A systematic proximity ligation approach to studying protein‐substrate specificity identifies the substrate spectrum of the Ssh1 translocon

Abstract Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein–protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins ‐ substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel‐lctiv (CELlular biotin‐Ligation for Capturing Transient Interactions in vivo), uses high‐throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel‐lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel‐lctiv can provide direct information on substrate specificity even for highly homologous proteins.

1. The Figure 1 legend indicates that the BirA tagged strains are mated with ~6000 AviTag yeast strains but results in Figure 2 pie chart account for 4812 total readouts. Presumably 1000 or more strains could not mate or did not produce viable diploids with the BirA tagged strains? It would be helpful to explain this differential. 2. If possible, suggest including more of the raw data (in supplementary) that supports the pie chart in Figure 2. Table S1 shows the 111 proteins that display preference for Ssh1 (out of 586 total interactors?) and the fold change (in rank order) for interaction preference. At a minimum, similar data on Sec61 preference and the list of positive interactors should be included. There may also be useful information in the relative biotinylation signal for each BirA and AviTag combination when significantly above background. This is presumably a readout of AviTag protein abundance, dwell time and orientation to BirA activity. The data could be useful to other investigators. 3. The authors might want to be more cautious in interpreting impact of the UPR on ssh1 phenotypes in the results and discussion. The Wilkinson et al 2002 paper referenced used very different conditions to detect UPR in ssh1 deletions strains. Jonikas et al 2009 does not detect a chronic UPR in ssh1 deletion cells and the conditions used in the current study seem more similar to the 2009 report. It seems more likely that deficits in translocating/localizing specific proteins causes the observed phenotypes instead of chronic UPR due to reduced ER levels of PDI.
The reported findings support their primary conclusions. The technology development and results are significant, highly relevant and will be of interest to a broad readership in cell and membrane biology.

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Evidence, reproducibility and clarity:
Evidence, reproducibility and clarity (Required) **Summary:** Cohen et al. presented a high-throughput approach to analyze protein-(putative) substrate interactions in yeast using BirA biotin ligase and its acceptor peptide AVI tag. Using this system, the authors identified the common and unique substrates of translocation pores, Sec61 and Ssh1. Interestingly, the differential substrates between Sec61 and Ssh1 seem to be explained by the degree of hydrophobicity in signal peptide sequences, which was also nicely demonstrated by an experiment showing that swapping the first three amino acids of substrate proteins is sufficient to convert the substrate specificity. While I appreciate that the approach is high-throughput and simple (does not require mass spectrometers), there are some technical comments to be addressed.
1. Why was BirA used to study transient interactions? Biotinylation through BirA is slow (that is why it takes several hours to label proximity proteins) and thus it may not be suitable for capturing transient interactions. Instead, TurboID would be more suitable as the biotinylation reaction is faster than BirA. A reasonable explanation using BirA is required. 2. One key question is whether biotinylated proteins identified by this method are substrates or proteins just proximal to Sec61 or Ssh1 due to close cellular localization (e.g. ER membrane) or same protein complex members. An experiment or analysis would be required to confirm that the proteins they identified are indeed potential substrates. 3. Along the same line, if proteins identified by this approach are bona fide substrates of Sec61 and Ssh1, proteins having signal peptides should be enriched in the candidate list of substrates. However, it does not look like that according to Figure 2A where the secretome proteins/total proteins ratio appears to be similar among the 4 categories (e.g., Ssh1 preferring,No preference,and Not interacting or excluded). The authors should comment on this. 4. Figures 1-2: They should comment on the reproducibility of the method. How many independent experiments were performed? If performed, how was reproducibility of results? 5. Figure 3: It is important to know the overlap of proteins commonly identified in both the interaction screening and protein localization assay. A Venn diagram that compares results between the two high-throughput assays would be useful. 6. Figure 4A (GO term): The authors mentioned that " the most consistent and repeating GO term group was those related to budding and polarity process. These include: "Establishment or maintenance of cell polarity"; "Development process involved in reproduction"; "Bipolar cellular bud site selection"; "Cell budding" and "Structural constituent of cell wall". Are protein sets in these functional categories similar or different? I am asking because GO enrichment analysis often provides apparently different functional categories but similar protein sets are included. **Referees cross-commenting** The comments from reviewer #1 are reasonable and would further strengthen the quality of the paper.

Significance (Required)
The approach is high-throughput and simple (does not require mass spectrometers).
The differential substrates between Sec61 and Ssh1 seem to be explained by the degree of hydrophobicity in signal peptide sequences, which was also nicely demonstrated by an experiment showing that swapping the first three amino acids of substrate proteins is sufficient to convert the substrate specificity.

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Cohen et al, 2022
Reviewer #1 1. The Figure 1 legend indicates that the BirA tagged strains are mated with ~6000 AviTag yeast strains but results in Figure 2 pie chart account for 4812 total readouts. Presumably 1000 or more strains could not mate or did not produce viable diploids with the BirA tagged strains? It would be helpful to explain this differential. We thank Reviewer #1 for pointing out this gap which occurred exactly as they have interpreted. We have now corrected the figure legend to say exactly how many strains were in the library (5330) and have clearly stated the attrition of strains.
2. If possible, suggest including more of the raw data (in supplementary) that supports the pie chart in Figure 2. Table S1 shows the 111 proteins that display preference for Ssh1 (out of 586 total interactors?) and the fold change (in rank order) for interaction preference. At a minimum, similar data on Sec61 preference and the list of positive interactors should be included. There may also be useful information in the relative biotinylation signal for each BirA and AviTag combination when significantly above background. This is presumably a readout of AviTag protein abundance, dwell time and orientation to BirA activity. The data could be useful to other investigators. This is a very good suggestion. We have now added a supplementary table (Supplementary  Table S2) with the interaction results for proteins that preferred Sec61 and proteins that did not show any preference.
3. The authors might want to be more cautious in interpreting impact of the UPR on ssh1 phenotypes in the results and discussion. The Wilkinson et al 2002 paper referenced used very different conditions to detect UPR in ssh1 deletions strains. Jonikas et al 2009 does not detect a chronic UPR in ssh1 deletion cells and the conditions used in the current study seem more similar to the 2009 report. It seems more likely that deficits in translocating/localizing specific proteins causes the observed phenotypes instead of chronic UPR due to reduced ER levels of PDI. We agree that as result of the different conditions it is difficult to compare our data to the Wilkinson et al 2002 paper. We have therefore adjusted the text to remove this interpretation.

Reviewer #2
1. Why was BirA used to study transient interactions? Biotinylation through BirA is slow (that is why it takes several hours to label proximity proteins) and thus it may not be suitable for capturing transient interactions. Instead, TurboID would be more suitable as the biotinylation reaction is faster than BirA. A reasonable explanation using BirA is required. We thank the reviewer for this comment which indeed also reflects our "process" of thinking. Originally, we did try to use TurboID to identify potential cargo proteins. We now have a very robust methodology to look at protein substrates by TurboID (see: https://www.biorxiv.org/content/10.1101TurboID (see: https://www.biorxiv.org/content/10. /2022 and so this would have obviously been the easier and faster method. However using this approach we mainly observed ribosome subunits and cytosolic proteins for Sec61 and very few, mostly cytosolic, proteins for Ssh1. Our interpretation of this is that since all biotinylation of TurboID strains Authors Revision Plan occurs in parallel there is "competition" from the highly abundant and strong interactors and this does not leave a possibility to detect the low-abundance and even more transient interactions that we would like to measure. It seems that although birA/AviTag are much slower, the specificity and singular ligation site that should be exposed also in cotranslational-translocation events, are more suitable for this specific experimental setup. We have now explained this also in the text.

2.
One key question is whether biotinylated proteins identified by this method are substrates or proteins just proximal to Sec61 or Ssh1 due to close cellular localization (e.g. ER membrane) or same protein complex members. An experiment or analysis would be required to confirm that the proteins they identified are indeed potential substrates. This is indeed an extremely important point and we have now carefully addressed it in the text. We are certain that the reviewer is right and that many of the biotinylated proteins are same complex members and cytosolic components that happen to be in proximity (maybe regulators?) just as the reviewer suggested. We now clearly write this in the results section. This is why we focused on signal peptide containing proteins. These proteins CAN NOT be complex members nor biotinylated simply due to proximal location on the ER membrane. This is since they reside inside the lumen of the ER if they are soluble or are inserted (if they contain also a transmembrane domain) with their tagged N' facing the lumen of the ER (So called Type I proteins). The only way such proteins could be biotinylated by the slow BirA on the cytosolic surface is if they passed through the pore of the translocon.
3. Along the same line, if proteins identified by this approach are bona fide substrates of Sec61 and Ssh1, proteins having signal peptides should be enriched in the candidate list of substrates. However, it does not look like that according to Figure 2A where the secretome proteins/total proteins ratio appears to be similar among the 4 categories (e.g., Ssh1 preferring, No preference, and Not interacting or excluded). The authors should comment on this. We thank Reviewer #2 for highlighting this point that was not clear from our text and figures. There is definitely an enrichment of Signal Peptide (SP) containing proteins amongst the proteins that we think are bona fide substrates however this was not visualized clearly. To highlight this point we have modified Figure 2 and added a bar graph showing the distribution of SP and TMD proteins within the potential secretome. This graph now highlights the enrichment of SP containing proteins in the groups of proteins that preferred Sec61 or Ssh1 in comparison to the group that did not show a preference. We also now add a citation from a new manuscript from the Hegde lab that suggests that indeed soluble SP containing proteins are the key clients for the translocon pore (https://pubmed.ncbi.nlm.nih.gov/36261528/). We have also added a section to the discussion as to why we do not see differential enrichment of SRP or its receptor on either pore although in the past this was suggested to be the key difference between the two translocons.
4. Figures 1-2: They should comment on the reproducibility of the method. How many independent experiments were performed? If performed, how was reproducibility of results?
Thank you for highlighting that this was not clarified enough -we have now extended the materials and methods section to make all of the above issues clear and apparent. In short, we performed 3 biological repetitions for each experiment and for each biological repeat we performed 3 technical repeats making our results altogether rely on 9 repeats. We then excluded proteins in two cases 1. If strains were missing in either of the collections (so there was no complete set to compare them) -this caused us to drop 661 strains. 2. In cases where the proteins were expressed at very low levels of extracted poorly in our assay -we defined this as the signal being ten standard deviations (or more) lower than the rest of the signals on the same membrane -this caused us to lose an additional 93 strains.
Importantly, the SD between all 9 repeats never rose above 3 (see graph below showing al strains arranged by order in library and the SD between all 9 repeats) and also now stated clearly in the text) hence we think that our method is highly reproducible 5. Figure 3: It is important to know the overlap of proteins commonly identified in both the interaction screening and protein localization assay. A Venn diagram that compares results between the two high-throughput assays would be useful.
We have indeed considered making this Venn diagram (See below). However, since the connection between the screens is not direct due to the fact that the protein localization is downstream to translocation events or maybe completely independent of it, we found that the number of specific proteins that are in both screens is low. However, there is a much larger overlap in joining processes and functions, therefore we decide to make the grouping showed in Figure 4B. We would prefer not to show this figure in the manuscript however we leave this to editorial decision.
6. Figure 4A (GO term): The authors mentioned that " the most consistent and repeating GO term group was those related to budding and polarity process. These include: "Establishment or maintenance of cell polarity"; "Development process involved in reproduction"; "Bipolar cellular bud site selection"; "Cell budding" and "Structural constituent of cell wall". Are protein sets in these functional categories similar or different? I am asking because GO enrichment analysis often provides apparently different functional categories but similar protein sets are included. Indeed, this reviewer is totally correct and this is also the case here to some extent. There is some level of overlap between the GO terms. However, in our case this overlap is quite small: Out of the 77 genes that have one of those GO terms assigned only 2 have all 4, 9 have 3 and 4 have 2 of the GO terms therefore we believe that in this case this issue with GO terms hierarchy and assignment is not redundant. We are happy to highlight this in the figure or text if this is deemed to be important.  (EMBOJ-2023-113385). We sent it back to the two initial referees and have now received their reports (please see below). Referee #1 has a remaining comment, which you should address, but I am happy to say that we can overall proceed towards publication in The EMBO Journal. Therefore, I would ask you to please also resolve a number of editorial issues that are listed in detail below. Please use the document that the data editors have added their comments to for any changes, both those to address the referee comment, as well as those to resolve the editorial issues (see below). If you have any further questions regarding the specific points listed below or the final manuscript version, please feel free to contact me.
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Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide ***************************************** Revision to The EMBO Journal should be submitted online within 90 days, unless an extension has been requested and approved by the editor; please click on the link below to submit the revision online before 23rd Apr 2023: Link Not Available **************************************** We have now received re-review reports from the two referees that initially appraised your manuscript for Review Commons. As you will see, you have addressed their concerns satisfactorily. Before I can finally accept the manuscript though, I have noticed some small editorial points that remain and need to be addressed. In this regard would you please: -state how the relevant error bars were calculated in the legend to figure 5A, -replace the second sentence in the figure legend for 5B to the suggested "Shown are Pdi1 and Mpd2 (two PDI family members that showed an interaction preference to Ssh1) and Mpd1 (a protein that showed an interaction preference for Sec61)", -consider the following suggestions for the fifth sentence of the introduction "Hence, there is a general need in cell biology for approaches able to describe the particular range of any given protein's functions in vivo, even when that protein belongs to a large and closely-related family.", -provide the synopsis image as a jpeg, tiff or a png file, -provide page numbers for the Table of Contents  We have now received re-review reports from the two referees that initially appraised your manuscript for Review Commons. As you will see, you have addressed their concerns satisfactorily. Before I can finally accept the manuscript though, I have noticed some small editorial points that remain and need to be addressed. In this regard would you please: -state how the relevant error bars were calculated in the legend to figure 5A, This has now been added to the text and a new article file has been uploaded to the system -replace the second sentence in the figure legend for 5B to the suggested "Shown are Pdi1 and Mpd2 (two PDI family members that showed an interaction preference to Ssh1) and Mpd1 (a protein that showed an interaction preference for Sec61)", Done and in new article file as above -consider the following suggestions for the fifth sentence of the introduction "Hence, there is a general need in cell biology for approaches able to describe the particular range of any given protein's functions in vivo, even when that protein belongs to a large and closely-related family.", Agreed -this is a great way of phrasing it. Now in the new text file.
-provide page numbers for the Table of Contents and use the nomenclature 'Appendix Figure S1' and so on; please also remove supplementary figure legends from the manuscript file and add them below each figure in the Appendix PDF, and I hope we did this right -if there is a problem please let us know. since there was only one sup Figure the appendix was a bit small.
-add the following sentence to the Disclosure and Competing Interests Statement "Maya Schuldiner is an EBM Member. This has no bearing on the editorial consideration of this article for publication." Added in the new text file Many thanks for all of your help Maya 17th Mar 2023 2nd Revision -Editorial Decision Dear Maya, I am pleased to inform you that your manuscript has been accepted for publication in the EMBO Journal.
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