpretation that Erd1 directly activates Vps74 to drive recycling. Much of the evidence is circumstantial--e.g., loss of Erd1 and loss of Vps74 confer similar phenotypes. Skepticism about the authors' interpretation centers around timing during the maturation process:

– Two of the authors (Highland and Fromme) recently published an analysis of PI4P dynamics, and they concluded that PI4P is a hallmark of the late Golgi, sharing "little overlap with markers of less mature Golgi compartments".

– Vps74 has been found to require PI4P for its recruitment, so Vps74 would be expected to reside at the late Golgi. Indeed, even though Vps74 peaks earlier than the late Golgi marker Sec7, it seems to arrive at about the same time as Sec7.

– By contrast, Erd1 clearly overlaps with early Golgi enzymes, which have mostly departed by the time that Sec7 arrives. It is therefore unclear whether Erd1 and Vps74 overlap for long enough to engage in a direct physical interaction that would drive Golgi enzyme recycling.

The major recommendation, intended to address the concern raised above is to visualize the maturation kinetics of Erd1 and Vps74 simultaneously.

Reviewer #2:

Based on bioinformatic, biochemical, morphological and genetic data, the authors propose a model of the mechanism of action of ERD1 according to which enzymes belonging to a selected group of glycosyltransferases are retained in the Golgi by recycling via COPI vesicles. According to this model the incorporation of enzymes in vesicles requires a sequential interaction of these enzymes first with ERD1 in the cis golgi and then with GOLPH3 in the medial trans Golgi. The first interaction has, in the proposed model, a preparatory or facilitating role for the second interaction, with GOLPH3, which mediates entry into COPI -dependent retrograde transport vesicles, and therefore the recycling, of the enzymes.

This is an interesting study in the rapidly developing area of the recycling mechanisms of glycosylation enzymes in Golgi. However, there are some weaknesses, which are outlined below.

The biochemical data on the binding between ERD1, VPS74 and Golgi enzymes are incomplete and should be completed with the demonstration that ERD1 and client enzymes interact directly, by means of the Y2H technique. This interaction is so far only demonstrated by co-precipitation of ERD1 with GOLPH3 and the enzymes, but these experiments do not exclude that ERD1 which interacts directly with GOLPH3, might interact indirectly with the enzymes, through its direct interaction with GOLPH3. Since the interaction between ERD1 and the client enzymes is at the heart of this study, it should be demonstrated with dedicated experiments.

On the technical plane, in the experiment in Figure 3C the efficiencies of co-ip in WT cells and in deltaERD1, once corrected for the inputs, do not seem very different. It would be useful to show inputs and IPs in the very same blot and to provide a quantification graph.

The localization of ERD1, GOLPH3 and enzymes are interpreted by the authors in the context of the ERD1/GOLPH3 synergy model briefly outlined above, and presented in figure 4. However, not all of the data are easily reconcilable with this model.

The main difficulty is that both ERD1 and its client enzymes appear and recycle in the cis cisterna while GOLPH3 resides in the medial and partly in the trans cisternae, but not in the cis cisterna. Moreover, the scheme presented by the authors in Figure 4 indicates that the recycling of the cis enzymes (eg, Mann9) begins before the appearance of GOLPH3 in the cis cisterna. This is in principle not compatible with the model, unless the authors propose that non-detectable traces of GOLPH3 might be present in the cis cisterna and activate the recycling mechanism there. If the authors show that this is the case, it might be acceptable to present their model in this study. However, given that our understanding of Golgi enzyme recycling is actually in its infancy, the underlying mechanism might be more complex. For example, ERD1 could mediate the recycling of client enzymes in the cis cisterna in synergy with a hitherto unknown adapter other than GOLPH3 (perhaps COPI itself?), and might also mediate the recycling of enzymes in synergy with GOLPH3 in the medial cisterna. This is possible because according to the scheme in figure 4 ERD1 shows a peak in the cis cisterna but is clearly present also in the medial cisterna. Furthermore, all ERD1-dependent enzymes should also be indirectly dependent on GOLPH3 since GOLPH3 depletion causes ERD1 loss and lysosomal degradation. ERD1 might thus be an adapter capable of acting at various levels of the transport system: in Golgi to ER recycling and in glycoenzyme recycling at both the cis and the medial cisterna. This model can explain the data probably better than that presented by the authors in Figure 4, but is more complicated and requires some more assumptions. It is up to the authors to decide whether they want to consider this hypothesis as an alternative model.

A question that would need to be addressed in this context is whether all the enzymes found by the authors to depend on ERD1 are also GOLPH3 customers and in particular whether they all contain the recognition motif (F / L) – (L / I / V ) -xx- (R / K) for GOLPH3 described by Banfield.

My recommendations to the authors have already been outlined above. In short, I think that the authors should:

Characterize the interaction between GOLPH3 and client enzymes by Y2H.

Discuss the weaknesses of their model as presented in figure 4 and, if they wish, mention the possibility of a more complex mechanism,

Clarify whether all ERD1 client enzymes contain the motif (F / L) – (L / I / V) -x-x- (R / K),

Discuss the earlier paper by Okamoto et al., (2008) that is not cited in this study.

In my opinion the authors should also discuss two recent papers (Rizzo et al; Parashurama et al., both in EMBO J), that are based on the concepts of recycling adapter and retention adapter and propose a model for the localization of GOLPH3-dependent enzymes in Golgi compartments in the context of cisternal maturation.

Reviewer #3:

The authors investigate the role of the yeast protein Erd1 in Golgi-dependent glycosylation. They conclude that Erd1 acts with Vps74, a known retention factor for Golgi glycosyltransferases, to direct the recycling of these enzymes in COPI-coated vesicles in the Golgi stack. The data are well presented and quantified, and the paper is clearly written. Addressing the role of Erd1, and insight into how glycosyltransferases are retained in the Golgi, are both interesting questions, but the authors data do not preclude alternative interpretations, and one or two aspects require resolution. These issues are summarized below:

1) A role for Erd1 in acting as a coreceptor with Vps74 to recycle glycosyltransferases is interesting but also raises some questions. Firstly, Erd1 is only found in fungi and not in metazoans, whereas Vps74 has metazoan orthologs that are known to play a role in glycosyltransferase recycling raising the question of why Erd1 is only needed in yeast. Secondly, there is published evidence for Erd1 acting as a channel/transporter for the movement of phosphate out of the Golgi lumen, and indeed the entire protein comprises a domain (the EXS domain) that is present in known phosphate transporters in plants and metazoans. Thus, careful dissection is required to determine if the effects seen are direct via an interaction with glycosylation enzymes, or if they are an indirect consequence of a perturbation of the Golgi lumen due to accumulation of inorganic phosphate. The fact that Erd1 was originally identified as having a defect in the retrieval of soluble ER residents from the Golgi suggests that the Golgi lumen may well be perturbed, possibly by a change in pH or cation content, and it is known that alterations in these features of the Golgi can affect glycosylation.

2) Perhaps the most striking finding is that Erd1 co-precipitates with Vps74 and that the presence of Erd1 is required for Vps74 to efficiently co-precipitate with the glycosyltransfereases that it is known to bind. However, the authors also show that the glycosyltransferases are destabilized by the loss of Erd1, and so the loss of apparent interaction may simply reflect the fact that there is less protein present to co-precipitate. Secondly, the authors show that Erd1 and Vps74 do not substantially co-localize, and so any tripartite complex would have to reflect a small sub-population of the proteins that briefly come together later in the Golgi stack during formation of COPI coated vesicles that are to be recycled. Finally, Vps74 is known to bind to COPI, and so if Erd1 also bound to COPI, then Vps74 and Erd1 may co-precipitate because they are held together via COPI, with Vps74 then bringing some glycosyltransferases into the complex.

3) Some aspects of the data may need resolving. Firstly, the authors provide clear images showing degradation of Och1p-GFP and Kre2-GFP in the vacuole in the absence of Erd1 (Figure 1F). However, the immunoblots in Figure 3D indicate that the levels of the intact proteins are unchanged in the absence of Erd1 which suggests that they are not destabilized and degraded. Secondly, the authors use the split ubiquitin system to provide evidence for an interaction between Erd1 and Vps74. As a control they remove the "cytoplasmic-tail" from Erd1, but they do not state how many residues were removed. The structural prediction for the EXS domain in Pfam suggests that the last membrane spanning helix of Erd1 would be very close to the C-terminus (Pfam entry PF03124), and the location of the truncation is not tested. Finally, the authors show some nice live cell imaging data to follow Golgi maturation. However, they do not directly compare Vps74 and Erd1. Such a comparison would be very helpful, especially as it seems from the other graphs that Erd1 is significantly depleted from the maturing cisterna before the time when there are substantial amounts of Vps74 present.

The prior publications on Erd1, the absence of an orthologue in mammals even though they have a Vps74 ortholog, and the potential role of COPI as a bridge between the two proteins, really necessitate a much more in depth and substantial analysis for a broad readership journal such as eLife. Ideally, in vitro reconstitution of binding with purified proteins would resolve many issues, but I appreciate that this may be technically challenging. Below I have suggested some things that could be done to strengthen the paper's conclusions, and at the very least these may be helpful to the authors to consider before resubmitting elsewhere:

1) Examination of the effect of other gene deletions that affect the ionic content of the Golgi such as deletion of Gdt1, Pmr1 or Stv1, on Och1p-GFP and Ktr2p-GFP levels and on glycosylation.

2) Resolution of the apparent contradiction of the effects of Erd1 deletion on Och1 and Ktr2 by microscopy and blotting assays. If available, antibodies to the endogenous proteins could be used to test their levels in wild type and mutants.

3) Does the mutation in the COPI binding site of Vps74 affect its Golgi localization? If not, the authors should check if this prevents the co-ip with Erd1.

4) It would be very valuable to add videos and graphs to follow the Golgi localization of Erd1 vs Vps74 to better reveal their spatial relationship over time.

5) Substantial new insight would be provided by determining what part of at least of the Erd1-dependent glycosyltransferases interacts with Erd1. This could be addressed by making chimeric proteins that contain only either the cytoplasmic tail, or the TMD, or the lumenal domain of a Erd1-dependent glycosyltransferase in the context of an Erd1-indepenent glycosyltransferase. The localization and co-ip of these chimeras could be then be tested.

6) The authors argue that the cytoplasmic tail of Erd1 interacts with Vps74. This could be tested biochemically as has been done for the tails of glycosyltransferases. If the authors keep the split-ubiquitin experiments they should confirm that the constructs are localized to the Golgi.

7) Are their glycosyltransferases that do no rely on Vps74? It would be useful to test if these are affected by loss of Erd1.

https://doi.org/10.7554/eLife.70774.sa1

Author response

Essential revisions:

The reason for these revisions is that the evidence about the role of Erd1 could be consistent with mechanisms different from the one presented in the manuscript. In the spirit of constructive feedback, the authors are encouraged to dig a bit deeper and potentially reconsider their conclusions.

1) Perform a simultaneous kinetic analysis of Erd1 and Vps74 to determine the extent of temporal overlap between these two proteins during maturation. This result will help to clarify the functional relationship between Erd1 and Vps74, and may point to a more complex model along the lines of the suggestion from Reviewer #2.

As per the reviewers’ request, we have now performed simultaneous kinetic analysis of Erd1 and Vps74 and included the data in Figure 4G. Kinetic analysis of Erd1 and Vps74 suggests a temporal overlap between the two, consistent with our model that arrival of Vps74 coincides with the packaging and departure of the cargos in COPI vesicles. Erd1 kinetics resemble earlyGolgi proteins, some of which have been previously reported to be Vps74 clients and have a similar overlap time with Vps74 as Erd1. The temporal overlap is also consistent with our kinetic analysis of Erd1 and Vps74 with early (Mnn9), medial (Gea2) and late (Sec7) Golgi markers (see Figure 4 A-F and Figure 4-supplement 1).

A related issue is that Erd1 seems to resemble glycosyltransferases in being a cargo of Vps74-mediated recycling. This point should be kept in mind when considering whether the Erd1-Vps74 interaction has a richer meaning.

We agree with the reviewers, that Erd1 indeed acts as a mediator and a cargo of Vps74dependent recycling is quite intriguing. By chaperoning the glycosyltransferase cargos throughout the recycling process, Erd1 appears to play a unique role in ensuring productive recycling of the cargos. To our knowledge, Erd1 is also the first example of a multi-pass membrane protein recycled via this pathway.

2) Provide evidence for direct interactions between Erd1 and glycosyltransferases. Such data are important to support a model in which Erd1 plays a receptor-like role in recycling.

As suggested by reviewer 2, we first tested the interaction between full length Erd1 and the glycosyltransferases using the split-Ub Y2H assay but did not observe a robust interaction. The nature of the Y2H assay requires the tag to be present on the cytosolic facing end of the proteins being tested for interaction. All glycosyltransferases are Type II single TM proteins, with the N terminus in the cytosol, and the C terminus in the lumen. To make sure the tagged constructs localized as expected, we tested the localization of N- and C-terminally GFP tagged glycosyltransferases, Kre2 and Och1. As shown in Author response image 1, while C-terminal tagged Kre2-GFP exhibits a punctate Golgi localization (right), N-terminal tagged GFP-Kre2 (left) is significantly retained in the ER (likely by interfering with COPII packaging). These observations clarified why the Y2H assay was unable to score the interaction between Erd1 and the glycosyltransferases.

Author response image 1

We also attempted to purify GST tagged full length Erd1 from yeast cells for in vitro binding experiments but were unable to obtain enough protein needed for the analysis. Finally, as an alternative strategy, we employed bimolecular fluorescence complementation (BiFC) to test interaction between Erd1 and glycosyltransferases with tags fused to the termini facing the lumen. This assay has been employed to score interactions between Vps74-Vps74 and Vps74Sac1 previously (Cai et al., JCB 2014, Wood et al., MBoC 2012). We observed reconstitution of fluorescence signal in the BiFC assay in strains expressing Och1-mNG1-10 and Erd1-mNG11. As expected, the BiFC signal co-localized with the cis-Golgi marker Mnn9-mCherry. We have included this data in Figure 3A. While we cannot rule out the possibility of an indirect interaction, these results in combination with other data presented in the manuscript, make a compelling case for a close interaction between Erd1 and its client glycosyltransferases.

3) Carefully evaluate the earlier work implicating Erd1 in fungal phosphate transport, especially in light of the statement by Reviewer #3 that the entire Erd1 protein is an EXS domain. A key paper here is reference 46 in the manuscript.

Proteins known to maintain the luminal ionic environment of the Golgi, such as Pmr1, are also required for normal glycosylation. It will be informative if the authors can present arguments that Erd1 is in a different category.

Erd1 has indeed been proposed to play a role in phosphate transport owing to the conserved EXS domain, and interactions reported by Snyder et al., 2017. We completely agree with the reviewers that the ionic environment of the Golgi is expected to affect normal glycosylation. We have now included multiple controls, that demonstrate that our observations on the role in Golgi glycosyltransferase recycling is distinct from just an effect on the ionic environment.

As requested by the reviewers, we compared the effects of Erd1 and Pmr1 (Golgi Ca2+ and Mn2+ transporter) on glycosylation and glycosyltransferase recycling. We observed that both erd1∆ and pmr1∆ mutants exhibit defects in glycosylation of Gas1 reporter (see Figure 2- supplement 2A). However, unlike erd1∆ mutant, the pmr1∆ mutant shows no defect in the localization and recycling of Kre2 (see Figure 2- supplement 2D). If the primary defect is an ionic imbalance, then supplementing the media with the key ion will be expected to rescue the observed glycosylation defects. Synder et al., had reported that erd1∆ mutant requires 1.5fold higher phosphate concentration in the media for 50% maximal growth as compared to wild type, and that Erd1 likely limits the export of Pi from wild type cells via exocytosis. However, supplementing the media with higher concentration of Pi (even at concentrations needed to support the growth of erd1∆ pho∆-5 mutant reported in Synder et al.,) did not rescue the glycosylation defects observed in erd1∆ (see Figure 2- supplement 2B). On the other hand, supplementing the media with Ca2+ (complete rescue) or Mn2+ (partial rescue) suppressed the glycosylation defect observed in pmr1∆ mutant (see Figure 2- supplement 2C). Therefore, while it is possible that Erd1 plays a role in phosphate homeostasis at the early Golgi, our data support an additional role of Erd1 (distinct from other ion transporters at the Golgi) in also being required for glycosyltransferase recycling.

4) Cite and discuss the earlier paper by Okamoto et al.

We thank the reviewers for pointing this out and apologize for the oversight. We have now cited the paper and included it in the discussion.

Reviewer #1:

Glycosylation in the Golgi is carried out by resident enzymes that recycle within the organelle while secretory proteins move forward in the maturing cisternae. The mechanism of Golgi enzyme recycling is incompletely understood, but it seems to involve retrograde COPI coated vesicles. In yeast, a subset of Golgi enzymes interact with the peripheral membrane protein Vps74, which is thought to serve as an adaptor for capturing Golgi enzymes in COPI vesicles. The current manuscript identifies the integral membrane protein Erd1 as a facilitator of Golgi enzyme recycling. Evidence is presented that Erd1 interacts with Golgi enzymes as well as Vps74, and that Erd1 is needed for normal Golgi enzyme localization. Therefore, Erd1 is an important new player in the maintenance of Golgi organization.

This study is of high quality. The data are generally strong, the experiments are logical, and multiple complementary approaches are used to answer the questions. A persuasive case is made that Erd1 plays a role in recycling Golgi enzymes.

Less persuasive is the interpretation that Erd1 directly activates Vps74 to drive recycling. Much of the evidence is circumstantial--e.g., loss of Erd1 and loss of Vps74 confer similar phenotypes. Skepticism about the authors' interpretation centers around timing during the maturation process:

– Two of the authors (Highland and Fromme) recently published an analysis of PI4P dynamics, and they concluded that PI4P is a hallmark of the late Golgi, sharing "little overlap with markers of less mature Golgi compartments".

– Vps74 has been found to require PI4P for its recruitment, so Vps74 would be expected to reside at the late Golgi. Indeed, even though Vps74 peaks earlier than the late Golgi marker Sec7, it seems to arrive at about the same time as Sec7.

– By contrast, Erd1 clearly overlaps with early Golgi enzymes, which have mostly departed by the time that Sec7 arrives. It is therefore unclear whether Erd1 and Vps74 overlap for long enough to engage in a direct physical interaction that would drive Golgi enzyme recycling.

We thank the reviewer for recognizing our work and the importance of our findings.

We find it quite interesting how Erd1, an integral membrane protein that localizes with its client early Golgi enzymes, and Vps74, a peripheral membrane protein that is recruited to medial Golgi compartments can cooperatively facilitate recycling. As reported in Highland et al., PI4P is indeed a hallmark of the late Golgi compartments. Additionally, previous work from Wood et al., has shown the important of PI4P binding for stable association of Vps74 with the Golgi, and the co-localization of Vps74 with medial Golgi markers. Our kinetic analysis of Vps74 association with the Golgi in the context of Golgi maturation agree with these previous findings and suggest more complex dynamics than just PI4P association. We find that Vps74 arrives at the Golgi after the medial Golgi marker, Gea2 when the PI4P levels are low, but perhaps enough to recruit Vps74. Vps74 peaks and leaves before peak Sec7 or PI4P signal. These observations suggest that in addition to PI4P, protein-protein interactions likely also play a role in regulating Vps74 recruitment and departure at the medial-late Golgi transition. Simultaneous analysis of Erd1 and Vps74 indicates a temporal overlap, and arrival of Vps74 marks the beginning of departure of Erd1.

The major recommendation, intended to address the concern raised above is to visualize the maturation kinetics of Erd1 and Vps74 simultaneously.

As suggested by the reviewer, we have now included simultaneous kinetic analysis of Erd1 and Vps74. This data is included in Figure 4G. As mentioned above, these results are consistent with our model.

Reviewer #2:

Based on bioinformatic, biochemical, morphological and genetic data, the authors propose a model of the mechanism of action of ERD1 according to which enzymes belonging to a selected group of glycosyltransferases are retained in the Golgi by recycling via COPI vesicles. According to this model the incorporation of enzymes in vesicles requires a sequential interaction of these enzymes first with ERD1 in the cis golgi and then with GOLPH3 in the medial trans Golgi. The first interaction has, in the proposed model, a preparatory or facilitating role for the second interaction, with GOLPH3, which mediates entry into COPI -dependent retrograde transport vesicles, and therefore the recycling, of the enzymes.

This is an interesting study in the rapidly developing area of the recycling mechanisms of glycosylation enzymes in Golgi. However, there are some weaknesses, which are outlined below.

The biochemical data on the binding between ERD1, VPS74 and Golgi enzymes are incomplete and should be completed with the demonstration that ERD1 and client enzymes interact directly, by means of the Y2H technique. This interaction is so far only demonstrated by co-precipitation of ERD1 with GOLPH3 and the enzymes, but these experiments do not exclude that ERD1 which interacts directly with GOLPH3, might interact indirectly with the enzymes, through its direct interaction with GOLPH3. Since the interaction between ERD1 and the client enzymes is at the heart of this study, it should be demonstrated with dedicated experiments.

On the technical plane, in the experiment in Figure 3C the efficiencies of co-ip in WT cells and in deltaERD1, once corrected for the inputs, do not seem very different. It would be useful to show inputs and Ips in the very same blot and to provide a quantification graph.

We thank the reviewer for the constructive suggestions.

As mentioned in the response to essential revisions above, we were unable to utilize the Y2H assay to score interaction between Erd1 and glycosyltransferases due to interference of the Nterminal Y2H tag with the Golgi localization of glycosyltransferases. By tagging the uminal ends, we were able to successfully employ a BiFC based approach to monitor interaction between Erd1 and Och1, as well as that between Erd1 and Vps74. These data are included in Figure 3A. We have also included the inputs and Ips from WT and erd1∆ mutant on the same blot and added a quantitation graph from multiple repeats of the experiment. These data are included in Figure 3D, 3E, Figure 3- supplement 1E.

The localization of ERD1, GOLPH3 and enzymes are interpreted by the authors in the context of the ERD1/GOLPH3 synergy model briefly outlined above, and presented in figure 4. However, not all of the data are easily reconcilable with this model.

The main difficulty is that both ERD1 and its client enzymes appear and recycle in the cis cisterna while GOLPH3 resides in the medial and partly in the trans cisternae, but not in the cis cisterna. Moreover, the scheme presented by the authors in Figure 4 indicates that the recycling of the cis enzymes (eg, Mann9) begins before the appearance of GOLPH3 in the cis cisterna. This is in principle not compatible with the model, unless the authors propose that non-detectable traces of GOLPH3 might be present in the cis cisterna and activate the recycling mechanism there. If the authors show that this is the case, it might be acceptable to present their model in this study. However, given that our understanding of Golgi enzyme recycling is actually in its infancy, the underlying mechanism might be more complex. For example, ERD1 could mediate the recycling of client enzymes in the cis cisterna in synergy with a hitherto unknown adapter other than GOLPH3 (perhaps COPI itself?), and might also mediate the recycling of enzymes in synergy with GOLPH3 in the medial cisterna. This is possible because according to the scheme in figure 4 ERD1 shows a peak in the cis cisterna but is clearly present also in the medial cisterna. Furthermore, all ERD1-dependent enzymes should also be indirectly dependent on GOLPH3 since GOLPH3 depletion causes ERD1 loss and lysosomal degradation. ERD1 might thus be an adapter capable of acting at various levels of the transport system: in Golgi to ER recycling and in glycoenzyme recycling at both the cis and the medial cisterna. This model can explain the data probably better than that presented by the authors in Figure 4, but is more complicated and requires some more assumptions. It is up to the authors to decide whether they want to consider this hypothesis as an alternative model.

A question that would need to be addressed in this context is whether all the enzymes found by the authors to depend on ERD1 are also GOLPH3 customers and in particular whether they all contain the recognition motif (F / L) – (L / I / V ) -xx- (R / K) for GOLPH3 described by Banfield.

While we present a simpler model, we agree with the reviewer that it is possible that the mechanism of protein recycling in the Golgi may be more complex for different cargos- with Erd1 assisting in Vps74-independent (for Golgi to ER recycling) and Vps74-dependent (for intra- Golgi recycling) steps. Indeed, Erd1 was identified in a screen for mutants defective in recycling of ER residents. Exploring these roles of Erd1 would require a more systematic approach in the future.

Using simultaneous kinetic analysis, we find that Erd1 and Vps74 do overlap (figure 4G). This interaction can also be captured by the BiFC and immunoprecipitation (figure 3A, 3B). Erd1 kinetics resemble early-Golgi proteins, some of which have been previously reported to be Vps74 clients and have a similar overlap time with Vps74 as Erd1. We think that these observations support a synergy model, but we agree with the reviewer about the possibility of a dual step role for Erd1.

As the reviewer correctly noted, since Erd1 itself requires Vps74 for its recycling, it is difficult to tease apart Erd1-only from Vps74 dependent clients. Indeed, all the glycosyltransferases that we tested and found to be dependent on Erd1, were also dependent on Vps74 for their recycling. This subset of glycoenzymes do appear to contain (F/L)-(I/L/V)-x-x-(R/K) sequence in their cytosolic tails previously proposed to bind Vps74 (figure 2- supplement 2E).

My recommendations to the authors have already been outlined above. In short, I think that the authors should:

Characterize the interaction between GOLPH3 and client enzymes by Y2H.

Discuss the weaknesses of their model as presented in figure 4 and, if they wish, mention the possibility of a more complex mechanism,

Clarify whether all ERD1 client enzymes contain the motif (F / L) – (L / I / V) -x-x- (R / K),

Discuss the earlier paper by Okamoto et al., (2008) that is not cited in this study.

In my opinion the authors should also discuss two recent papers (Rizzo et al; Parashurama et al., both in EMBO J), that are based on the concepts of recycling adapter and retention adapter and propose a model for the localization of GOLPH3-dependent enzymes in Golgi compartments in the context of cisternal maturation.

We thank the reviewer for the feedback. As listed above, we have addressed all the reviewer comments. We have also discussed the work published by Okamoto et al., Rizzo et al., and Pothukuchi…Parashuraman et al., in the text.

Reviewer #3:

The authors investigate the role of the yeast protein Erd1 in Golgi-dependent glycosylation. They conclude that Erd1 acts with Vps74, a known retention factor for Golgi glycosyltransferases, to direct the recycling of these enzymes in COPI-coated vesicles in the Golgi stack. The data are well presented and quantified, and the paper is clearly written. Addressing the role of Erd1, and insight into how glycosyltransferases are retained in the Golgi, are both interesting questions, but the authors data do not preclude alternative interpretations, and one or two aspects require resolution. These issues are summarized below:

1) A role for Erd1 in acting as a coreceptor with Vps74 to recycle glycosyltransferases is interesting but also raises some questions. Firstly, Erd1 is only found in fungi and not in metazoans, whereas Vps74 has metazoan orthologs that are known to play a role in glycosyltransferase recycling raising the question of why Erd1 is only needed in yeast. Secondly, there is published evidence for Erd1 acting as a channel/transporter for the movement of phosphate out of the Golgi lumen, and indeed the entire protein comprises a domain (the EXS domain) that is present in known phosphate transporters in plants and metazoans. Thus, careful dissection is required to determine if the effects seen are direct via an interaction with glycosylation enzymes, or if they are an indirect consequence of a perturbation of the Golgi lumen due to accumulation of inorganic phosphate. The fact that Erd1 was originally identified as having a defect in the retrieval of soluble ER residents from the Golgi suggests that the Golgi lumen may well be perturbed, possibly by a change in pH or cation content, and it is known that alterations in these features of the Golgi can affect glycosylation.

2) Perhaps the most striking finding is that Erd1 co-precipitates with Vps74 and that the presence of Erd1 is required for Vps74 to efficiently co-precipitate with the glycosyltransfereases that it is known to bind. However, the authors also show that the glycosyltransferases are destabilized by the loss of Erd1, and so the loss of apparent interaction may simply reflect the fact that there is less protein present to co-precipitate. Secondly, the authors show that Erd1 and Vps74 do not substantially co-localize, and so any tripartite complex would have to reflect a small sub-population of the proteins that briefly come together later in the Golgi stack during formation of COPI coated vesicles that are to be recycled. Finally, Vps74 is known to bind to COPI, and so if Erd1 also bound to COPI, then Vps74 and Erd1 may co-precipitate because they are held together via COPI, with Vps74 then bringing some glycosyltransferases into the complex.

3) Some aspects of the data may need resolving. Firstly, the authors provide clear images showing degradation of Och1p-GFP and Kre2-GFP in the vacuole in the absence of Erd1 (Figure 1F). However, the immunoblots in Figure 3D indicate that the levels of the intact proteins are unchanged in the absence of Erd1 which suggests that they are not destabilized and degraded. Secondly, the authors use the split ubiquitin system to provide evidence for an interaction between Erd1 and Vps74. As a control they remove the "cytoplasmic-tail" from Erd1, but they do not state how many residues were removed. The structural prediction for the EXS domain in Pfam suggests that the last membrane spanning helix of Erd1 would be very close to the C-terminus (Pfam entry PF03124), and the location of the truncation is not tested. Finally, the authors show some nice live cell imaging data to follow Golgi maturation. However, they do not directly compare Vps74 and Erd1. Such a comparison would be very helpful, especially as it seems from the other graphs that Erd1 is significantly depleted from the maturing cisterna before the time when there are substantial amounts of Vps74 present.

We thank the reviewer for the comments. We acknowledge that the role of Erd1 in phosphate transport is likely an important function. However, as mentioned above in our response to essential revision, we have now included data to show the effect on protein recycling upon the loss of Erd1 cannot be reconciled by just changes in the Golgi ionic environment, and this effect is distinct from that due to the loss of Pmr1 (Figure 2- supplement 2). Our observations also suggest that like Erd1 in yeast, other transmembrane proteins may play similar roles in GOLPH3 dependent recycling in metazoans. We were also surprised to observe that despite the recycling defect observed in erd1∆ and vps74∆ mutants, we saw a modest reduction in steady state levels of tagged Kre2 or Och1 on western blots. This may be reflective of upregulated biosynthesis or slower degradation of these constructs in the vacuolar lumen. We also confirmed that tagged Och1 can complement the temperature sensitivity of och1∆ mutant and is functional (Figure 4- supplement 1D). Reduced co-immunoprecipitation of Vps74 and Och1/Kre2 in the absence of Erd1 may be exacerbated by the fact that a significant fraction of Och1/Kre2 are sequestered away from the Golgi. However, this is in part mediated because of a defect in productive recycling complex formation required to maintain these enzymes at the Golgi, highlighting the importance of Erd1’s role in the pathway.

We have now included a simultaneous kinetic analysis of Vps74 and Erd1 and show that there is overlap between the two (figure 4G). Additionally, using split-fluorescent protein complementation to monitor protein-protein interactions, we show that Erd1 interacts with wild type Vps74, as well as Vps74 R6-8A COPI binding mutant, suggesting the interaction between Erd1 and Vps74 is not bridged via COPI binding (figure 3A).

The C-terminal truncation of Erd1 employed in the Y2H assay, was initially generated based on the original truncation mutation identified in the ‘erd’ screen by Hardwick et al., along with the cytosolic region prediction on the Uniprot database and lacks the last 100 aa. Based on structural modeling of Erd1 using AlphaFold, we agree that truncation likely disrupts the intramembrane helices of the EXS domain. We subsequently also generated truncations of Cterminal 10 and 50 aa to test Y2H interaction with Vps74, but they all affected protein stability. It is unclear if they are degraded because of loss of interaction with Vps74 or structural instability and cannot be conclusively used to inform of the interaction region with Vps74 and have not been included in the revised manuscript. How Erd1 interacts with Vps74 will require more in-depth analysis in the future.

The prior publications on Erd1, the absence of an orthologue in mammals even though they have a Vps74 ortholog, and the potential role of COPI as a bridge between the two proteins, really necessitate a much more in depth and substantial analysis for a broad readership journal such as eLife. Ideally, in vitro reconstitution of binding with purified proteins would resolve many issues, but I appreciate that this may be technically challenging. Below I have suggested some things that could be done to strengthen the paper's conclusions, and at the very least these may be helpful to the authors to consider before resubmitting elsewhere:

1) Examination of the effect of other gene deletions that affect the ionic content of the Golgi such as deletion of Gdt1, Pmr1 or Stv1, on Och1p-GFP and Ktr2p-GFP levels and on glycosylation.

2) Resolution of the apparent contradiction of the effects of Erd1 deletion on Och1 and Ktr2 by microscopy and blotting assays. If available, antibodies to the endogenous proteins could be used to test their levels in wild type and mutants.

3) Does the mutation in the COPI binding site of Vps74 affect its Golgi localization? If not, the authors should check if this prevents the co-ip with Erd1.

4) It would be very valuable to add videos and graphs to follow the Golgi localization of Erd1 vs Vps74 to better reveal their spatial relationship over time.

Please see our detailed response above that addresses these points.

5) Substantial new insight would be provided by determining what part of at least of the Erd1-dependent glycosyltransferases interacts with Erd1. This could be addressed by making chimeric proteins that contain only either the cytoplasmic tail, or the TMD, or the lumenal domain of a Erd1-dependent glycosyltransferase in the context of an Erd1-indepenent glycosyltransferase. The localization and co-ip of these chimeras could be then be tested.

6) The authors argue that the cytoplasmic tail of Erd1 interacts with Vps74. This could be tested biochemically as has been done for the tails of glycosyltransferases. If the authors keep the split-ubiquitin experiments they should confirm that the constructs are localized to the Golgi.

7) Are their glycosyltransferases that do no rely on Vps74? It would be useful to test if these are affected by loss of Erd1.

We have attempted to generate chimeric proteins by swapping the cytoplasmic or transmembrane domain of an Erd1-dependent and independent glycosyltransferase. However, the chimeric protein was unstable and degraded even in wild type cells. While we cannot address the comments about binding determinants with our current toolset, this will be the focus our investigations in the future. All the glycosyltransferases that we have tested that rely on Erd1, also rely on Vps74, so we are currently unable to test the effect of Erd1 or Vps74 alone.

https://doi.org/10.7554/eLife.70774.sa2

Article and author information

Author details

  1. Richa Sardana

    1. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    2. Department of Molecular Medicine, Cornell University, Ithaca, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review and editing
    For correspondence
    sardana@cornell.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5861-9648
  2. Carolyn M Highland

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Contribution
    Investigation, Visualization, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4029-0113
  3. Beth E Straight

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Christopher F Chavez

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. J Christopher Fromme

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Contribution
    Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8837-0473
  6. Scott D Emr

    Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – review and editing
    For correspondence
    sde26@cornell.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5408-6781

Funding

Cornell University Research Grant (CU3704)

  • Scott D Emr

National Institutes of Health (R35GM136258)

  • J Christopher Fromme

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

Acknowledgements

We thank Ashley Yu for assistance with cloning, and all the members of the Emr laboratory for helpful discussions.

Senior Editor

  1. Vivek Malhotra, The Barcelona Institute of Science and Technology, Spain

Reviewing Editor

  1. Benjamin S Glick, The University of Chicago, United States

Reviewers

  1. Benjamin S Glick, The University of Chicago, United States
  2. Alberto Luini, National Research Council, Italy

Publication history

  1. Received: May 28, 2021
  2. Accepted: November 17, 2021
  3. Version of Record published: November 25, 2021 (version 1)

Copyright

© 2021, Sardana 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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