Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts

  1. Mirjam Pennauer
  2. Katarzyna Buczak
  3. Cristina Prescianotto-Baschong
  4. Martin Spiess

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Abstract

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.

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  1. Version published to 10.1083/jcb.202106100
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    Reply to the reviewers

    Response/revision plan

    (Point-by-point response)

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Pennauer et al is the first to systematically investigate the role of class I&II Arfs using a knockout approach. It builds on earlier work by the Kahn lab who used an RNAi approach (Volpicelli-Daley et al. 2005) and is complementary to the overexpression approach used by the Hauri lab (Ben-Tekaya et al, 2010). The work is elegant and the data are strong. I am strongly in favor of publishing this work and my comments are technical in nature (2-5) and a request for some text changes (1). have the following comments for improvements:

    1- When it comes to evaluating the role of depletions of Arfs on cell fitness, it would be better to use a non-transformed cell line. I am not asking the authors to go through the painstaking process of generating knockout cell lines in RPE1 cells for instance. Rather, I suggest that the authors make the reader aware that conclusions about cell survival have to be taken with care due to the use of a transformed cell line.

    We will add this valid point to the Discussion.

    2- Why do Arf1 and Arf4 ko cells grow more slowly. Is it a higher rate of cell death? Is it a block in a certain phase of the cell cycle. Given the link of the Golgi to G2-M entry, I think that an analysis of the cell cycle distribution would add more depth to these data. If the cell cycle distribution is unaffected, then I would conclude that that the difference in doubling time are due to reduced cell survival. If there is an effect on the cell cycle distribution, then the conclusion of the authors is safe that no single Arf is required for survival

    We plan to analyze cell cycle distribution.

    3- It is not clear to me how many cells were quantified in Figure 2D-F. I suppose that each dot represents a cell. In this case, the number of cells quantified is a bit low. Such a quantification of fluorescence intensities in two channels in the same region is a simple task and I think it should be no problem obtaining at least 100 cells per condition.

    We will add the number of cells analyzed to the figure legends: At least 40 Golgis were quantified in each experiment. thus >100 in total.

    4- Is the drop in the ratio of beta-COP/GM130 in Arf1 depleted cells reflecting reduced recruitment to the Golgi? Because the Golgi is bigger, it might be reflecting a reduced density in the number of coatomer molecules per surface area. If it is due to reduced recruitment, then the ratio of membrane/cytosolic betaCOP should be altered. This of course requires to show that the knockout does not affect total levels of coatomer. I think that such fractionation experiments would be a valuable addition to the manuscript and increase the depth of the data.

    We are currently performing immunoblot analysis to determine bCOP levels.

    In the Figure below, we have plotted the total intensity of GM130 or bCOP per Golgi from our immunoflurescence data. Total intensity of GM130 significantly increased in the cell lines lacking Arf1, consistent with the increase in Golgi volume. The amount of bCOP at the Golgi remained constant, resulting in reduced bCOP/GM130 ratio. Deletion of Arf1 thus results in reduced rate of coat recruitment that is compensated by an increase in Golgi mass. In the simplest model, reduced formation of Golgi-exit carriers causes Golgi growth until exit carrier formation allows for the required flux.

    We propose to include this data in the revised manuscript.

    FIGURE

    5- The finding that Arf4-ko cells exhibit a defect on retrieval of ER-resident proteins is exciting, and in my opinion, it is the most significant finding in this manuscript. How can this be reconciled with the lack of an ARf4 ko effect on coatomer recruitment to the Golgi. Looking carefully at the data, I see that in 2 out of 3 experiments, Arf4 ko reduced the betaCOP/GM130 ratio. This is why I think it is crucial to perform more experiments and add more cells to increase the confidence in the data. Reduced retrieval of ER chaperones is frequently found in tumors and we still don't understand the reason behind this. Therefore, this finding is of significance beyond the community of cell biologists.

    We plan to repeat quantitation with COPI for better statistical validity.

    6- I find Figure 6A confusing. Why do Arf1 overexpressing parental HeLa cells exhibit less Arf1 than control cells?

    In order not to overload the immunoblot of Arf overexpressing lysates, a smaller aliquot (1/20) was loaded. We will indicate this directly below the blots to make this more obvious in the revised figure.

    7- Why was the following condition not tested: Arf4ko cells with Arf1 overexpression. Given the importance of Arf1 in retrograde (Golgi-to-ER) trafficking, I would expect a partial rescue of the retrieval of ER chaperones.

    We will to do this experiment.

    Reviewer #1 (Significance (Required)):

    **Significance of the work:**

    The paper is important because it is the first to examine the role of Arfs using a knockout approach. Another very important finding is that Arf4 depleted cells exhibit problems with retrieval of ER chaperones. This is a very novel finding and to the best of my knowledge

    **Audience:**

    The primary audience is of course the community working on membrane trafficking, organelle biology and proteostasis. However, I think that the data on the role of Arf4 in retrieval of ER chaperones might be of relevance for cancer biologists. Secretion of ER chaperones is frequently found in many tumors and we still do not understand why this is happening and what the significance thereof is.

    **My own expertise:**

    Export from the endoplasmic reticulum Golgi fragmentation in cancer cell migration Rho GTPases Kinase signaling Pseudoenzymes Cell migration of breast cancer cells Proteostasis in multiple myeloma

    **Referee Cross-commenting**

    Just a follow-up comment from my side:

    I agree that it has not been unequivocally established that Arf1 is the main/sole of retrograde transport. However, even less established is the role of Arf4 in this process. The authors show that it is mainly Arf1 depletion that reduces the amount of COPI at the Golgi (ratio of COPI/GM130). Thus, I remain very surprised that it is actually the Arf4 depletion that results in reduced retrieval.

    What is the significance of having less COPI at the Golgi in Arf1-ko cells? Certainly, the Golgi is not more "leaky". Does the level of COPI at the Golgi not reflect the strength of retrograde trafficking? Maybe there is no less COPI at the Golgi, and it only appears to be less, because the Golgi is bigger. This is why a simple fractionation experiment would be good. Something like making a cytosol and a microsome fraction and looking at the ratio of COPI (Cyt/Mem).

    If both reviewers think it is too much, or unlikely to work, then I am happy to drop this point.

    Below are my comments to the evaluations by the other two reviewers:

    1- I agree with most comments that the two other reviewers made. Some of them are actually overlapping with mine (e.g. the use of a cell line other than HeLa).

    2- I am not sure whether the impact of the paper would improve by adding data on Arf6.

    3- To the comment on Golgi polarity. Maybe we could be more specific here and say that it would be sufficient to show that a trans-Golgi protein and a cis-Golgi protein can be separated by fluorescence microscopy, or whether we alternatively want them to actually do it by immunogold labeling for EM (which is more difficult).

    4- I agree with reviewer 2 that the work proposed needs 1-3 months. I think reviewer 3 is a bit too optimistic with 1 month, because her/his comment on using a cell line other than HeLa cannot be addressed in just a month.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Pennauer uses HeLa cells and CRISPR/Cas9 to delete the 5 members of the class I and class II ARF family of small regulatory GTPases either individually or in combinations. The characterization of the KO cells is excellent and convincingly demonstrates that true KOs were generated. The quality of the data presented is high. Using the KO cells she documents minor alterations in Golgi architecture and the recruitment of vesicular coats in cells deleted of all ARFs except ARF4. In contrast, there is a significant lack of retention/recycling to the ER of KDEL-containing ER proteins in ARF4 KO cells, with numerous ER chaperones now released into the medium (the ARF4 KO secretome). This is a well-done study that showcases the ability of ARF4 alone to sustain cellular life (quite a surprise to this reviewer). Yet, the characterization of the phenotypes is somewhat minimal and the conclusions would be more robustly supported by additional experiments. Specifically:

    1. The authors completely ignore class III ARF6 and this paper would be much more comprehensive and informative if analysis of that ARF was also included (ARF6 has been seen at the Golgi and also mediates endosomal trafficking that intersects with the TGN).

    In agreement with the reviewers' consensus in cross-commenting, we consider Arf6ko to be beyond the scope of this study.

    Although the overall Golgi architecture seems to be largely conserved, it remains essential to test whether Golgi polarity is similarly maintained, and such data would significantly expand the significance of the reported findings

    We have performed super-resolution microscopy of wild-type and Arf1ko Golgis for GM130 and TGN46 as cis- and trans-Golgi markers, respectively, showing that polarity is still intact for Arf1ko, the morphologically most affected knockout cell line. We plan to include the following Figure in the revised manuscript.

    FIGURE

    Golgi complexes were imaged by superresolution microscopy for GM130 (green) and TGN46 (red), and displayed as maximum intensity projections, or tomographic 2D slices. Scale bar, 3 μm.

    Since there is a defect in retrieval of KDEL-proteins, it would be important to show the intracellular localization of the KDEL-R in the cells (especially in the ARF4 KO cells that don't retrieve KDEL-GFP) - is the receptor degraded, stuck in some specific place - knowing that would increase the impact of this study and provide a mechanistic explanation for the observed phenotype

    We plan to perform immunoblot analysis for KDELR to test for changes in levels in Arf deletion cells, and immunofluorescence microscopy to analyze changes in KDELR localization.

    The rescue experiments in Figure 6 are good as far as they go, but this experiment would be much more informative if in addition to the same class rescue, the other class ARFs (at least one!) were also characterized.

    We will to do this experiment.

    This is maybe a little too much to ask, but since the authors propose a mechanistic explanation for the ARF4 KO KDEL phenotype as being due to different effectors recruited by this ARF (in this case different COPI isotypes - this study would increase in impact by actually testing this mechanisms by assessing whether ARF4Q71L mutant preferentially bound any particular isotype of COPI or even try to do mass spec to identify relevant effectors for this extremely interesting ARF.

    We also think that this additional analysis is beyond the scope of this study.

    The Discussion is a very limited and would be more impactful by adding some discussion of organismal effects of ARF deletions (many are embryonic lethal while cells seems to live quite happily) or mutations (links to cancer come to mind here), as well as some mention of data from yeast ARF (what is and isn't essential in those cells). As is, the authors miss an opportunity to highlight the importance of their findings as they relate to current knowledge of ARFology.

    We agree to add a discussion of information on embryonic lethality and disease.

    Reviewer #2 (Significance (Required)):

    This is an important paper for the ARF field and people interested in ARF signaling will be glad to read about the findings and perhaps also use the developed KO cell lines - this is a significant advancement. The impact would be even higher if some of the experiments suggested above were incorporated into the manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This paper describes the application of CRISPR/Cas9 to systematically delete from HeLa cells all four Arf genes, either singly or in combination. The authors find it is possible to generate a number of double deletions (notably one lacking both Class I Arfs), and a triple deletion lacking all but Arf4. The authors characterise the structure of the Golgi in these mutants as well as retention of ER residents. The work is a comprehensive study of an exceptionally high technical standard. There is excellent validation of the deletions, and then the application of a wide range of methods including immunofluorescence, electron microscopy and mass-spectrometry, all with careful and extensive quantitation. The finding that cells can survive without Class I Arfs is interesting and unexpected, as is the fact that Arf4 alone is sufficient. This work will provide an excellent platform for future studies on Arf protein function in human cells. There are of course many questions that arise from these findings, but given the scope and quality of the work they would seem better left for future publications. There is one experiment that could be added, and some additions needed to the text for clarity and minor adjustments to the figures (all listed below), but if these are addressed, this would be a high quality paper of wide audience to a cell biological audience.

    **Specific comments:**

    1. Have the authors tested the levels and/or localisation of the KDEL receptor in the various lines? This is not essential, but if it were easily done, it would add to the work on ER resident secretion.

    We plan to perform immunoblot analysis for KDELR to test for changes in levels in Arf deletion cells, and immunofluorescence microscopy to analyze changes in KDELR localization.

    1. The work is entirely done in HeLa cells. The authors should note that the situation might be different in other cells types and cell lines. For instance, the DepMap CRISPR database suggests that quite a lot of cell lines are strongly affected by loss of Arf1.

    We agree to add a discussion on known effects in other tissues.

    1. Figure 2. Please show single channels as grey scale, and only merge as RGB. This is easier to see, especially for the colour blind. Likewise, Figure 3D would be clearer in greyscale rather than green, and 6B better in grey than in red.

    We will make these changes.

    1. Figure 5C. A brief comment is needed as to why it might be that BiP and calreticulin are not so efficiently secreted when Arf5 is knocked out in addition to Arf4.

    This was a mistake in labeling that lane and will be corrected. It should read "Arf3+5ko" not "Arf4+5ko. Thank you for pointing this out.

    1. Discussion:

    a) The authors should relate these studies to work in other species. For instance, in yeast reduction of Arf levels causes the Golgi to enlarge (PubMed ID 9487133).

    We can discuss this.

    b) Some more discussion is needed of the fact that Arfs may not all act in the same part of the Golgi, which could explain some of the differences observed between the various deletions.

    We can add this point in the discussion.

    Reviewer #3 (Significance (Required)):

    The Arf GTPases have been studied extensively for over 30 years as major regulators of Golgi function. They are essential for the recruitment to Golgi membranes of both COPI and clathrin/AP-1 coats, as well as various other proteins that regulate Golgi function. In addition, they have been reported to have roles in viral replication, and even other cellular processes such as lipid droplet formation and mitochondrial division. In humans there are four Arfs, Arf1 and Arf3 (Class I Arfs), and Arf4 and Arf5 (Class II Arfs). All are present on the Golgi, but their precise individual roles have remained unclear. Attempts have been made to deplete individual Arfs using RNAi, but incomplete knockdowns have made the results hard to interpret.

    **Referee Cross-commenting**

    There is probably no need for a prolonged debate about this, but I agree that the importance of Arf4 is striking, but it reflects the nature of this work that CRISPR has finally allowed these sorts of questions to be addressed unequivocally. COPI is also involved in recycling of Golgi resident enzymes, and it may be that Arf1 acts in this role.

    If the authors check levels of COPI by blotting, and measure the intensity over the Golgi by quantitative IMF, that will reveal whether stability or membrane association if affected without fractionation which is probably less reliable.

    If they want to do some extra experiments, then it would be quite easy to check the levels of some Golgi enzymes, or look at lectin binding as a proxy for glycosylation enzyme levels.

    Overall, I agree with the positive comments of Reviewers 1 and 2, and it good that we all recognise the quality and importance of the work. However, I feel that one or two of their requests go beyond the scope of a single publication, or would add rather little for a lot of additional work. It is of course easy to propose experiments that someone else has to do!

    **[On] Reviewer 1:**

    Point 4. I agree that it would be useful to perform a blot to determine if the levels of coatomer are effected in the various KO lines. I am not sure if Reviewer 1 is also proposing fractionation to determine cytosol vs membrane ratio, but if so, then this would be less useful as peripheral membrane proteins tend to fall off membranes during fractionation and so such analysis is generally questionable. A blot, and clarification of the way the COPI/GM130 ratio is determined, would answer the key points in a relatively straightforward way.

    Point 5. I agree that the defect in retrieval of ER residents in Arf4-KO is striking, but it a clear effect even if the reviewer does not understand it themselves! It does not seem so surprising to me, given that Arf4 is likely to act on the early Golgi were such retrieval occurs from. However, the experiment suggested by myself and Reviewer 32 of checking the levels and localisation of the KDEL-receptor would seem to me a good first step to addressing possible mechanism, and certainly sufficient for an initial publication.

    Point 7. I am not sure that it has been unequivocally established that Arf1 is important in retrograde traffic. The reality is that many labs have taken Arf1 as being representative of all others and so concentrated biochemical and in vivo studies on this protein. This paper is really important as it highlights the need to investigate both Class I and Class II Arfs, and to bear in mind that their roles in vivo may well be more distinct than their in vitro properties would lead one to suspect. Perhaps, the simplest explanation for this is that the GEFs that activate them have a strong preference for one or the other.

    Follow up Comment 1. I was not suggesting that the authors repeat all this in a cell line other than HeLa cells, as this is clearly impractical. HeLa cells are widely used, and so the findings are useful, and whilst it seems certain that some other cell lines would give different data (and indeed the DepMap data show this), then testing one other line would not change the conclusions much. All I wanted the authors to do is to clearly state in the text that what they see in HeLa cells may well be different in other cell lines. This does not detract from the fact that their HeLa cells will provide an excellent platform for focused studies on the role of individual Arfs.

    Follow up Comment 2. I agree that Arf6 is not relevant to this paper (as discussed in detail below).

    Follow up Comment 3. agree that a simple IMF experiment would suffice to check polarity and immuno-EM is technically very demanding and would add little in this context. The authors have already shown that the Golgi forms stacks in the KO cell lines, and I cannot see how this could occur without the stack being polarised - it has to form at one end and then mature to the other. In addition, after decades of working on the Golgi I have yet to see a credible report of a change to cells causing a loss of Golgi polarity, but maintaining a stacked structure. If the Golgi is not polarised it could not form a stack.

    Follow up Comment 4. I agree that one month is perhaps too short to look at KDEL-R, COPI levels and checking polarity by IMF. As noted above, I am NOT suggesting that they repeat all this in a different cell line.

    **[On] Reviewer 2.**

    Point 1. I agree with Reviewer 1 that the authors are correct to ignore Arf6. It is a completely different GTPase with a distinct function in a different part of the cell. The family of Arf1-Arf5 arose in metazoans from a single Arf, but Arf6 had already split away from the Arf1-5 family in the last eukaryotic common ancestor, as Arf6 is present in plants and yeasts. There is overwhelming evidence that Arf1-Arf5 are partially redundant and this has hampered their study. Arf6 does not share these roles. The fact that it is acts on endosomes and has been reported to be on the Golgi (which is not widely agreed), is also true of many other GTPases. Indeed, other distant relatives of Arf1-5 are actually on the Golgi (Arl1, Arl5 etc), but these are also not relevant as like Arf6 they do not bind coat proteins and other major effectors of Arf1-5.

    Point 2. As noted above, it is hard to see how polarity could be affected given that a Golgi stack is formed, but, at most, a simple application of IMF would seem sufficient to confirm this.

    Point 3. Agreed.

    Point 5. I agree with the reviewer that this is (much!) too much to ask for an initial publication. Various labs have already reported analysis of the effectors of Class II Arfs and they tend to overlap with Class I. Moreover, it is quite possible that the difference of role in vivo reflects differing interactions with regulators.

    Point 6. Agreed.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper describes the application of CRISPR/Cas9 to systematically delete from HeLa cells all four Arf genes, either singly or in combination. The authors find it is possible to generate a number of double deletions (notably one lacking both Class I Arfs), and a triple deletion lacking all but Arf4. The authors characterise the structure of the Golgi in these mutants as well as retention of ER residents. The work is a comprehensive study of an exceptionally high technical standard. There is excellent validation of the deletions, and then the application of a wide range of methods including immunofluorescence, electron microscopy and mass-spectrometry, all with careful and extensive quantitation. The finding that cells can survive without Class I Arfs is interesting and unexpected, as is the fact that Arf4 alone is sufficient. This work will provide an excellent platform for future studies on Arf protein function in human cells. There are of course many questions that arise from these findings, but given the scope and quality of the work they would seem better left for future publications. There is one experiment that could be added, and some additions needed to the text for clarity and minor adjustments to the figures (all listed below), but if these are addressed, this would be a high quality paper of wide audience to a cell biological audience.

    Specific comments:

    1. Have the authors tested the levels and/or localisation of the KDEL receptor in the various lines? This is not essential, but if it were easily done, it would add to the work on ER resident secretion.

    2. The work is entirely done in HeLa cells. The authors should note that the situation might be different in other cells types and cell lines. For instance, the DepMap CRISPR database suggests that quite a lot of cell lines are strongly affected by loss of Arf1.

    3. Figure 2. Please show single channels as grey scale, and only merge as RGB. This is easier to see, especially for the colour blind. Likewise, Figure 3D would be clearer in greyscale rather than green, and 6B better in grey than in red.

    4. Figure 5C. A brief comment is needed as to why it might be that BiP and calreticulin are not so efficiently secreted when Arf5 is knocked out in addition to Arf4.

    5. Discussion:

    a) The authors should relate these studies to work in other species. For instance, in yeast reduction of Arf levels causes the Golgi to enlarge (PubMed ID 9487133).

    b) Some more discussion is needed of the fact that Arfs may not all act in the same part of the Golgi, which could explain some of the differences observed between the various deletions.

    Significance

    The Arf GTPases have been studied extensively for over 30 years as major regulators of Golgi function. They are essential for the recruitment to Golgi membranes of both COPI and clathrin/AP-1 coats, as well as various other proteins that regulate Golgi function. In addition, they have been reported to have roles in viral replication, and even other cellular processes such as lipid droplet formation and mitochondrial division. In humans there are four Arfs, Arf1 and Arf3 (Class I Arfs), and Arf4 and Arf5 (Class II Arfs). All are present on the Golgi, but their precise individual roles have remained unclear. Attempts have been made to deplete individual Arfs using RNAi, but incomplete knockdowns have made the results hard to interpret.

    Referee Cross-commenting

    There is probably no need for a prolonged debate about this, but I agree that the importance of Arf4 is striking, but it reflects the nature of this work that CRISPR has finally allowed these sorts of questions to be addressed unequivocally. COPI is also involved in recycling of Golgi resident enzymes, and it may be that Arf1 acts in this role.

    If the authors check levels of COPI by blotting, and measure the intensity over the Golgi by quantitative IMF, that will reveal whether stability or membrane association if affected without fractionation which is probably less reliable.

    If they want to do some extra experiments, then it would be quite easy to check the levels of some Golgi enzymes, or look at lectin binding as a proxy for glycosylation enzyme levels.

    Overall, I agree with the positive comments of Reviewers 1 and 2, and it good that we all recognise the quality and importance of the work. However, I feel that one or two of their requests go beyond the scope of a single publication, or would add rather little for a lot of additional work. It is of course easy to propose experiments that someone else has to do!

    Reviewer 1:

    Point 4. I agree that it would be useful to perform a blot to determine if the levels of coatomer are effected in the various KO lines. I am not sure if Reviewer 1 is also proposing fractionation to determine cytosol vs membrane ratio, but if so, then this would be less useful as peripheral membrane proteins tend to fall off membranes during fractionation and so such analysis is generally questionable. A blot, and clarification of the way the COPI/GM130 ratio is determined, would answer the key points in a relatively straightforward way.

    Point 5. I agree that the defect in retrieval of ER residents in Arf4-KO is striking, but it a clear effect even if the reviewer does not understand it themselves! It does not seem so surprising to me, given that Arf4 is likely to act on the early Golgi were such retrieval occurs from. However, the experiment suggested by myself and Reviewer 3 of checking the levels and localisation of the KDEL-receptor would seem to me a good first step to addressing possible mechanism, and certainly sufficient for an initial publication.

    Point 7. I am not sure that it has been unequivocally established that Arf1 is important in retrograde traffic. The reality is that many labs have taken Arf1 as being representative of all others and so concentrated biochemical and in vivo studies on this protein. This paper is really important as it highlights the need to investigate both Class I and Class II Arfs, and to bear in mind that their roles in vivo may well be more distinct than their in vitro properties would lead one to suspect. Perhaps, the simplest explanation for this is that the GEFs that activate them have a strong preference for one or the other.

    Follow up Comment 1. I was not suggesting that the authors repeat all this in a cell line other than HeLa cells, as this is clearly impractical. HeLa cells are widely used, and so the findings are useful, and whilst it seems certain that some other cell lines would give different data (and indeed the DepMap data show this), then testing one other line would not change the conclusions much. All I wanted the authors to do is to clearly state in the text that what they see in HeLa cells may well be different in other cell lines. This does not detract from the fact that their HeLa cells will provide an excellent platform for focused studies on the role of individual Arfs.

    Follow up Comment 2. I agree that Arf6 is not relevant to this paper (as discussed in detail below).

    Follow up Comment 3. agree that a simple IMF experiment would suffice to check polarity and immuno-EM is technically very demanding and would add little in this context. The authors have already shown that the Golgi forms stacks in the KO cell lines, and I cannot see how this could occur without the stack being polarised - it has to form at one end and then mature to the other. In addition, after decades of working on the Golgi I have yet to see a credible report of a change to cells causing a loss of Golgi polarity, but maintaining a stacked structure. If the Golgi is not polarised it could not form a stack.

    Follow up Comment 4. I agree that one month is perhaps too short to look at KDEL-R, COPI levels and checking polarity by IMF. As noted above, I am NOT suggesting that they repeat all this in a different cell line.

    Reviewer 2.

    Point 1. I agree with Reviewer 1 that the authors are correct to ignore Arf6. It is a completely different GTPase with a distinct function in a different part of the cell. The family of Arf1-Arf5 arose in metazoans from a single Arf, but Arf6 had already split away from the Arf1-5 family in the last eukaryotic common ancestor, as Arf6 is present in plants and yeasts. There is overwhelming evidence that Arf1-Arf5 are partially redundant and this has hampered their study. Arf6 does not share these roles. The fact that it is acts on endosomes and has been reported to be on the Golgi (which is not widely agreed), is also true of many other GTPases. Indeed, other distant relatives of Arf1-5 are actually on the Golgi (Arl1, Arl5 etc), but these are also not relevant as like Arf6 they do not bind coat proteins and other major effectors of Arf1-5.

    Point 2. As noted above, it is hard to see how polarity could be affected given that a Golgi stack is formed, but, at most, a simple application of IMF would seem sufficient to confirm this.

    Point 3. Agreed.

    Point 5. I agree with the reviewer that this is (much!) too much to ask for an initial publication. Various labs have already reported analysis of the effectors of Class II Arfs and they tend to overlap with Class I. Moreover, it is quite possible that the difference of role in vivo reflects differing interactions with regulators.

    Point 6. Agreed.

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    Referee #2

    Evidence, reproducibility and clarity

    Pennauer uses HeLa cells and CRISPR/Cas9 to delete the 5 members of the class I and class II ARF family of small regulatory GTPases either individually or in combinations. The characterization of the KO cells is excellent and convincingly demonstrates that true KOs were generated. The quality of the data presented is high. Using the KO cells she documents minor alterations in Golgi architecture and the recruitment of vesicular coats in cells deleted of all ARFs except ARF4. In contrast, there is a significant lack of retention/recycling to the ER of KDEL-containing ER proteins in ARF4 KO cells, with numerous ER chaperones now released into the medium (the ARF4 KO secretome). This is a well-done study that showcases the ability of ARF4 alone to sustain cellular life (quite a surprise to this reviewer). Yet, the characterization of the phenotypes is somewhat minimal and the conclusions would be more robustly supported by additional experiments. Specifically:

    1. The authors completely ignore class III ARF6 and this paper would be much more comprehensive and informative if analysis of that ARF was also included (ARF6 has been seen at the Golgi and also mediates endosomal trafficking that intersects with the TGN).
    2. Although the overall Golgi architecture seems to be largely conserved, it remains essential to test whether Golgi polarity is similarly maintained, and such data would significantly expand the significance of the reported findings
    3. Since there is a defect in retrieval of KDEL-proteins, it would be important to show the intracellular localization of the KDEL-R in the cells (especially in the ARF4 KO cells that don't retrieve KDEL-GFP) - is the receptor degraded, stuck in some specific place - knowing that would increase the impact of this study and provide a mechanistic explanation for the observed phenotype
    4. The rescue experiments in Figure 6 are good as far as they go, but this experiment would be much more informative if in addition to the same class rescue, the other class ARFs (at least one!) were also characterized.
    5. This is maybe a little too much to ask, but since the authors propose a mechanistic explanation for the ARF4 KO KDEL phenotype as being due to different effectors recruited by this ARF (in this case different COPI isotypes - this study would increase in impact by actually testing this mechanisms by assessing whether ARF4Q71L mutant preferentially bound any particular isotype of COPI or even try to do mass spec to identify relevant effectors for this extremely interesting ARF.
    6. The Discussion is a very limited and would be more impactful by adding some discussion of organismal effects of ARF deletions (many are embryonic lethal while cells seems to live quite happily) or mutations (links to cancer come to mind here), as well as some mention of data from yeast ARF (what is and isn't essential in those cells). As is, the authors miss an opportunity to highlight the importance of their findings as they relate to current knowledge of ARFology.

    Significance

    This is an important paper for the ARF field and people interested in ARF signaling will be glad to read about the findings and perhaps also use the developed KO cell lines - this is a significant advancement. The impact would be even higher if some of the experiments suggested above were incorporated into the manuscript.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Pennauer et al is the first to systematically investigate the role of class I&II Arfs using a knockout approach. It builds on earlier work by the Kahn lab who used an RNAi approach (Volpicelli-Daley et al. 2005) and is complementary to the overexpression approach used by the Hauri lab (Ben-Tekaya et al, 2010). The work is elegant and the data are strong. I am strongly in favor of publishing this work and my comments are technical in nature (2-5) and a request for some text changes (1). have the following comments for improvements:

    1- When it comes to evaluating the role of depletions of Arfs on cell fitness, it would be better to use a non-transformed cell line. I am not asking the authors to go through the painstaking process of generating knockout cell lines in RPE1 cells for instance. Rather, I suggest that the authors make the reader aware that conclusions about cell survival have to be taken with care due to the use of a transformed cell line.

    2- Why do Arf1 and Arf4 ko cells grow more slowly. Is it a higher rate of cell death? Is it a block in a certain phase of the cell cycle. Given the link of the Golgi to G2-M entry, I think that an analysis of the cell cycle distribution would add more depth to these data. If the cell cycle distribution is unaffected, then I would conclude that that the difference in doubling time are due to reduced cell survival. If there is an effect on the cell cycle distribution, then the conclusion of the authors is safe that no single Arf is required for survival

    3- It is not clear to me how many cells were quantified in Figure 2D-F. I suppose that each do represents a cell. In this case, the number of cells quantified is a bit low. Such a quantification of fluorescence intensities in two channels in the same region is a simple task and I think it should be no problem obtaining at least 100 cells per condition.

    4- Is the drop in the ratio of beta-COP/GM130 in Arf1 depleted cells reflecting reduced recruitment to the Golgi? Because the Golgi is bigger, it might be reflecting a reduced density in the number of coatomer molecules per surface area. If it is due to reduced recruitment, then the ratio of membrane/cytosolic betaCOP should be altered. This of course requires to show that the knockout does not affect total levels of coatomer. I think that such fractionation experiments would be a valuable addition to the manuscript and increase the depth of the data.

    5- The finding that Arf4-ko cells exhibit a defect on retrieval of ER-resident proteins is exciting, and in my opinion, it is the most significant finding in this manuscript. How can this be reconciled with the lack of an ARf4 ko effect on coatomer recruitment to the Golgi. Looking carefully at the data, I see that in 2 out of 3 experiments, Arf4 ko reduced the betaCOP/GM130 ratio. This is why I think it is crucial to perform more experiments and add more cells to increase the confidence in the data. Reduced retrieval of ER chaperones is frequently found in tumors and we still don't understand the reason behind this. Therefore, this finding is of significance beyond the community of cell biologists.

    6- I find Figure 6A confusing. Why do Arf1 overexpressing parental HeLa cells exhibit less Arf1 than control cells?

    7- Why was the following condition not tested: Arf4ko cells with Arf1 overexpression. Given the importance of Arf1 in retrograde (Golgi-to-ER) trafficking, I would expect a partial rescue of the retrieval of ER chaperones.

    Significance

    Significance of the work:

    The paper is important because it is the first to examine the role of Arfs using a knockout approach. Another very important finding is that Arf4 depleted cells exhibit problems with retrieval of ER chaperones. This is a very novel finding and to the best of my knowledge

    Audience:

    The primary audience is of course the community working on membrane trafficking, organelle biology and proteostasis. However, I think that the data on the role of Arf4 in retrieval of ER chaperones might be of relevance for cancer biologists. Secretion of ER chaperones is frequently found in many tumors and we still do not understand why this is happening and what the significance thereof is.

    My own expertise:

    Export from the endoplasmic reticulum Golgi fragmentation in cancer cell migration Rho GTPases Kinase signaling Pseudoenzymes Cell migration of breast cancer cells Proteostasis in multiple myeloma

    Referee Cross-commenting

    Just a follow-up comment from my side:

    I agree that it has not been unequivocally established that Arf1 is the main/sole of retrograde transport. However, even less established is the role of Arf4 in this process. The authors show that it is mainly Arf1 depletion that reduces the amount of COPI at the Golgi (ratio of COPI/GM130). Thus, I remain very surprised that it is actually the Arf4 depletion that results in reduced retrieval.

    What is the significance of having less COPI at the Golgi in Arf1-ko cells? Certainly, the Golgi is not more "leaky". Does the level of COPI at the Golgi not reflect the strength of retrograde trafficking? Maybe there is no less COPI at the Golgi, and it only appears to be less, because the Golgi is bigger. This is why a simple fractionation experiment would be good. Something like making a cytosol and a microsome fraction and looking at the ratio of COPI (Cyt/Mem).

    If both reviewers think it is too much, or unlikely to work, then I am happy to drop this point.

    Below are my comments to the evaluations by the other two reviewers:

    1- I agree with most comments that the two other reviewers made. Some of them are actually overlapping with mine (e.g. the use of a cell line other than HeLa).

    2- I am not sure whether the impact of the paper would improve by adding data on Arf6.

    3- To the comment on Golgi polarity. Maybe we could be more specific here and say that it would be sufficient to show that a trans-Golgi protein and a cis-Golgi protein can be separated by fluorescence microscopy, or whether we alternatively want them to actually do it by immunogold labeling for EM (which is more difficult).

    4- I agree with reviewer 2 that the work proposed needs 1-3 months. I think reviewer 3 is a bit too optimistic with 1 month, because her/his comment on using a cell line other than HeLa cannot be addressed in just a month.

  6. Version published to 10.1101/2021.04.19.440443v1 on bioRxiv