Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

  1. Danièle Stalder
  2. Igor Yakunin
  3. Conceição Pereira
  4. Jessica Eden
  5. David C. Gershlick

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Abstract

ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT 374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localization. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT 374-376 motif. Taken together, our results suggest that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.

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  5. Version published to 10.1091/mbc.e23-09-0376
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    Summary: This manuscript describes molecular mechanisms by which ACBD3 is recruited to the Golgi complex. ACBD3 recruits PI4KIIIb which is required to generate PI4P, a phosphoinositide which is key for the recruitment of essential Golgi proteins and hence is key to Golgi identity. The authors have used a combination of mass spectrometry, high quality fluorescence imaging, transient CRISPR knockdowns, and biochemical approaches such as IPs to identify the key determinant for recruitment of ACBD3 to the Golgi complex. They map the interaction between ACBD3 and the Golgi as a unique region (UR) upstream of its GOLD domain, identifying, in particular, an MWT motif as key for this recruitment. Using mass spectrometry they identify several novel interactors of ACBD3 as well as some established binding partners. Knockdown of these interactors reveal a key role for the SNARE, SCFD1, where reduced levels lead to complete loss of ACBD3 localisation to the Golgi without apparent disruption of Golgi structure. They further validate this interaction and that of another SNARE (Sec22b), which is part of the same SNARE complex as SCFD1, mapping the interaction to the longin domain of Sec22b. Surprisingly however they demonstrate that the UR domain does not mediate the interaction between ACBD3 and these SNAREs suggesting an alternative mechanism of recruitment. Previously identified ACBD3 interactors, Golgi proteins giantin and golgin-45 were also identified in the mass spectrometry screen and the authors demonstrate that these two proteins can recruit ACBD2 to the Golgi and this is dependent on the MWT motif identified in the UR domain. By knocking down SCFD1, they show reduced recruitment of ACBD3 leading them to propose a model of sequential recruitment of ACBD3 by SCFD1 followed by interactions with the golgins.

    Major points: This study is a well-executed and rigorous study of the molecular requirements for the recruitment of ACBD3 to the Golgi. The experimental approaches are state-of-the-art and the data are clean and convincing. The only caveat, raised by the authors themselves, is their interpretation that there are two sequential steps for Golgi recruitment of ACBD3. While they show that loss of SCFD1 reduces the interaction of ACBD3 with giantin and golgin 45, their model depends on doing the reverse experiment, i.e. assessing the effects of knocking down either giantin or golgin-45. This is especially relevant given the demonstration that golgin-45 is sufficient to recruit ACBD3 to mitochondria. It may well be that recruitment involves a tripartite complex, which is not uncommon in vesicular transport mechanisms Giantin is not an essential protein do it should be feasible to perform this experiment. The authors are equipped in the quantitative fluorescence microscopy which would be required and which would help resolve whether sequential or redundant mechanisms are required for ACBD3 recruitment.

    We thank the reviewer for the positive comments and are glad that they consider our study "well-executed and rigorous". We totally agree with the reviewer that our conclusions regarding the sequential aspect of the recruitment of ACBD3 in the original submission could be better supported. We have worked to strengthen this in our resubmission. As the reviewer states, this limitation was already discussed in the original submission. To further support our model, we have performed the experiment suggested by the reviewer, in which we test the effects of knocking down both giantin and golgin45 (double knockdown) on the binding of ACBD3 to SCFD1.

    The results of this experiment further support our sequential model with little to no effect of loss of the Golgins on ACBD3. As we already knew, a large effect of SCFD1 KO on the binding of the Golgins to ACBD3 was also observed here. We should note that this was performed in a different cell line than before (HeLa cells rather than HEK cells), as the efficiency of multiple knockdowns was much lower in HEK cells, as determined by qPCR. Taken together, the new data in Figure 7 supports a sequential model for Golgi recruitment. We also agree that other, less likely models could explain our data and have included this openly in the discussion. In conclusion, we thank the reviewer for their comments and have revised the manuscript with a new experiment with the relevant repeats, which supports our model.

    Reviewer #1 (Significance):

    Significance PI4P is a phosphoinositide that is important for the recruitment of Golgi proteins. As with most PIs it is likely to act by coincidence detection in that Golgi associated proteins will recognise PI4P as well as other factors on Golgi membranes. This results in different local membrane environments which will be specific for particular functions. PI4KIII__b_ is key for PI4P production although the absolute levels of PI4P are likely to be determined by a balance of lipid kinases and phosphatases. However, since ACBD3 is key for the recruitment of PI4KIII__b, it is important to understand the molecular mechanisms by which it is recruited. The manuscript thus makes a significant contribution to understanding one of the underlying mechanisms for PI4KIII__b _recruitment although, as indicated above, stops short of establishing a clear model for the roles SCDF1 and Sec22b versus golgin 45 and giantin. For the future it will be of interest to determine why either a sequential or a redundant mechanism is required for the recruitment of ACBD3 as a scaffold protein.

    We thank the reviewer for this set of positive comments on the manuscript and for agreeing that this is a significant contribution. Our revised version further supports our sequential model of ACBD3 recruitment to the Golgi apparatus, and the comments here have helped us further to strengthen the quality and clarity of the manuscript.

    Reviewer #2 (Evidence, reproducibility and clarity):

    Summary This is a very interesting and potentially important paper for the field of membrane biology and membrane trafficking, in which the authors have studied the molecular mechanisms by which ACBD3 (and consequently PI4KIIIb) is recruited to the cis-Golgi membranes. The authors suggest that this recruitment is based on a two-step process, mediated by interactions to, on the one hand, SCFD-1 (SLY1) and, on the other hand, two redundant golgins (golgin-45 and giantin).

    We once again thank the reviewer for the positive comments and are glad that they consider our manuscript important.

    Comments:- Pg.1 : arfaptins, as far as I know, have not been shown to be involved in intra-golgi trafficking but rather in Golgi export (see e.g. ref. 12)

    We thank the reviewer for pointing this out. We have corrected the text accordingly.

    • Pg. 1: reigon --> region

    We thank the reviewer for noticing this typo. We have corrected the text accordingly.

    • Arf1 also recruits PI4KIIIb right?

    This is correct. The De Matteis lab has shown that PI4KIIIβ associates with the Golgi complex in an Arf1-dependent manner (Godi et al. 1999). We think this is excellent work. However, Arf1 is somewhat of a master regulator of the Golgi, affecting the recruitment and localisation of many different Golgi proteins. It has also previously been reported that Arf1 does not directly interact with PI4KIIIβ (Klima et al. 2016). Overall, the molecular relationship between Arf1 and the kinase remains unclear. We do not exclude, however, that there are factors other than ACBD3 important for recruiting and regulating PI4KIIIβ levels at the Golgi. We have changed the wording in the manuscript to reflect that there are multiple ways that PI4KIIIβ is recruited to the Golgi apparatus.

    Fig. S1: the information about the number of cells per experiment is missing. Also, please add the information about what exactly is represented in the box plots (is it the distribution of the mean value of R per experiment? or the total distribution on a cell-by-cell basis of a representative experiment?)

    For each experiment, a minimum of 100 cells per condition were imaged. The Pearson's correlation was then calculated, and the average was taken for each biological repeat. The plot in Fig. S1B represents 3 independent biological repeats. We have included this information in the revised manuscript.

    • The definition of Avg. Golgi int/avg. cell int. (a.u.) in Fig 1E,F is a bit difficult to understand to me. If I understand correctly, the total fl. int in the Golgi mask was computed and divided by the area of the Golgi mask (this is the av. Golgi intensity). A similar computation is done for the entire cell (including the Golgi), i.e., total fl. intensity in the cell mask is computed and divided by the area of the cell mask. Then the two av. intensities are divided (ratio = av. Golgi int / av. cell int.). This ratio, for a protein that is enriched in the Golgi area, should be larger than 1. For a protein that is equally distributed all over the cell, it should be 1, and for a protein that is excluded from the Golgi area, smaller than 1. Then to this value, the authors subtract the value of the ratio found for an inert construct (GFP of Halo alone), which I imagine should have an original ratio value of the order of 1, and hence, after this subtraction, norm. ratio values larger than 0 mean that they are more enriched at the Golgi area than GFP/HaloTag themselves. Is this correct? In principle, I don't see anything entirely wrong with this way of thought, but I just found it a bit difficult to understand, and in general one has to be careful when computing rations (quotients) and then subtract another ratio. Also, the units are not a.u., the value is dimensionless, what is "arbitrary" is the definition of 0 value and the based on this definition, also the actual value. I think it would probably be much clearer for the readers to compute somthing like the relative enrichment in the Golgi area as compared to the rest of the cell (excluding the Golgi area). That is, a value r'=(Int. Golgi mask / Area Golgi mask) / [(Int. Cell mask - Int. Golgi mask)/(Area cell mask - Area Golgi mask)]. This can be computed directly or defining a mask that is the cell mask - the Golgi mask. Also, some maths (unless I made a mistake) give that this r'= r (1-aG)/(1-r aG); where r is the ratio (before subtraction) defined by the authors, and aG=Area cell mask/Area Golgi mask. In any case, I'd suggest the authors to either adopt this other quantitation (without subtraction of the GFP/HAloTAG), which gives directly the fold-enrichment in the intensity density in the Golgi area with respect to the rest of the cell; or explain in more detail the maths of the value they are plotting now.

    We thank the reviewer for these well-reasoned and thoughtful suggestions for our imaging analysis. These are issues that we have also considered when quantifying this dataset. At the heart of it, the second method of calculation (Golgi/outside of Golgi), results in a non-linear distribution, as the pool of proteins re-distribute from inside the Golgi to the cytosol. This is why we have chosen to use the first method of Golgi/total, as it provides a linear distribution.

    The reviewer is also correct that the GFP (inert protein) ratio is 1 without adjustment. We have chosen to normalise to GFP/HaloTag (inert protein) as we think this is the clearest way of conveying our conclusions from these experiments. We have included the non-normalised graph here for the reviewer to see; however we thought that this conveys the key result less clearly. Overall, we agree this was poorly communicated in the manuscript and we have clarified it in the revised version.

    • Fig. 1C&F: Besides the MWT mutant, the FKE mutant also seems to have a somewhat compromised Golgi localization. Have the authors followed on that, or what is the reason that they have just focused on the MWT mutant?

    In contrast to the MWT mutant, the FKE mutant does not affect ACBD3 localisation significantly. In addition, when having a close look at the pdb structure of the GOLD domain of ACBD3 with 3A protein of Aichivirus A (5LZ3), the MWT patch, in particular residues M and T, make clear contact with protein 3A, which is not the case for FKE residues. Therefore we focused on the MWT residues, which we hypothesised to interact with a Golgi resident protein which competes with protein 3A to interact with ACBD3.

    • Very minor point, and without wanting to sound pedant at all, but I think (I might be wrong of course, so apologies if I am) that the plural of apparatus in latin is not apparati, but apparatus (fourth declination). So, I'd change the word in page 2 (or just rephrase the sentence: e.g. "resulting in Golgi fragmentation"). But of course, I'd leave this to the authors' discretion.

    We thank the reviewer for this precision, do not consider it pedantic, and have made the suggested change to the text.

    • Fig. 3A: have the authors tried or been able to perform IF of the endogenous SCFD1 protein?

    As suggested by the reviewer, we attempted to perform IF of endogenous SCFD1, as shown below. Despite trying several different antibodies, we were not satisfied that we were detecting real SCFD1 signal as there was no change in this staining upon SCFD1 CRISPR KO. Please see an example of this IF below (ProteinTech, 12569-1-AP). We have contacted the antibody manufacturers to inform them of this issue.

    • Similarly to what has been done for other panels, could you quantify Fig. 3C? Are PI4KIIIb protein levels affected upon the different KOs?

    As suggested by the reviewer, we are now showing in Figure S2D the percentage of cells with a partial or total loss of PI4KIIIβ at the Golgi in CRISPR-Cas9 KO cells of either PI4KIIIβ, ACBD3 or SCFD1. 3 independent biological repeats were performed and approximately 150 cells were quantified (~50 cells per condition). The results show that the PI4KIIIβ antibody used (BD Bioscience, 611816) is specific (93.22% of cells lose the antibody signal) and that ACBD3 and SCFD1 KO affects PI4KIIIβ recruitment to the Golgi in 88% and 73% of the cells, respectively._-

    The last paragraph of the "SCFD1 and ACBD3 interact upstream of PI4KIIIβ recruitment to the Golgi apparatus" section reads a bit odd placed there. I think it is more appropriate for the discussion or for the intro part on SCFD1.

    Many thanks to the reviewer for pointing this out. We simplified that paragraph to describe the relationship between SCFD1 and SEC22B.

    • I am confused on Fig. 5A/B. The labels in the blots show that 390-528 (without UR) does not bind sec22 or scfd1, but the 368-529 does? Or I guess, judging by the MW seen in the middle blots, that there's some error in the labelling?

    Many thanks to the reviewer for noticing this, which was clearly a labelling error. We corrected this accordingly in Figures 5A and B. We apologise for this oversight.

    also, the IP efficiency of the MWT mutant in the panel A blot is quite low, still sec22 seems to be very efficiently pulled down. Can the authors comment on that please? Would co-IPing against endogenous sec22 and scfd1 would work (so you don't need to rely on HaloTag+ligand?)

    We know that the MWT residues of ACBD3 are important for recruiting ACBD3 to the Golgi (Figure 1C and F). We also know that ACBD3 interacts with SEC22B and SCFD1 (Figure 3B and 4A) and that SCFD1 is important for ACBD3 Golgi recruitment. Therefore we initially speculated that ACBD3 interacts with SEC22B and SCFD1 through the MWT residues. However, as the reviewer points out, Figure 5 shows the opposite. Mutating MWT residues makes the interaction of ACBD3 with SEC22B and SCFD1 stronger. For this reason, we hypothesised that another player(s) also contributes to ACBD3 recruitment through interactions with the MWT residues. We have shown that the second recruitment factors are the 2 golgins, golgin-45 and giantin (Figure 6C). In short, whilst we agree that the IP efficiency is low, the binding is actually stronger, supporting our conclusions. No interaction of ACBD3 with endogenous SEC22B could be detected due to a lack of a sufficiently sensitive antibody (we tried Abcam ab181076 and ProteinTech 14776-1 AP).

    • I really like the experiment 6B. Have the authors tested whether SEC22 is also recruited to mitochondria in those conditions? But not SCFD1?

    We thank the reviewer for the positive comment. We have performed the suggested experiment and are now including this as an additional figure (Figure S3). Ectopic expression of golgin-45 targeted to the mitochondria is not sufficient to redistribute SCFD1-HaloTag or HaloTag-SEC22B to the mitochondria (Figure S3A and B, respectively). We, therefore, speculate that the fraction of ACBD3 that gets redirected in Figure 6B must be the small fraction of ACBD3 that is spontaneously in an open conformation and compatible for interaction with golgin-45.

    • The results shown in Fig 7 might show a partial depletion in the interactions, but to be fully trusted they would need to be quantified and a statistical test used to compare the values. I think this part is important to show very clearly, because even with low binding to golgins (remember, single knockouts do not prevent Golgi localization of ACBD3), one could expect that ACBD3 still localized to the Golgi but it does not in the absence of SCFD1 as shown in this paper. A prediction of the proposed model is that in cells depleted of the two Golgins, SCFD1 and ACBD3 should still bind to one another, right? Did the authors test this?

    We fully agree with the reviewer. As discussed in the replies to reviewer 1, we have repeated this experiment, including both sets of KO. This was not trivial, as a double transient KO is technically challenging and involves validation with qPCR and switching cell types (HEK cells to HeLa). The new data supports our current model and suggests some additional regulatory mechanisms at play.

    • The model presented here (fig 8) seems to suggest that only the conformational variation of ACBD3 that binds Golgins is able to recruit (bind) PI4KIIIb. Is this known, or is there any experimental evidence for that?

    HDX-MS experiments show that the ACBD and GOLD domains undergo conformational changes in the presence of 3A proteins (McPhail et al. 2017). Demonstrating this would require a complicated reconstitution experiment which is technically very challenging and would involve purifying various complex proteins, including SNAREs, SM proteins and golgins. This could perhaps be the subject of several future studies.

    • Have the authors thought about testing the FKE mutant in the experiemnts shown in Fig. 5?

    As mentioned above, since the FKE residues are not making any contact with the protein 3A and since the loss of ACBD3 recruitment to the Golgi is not statistically significant (Figure 1F), we haven't tested the FKE mutant for the binding to SEC22B and SCFD1. We do, however, agree with the reviewer that there might be something interesting happening here. We would like to experimentally interrogate this in future studies and develop more sensitive assays to test if there is a significant effect with the FKE mutant.

    In general, I think the title might be a bit misleading because of the use of PI4Kiiib. I understand what the authors mean, but because they have not thoroughly tested PI4Kiiib recruitment in their experiments, I think they should focuse rather on the mechanism of recruitment of ACBD3 the authors have found.

    We thank the reviewer for their advice regarding the manuscript title, and this is something that we have discussed internally. We chose that title as it highlights the key mechanistic impact of our findings and note that we did include a figure on the recruitment of PI4KIIIβ. However, we remain open to discussing this with advice from the journal editorial team.

    Reviewer #2 (Significance):

    I think, as said above, that this is potentially an important paper for the field of membrane trafficking and membrane biology. Most of the experiments are in general well performed and well controlled, and the paper is clearly written and follows a logical line.

    We once again thank the reviewer for their comments and overall thoughtful and considered review. We believe that the suggestions here have improved the manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity):

    Stalder and colleagues report experiments designed to identify interactors of the Golgi-localized protein ACBD3 (a.k.a. GCP60), and to delineate mechanisms that allow ACBD3 to localize at Golgi compartments. ACBD3 is a 528aa protein with diverse previously reported interactions and functions, both in normal physiology and as a host factor in viral assembly processes. Stalder et al. first map which domains of ACBD3 are required for Golgi localization in HeLa cells, concluding that residues 368-528 are sufficient for localization. This region includes a GOLD (GOLgi Dynamics) domain previously reported to interact with Golgin tethering proteins. Alanine scanning identifies the motif MWT just upstream of the GOLD motif as necessary for Golgi localization. Acute CRISPR knockout identifies two Golgins, Golgin45 and Giantin, as necessary for ACBD3 Golgi localization, and IP indicates that the MWT motif breaks this interaction. These data are a bit scattered around the paper but taken together are reasonably persuasive, particularly when viewed in context with published work. This reader would have found the manuscript easier to follow had the Golgin and MWT motif data been presented en bloc.

    We thank the reviewer for these comments and have considered presenting and rewriting the data as the reviewer suggested. On reflection, we have decided to present it in the original order. We feel that this allows us to highlight the two independent mechanisms individually, bringing them together at the end. In addition, as the experiments were performed in the order presented, it allows for more appropriate controls for each experiment rather than trying to combine them. We hope the reviewer accepts our preferred order.

    In a second set of experiments, IP-mass spec is used to identify ACBD3 interactors that might assist in the protein's localization. The MS data presented are filtered to exclude proteins not already identified as Golgi-localized. This is, I think, a mistake. Even if the authors choose to focus on known Golgi interactors as candidates for a localization function, the biological functions of ACBD3 are far from fully understood, and the full dataset would be of value to both cell biologists and virologists.

    We agree with the reviewer that there are many interesting mysteries surrounding ACBD3 and have therefore included an additional table (table S1) in the revised manuscript, showing the dataset of newly identified ACBD3 interactors before applying the Golgi localisation filter.

    Hits in the filtered dataset include the R-SNARE Sec22B, and the SNARE chaperone Sly1/SCFD1. Acute CRISPR inactivation of Sec22 decreases ACBD3 localization to the Golgi and SCFD1 inactivation more or less abolishes localization. Co-IP experiments are used to argue that ACBD3 interacts with the N-terminal regulatory Longin domain of SEC22B, as well as with SCFD1. The Sec22 data are more detailed and persuasive. No experiments with purified proteins are presented to establish that the detected interactions are direct rather than mediated through a bridging factor or factors. Importantly, SCFD1 is likely to have multiple different client SNARE complexes that operate at different stages of ER and Golgi traffic. Hence its inactivation is likely to be pleiotropic and consequently phenotypes arising must be interpreted with caution.

    We completely agree that studying membrane trafficking in an interconnected system is challenging. We also agree that direct binding experiments in reconstituted systems would be key to proving our model. Our data uses multiple different experimental approaches, including co-localisation, co-immunoprecipitation, CRISPR-KO, and biochemistry, to support our model. In the future, we agree full reconstitution would be necessary to examine this further, and we hope that either ourselves or others can do this in further studies.

    Lastly, the authors perform IP experiments which show that ACBD3-Golgin co-IP efficiency is lower in cells with acute inactivation of SCFD1. This epistatic relationship is used to argue for a sequential model of recruitment with SCFD1 and perhaps client SNARE proteins operating upstream of ACBD3-Golgin interaction. This argument is not persuasive because we do not know whether SCFD1 and its downstream activities increase the rate of ACBD3-Golgin complex asssembly, or alternatively stabilizes ACBD3-Golgin complexes, decreasing the rate of their dissociation.

    We agree with this weakness in our original submission, and it is a comment shared among all reviewers. Overall, we feel that we have chosen the model that best summarises our data. We, of course, accept that there are still components of this pathway that need clarification and are open for further study. This includes the issue raised here by the reviewer, as well as the intriguing observation that both golgins are transcriptionally upregulated upon SCFD1 KO in HeLa cells. In the revised manuscript, we have more clearly laid out the weaknesses of our model in the discussion and suggested future experiments to help clarify some of these issues. We have also modified the model to reflect some of these potential additional regulatory mechanisms.

    In general the methods are fairly clear but that there is room for improvement. The "high throughput" imaging pipeline is not clearly described.

    We agree with the reviewer, and apologise for not clearly explaining this. We feel that this unbiased approach of quantification is particularly rigorous and we have clarified this in the methods section of the updated manuscript.

    Each figure legend should specify the microscopy methods used, and for each result the number of biological replicates and cells analyzed should be specified.

    We agree with the reviewer and have included these details appropriately in the revised manuscript.

    The statistical methods (Student, Tukey, etc.) used for each experiment should be specified. Saying that statistics were calculated using Python 3.7 is useless without additional details. e.g. at least the libraries and codebase used should be indicated or deposited.

    We agree with the reviewer and have updated the manuscript accordingly. In short, all comparisons were made using either Student's t-test or Multiple Comparison of Means - Tukey HSD, FWER=0.05. These were conducted in Python 3.9 using pandas, matplotlib, seaborn and scipy. We used the MultiComparison function in scipy, and the comp.tukeyhsd for the post-hoc adjustment.

    Many figure labels (e.g. Fig. 2) use absurdly small fonts.

    We apologise for this. We believe that this is because we submitted it with in-line formatting. Our resubmission has full-page figures, and we feel the text is clearer now.

    The mass spec hits obtained should be provided both with and without exclusion of non-Golgi-localized proteins.

    We agree with the reviewer. Please see the new Table S1.

    Reviewer #3 (Significance):

    In general I think this is a useful and well controlled set of experiments producing useful insights. However, the interpretations need to be more carefully considered, and alternative interpretations must laid out as clearly as possible. Specifying the limitations of the study will make it more, not less, useful to the field. If the authors want to make the case more robustly that the interactions described are mediated through direct binding, or that the operation of SCFD1 and Golgins operate sequentially to recruit ACBD3, additional wet bench work will be required which will of course take time to complete.

    We once again thank the reviewer for the thoughtful and critical comments. These have helped to strengthen the manuscript. We have performed the additional bench work requested by the reviewer, which has further supported the paper and our model.

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    Referee #3

    Evidence, reproducibility and clarity

    Stalder and colleagues report experiments designed to identify interactors of the Golgi-localized protein ACBD3 (a.k.a. GCP60), and to delineate mechanisms that allow ACBD3 to localize at Golgi compartments. ACBD3 is a 528aa protein with diverse previously reported interactions and functions, both in normal physiology and as a host factor in viral assembly processes. Stalder et al. first map which domains of ACBD3 are required for Golgi localization in HeLa cells, concluding that residues 368-528 are sufficient for localization. This region includes a GOLD (GOLgi Dynamics) domain previously reported to interact with Golgin tethering proteins. Alanine scanning identifies the motif MWT just upstream of the GOLD motif as necessary for Golgi localization. Acute CRISPR knockout identifies two Golgins, Golgin45 and Giantin, as necessary for ACBD3 Golgi localization, and IP indicates that the MWT motif breaks this interaction. These data are a bit scattered around the paper but taken together are reasonably persuasive, particularly when viewed in context with published work. This reader would have found the manuscript easier to follow had the Golgin and MWT motif data been presented en bloc.

    In a second set of experiments, IP-mass spec is used to identify ACBD3 interactors that might assist in the protein's localization. The MS data presented are filtered to exclude proteins not already identified as Golgi-localized. This is, I think, a mistake. Even if the authors choose to focus on known Golgi interactors as candidates for a localization function, the biological functions of ACBD3 are far from fully understood, and the full dataset would be of value to both cell biologists and virologists. Hits in the filtered dataset include the R-SNARE Sec22B, and the SNARE chaperone Sly1/SCFD1. Acute CRISPR inactivation of Sec22 decreases ACBD3 localization to the Golgi and SCFD1 inactivation more or less abolishes localization. Co-IP experiments are used to argue that ACBD3 interacts with the N-terminal regulatory Longin domain of SEC22B, as well as with SCFD1. The Sec22 data are more detailed and persuasive. No experiments with purified proteins are presented to establish that the detected interactions are direct rather than mediated through a bridging factor or factors. Importantly, SCFD1 is likely to have multiple different client SNARE complexes that operate at different stages of ER and Golgi traffic. Hence its inactivation is likely to be pleiotropic and consequently phenotypes arising must be interpreted with caution.

    Lastly, the authors perform IP experiments which show that ACBD3-Golgin co-IP efficiency is lower in cells with acute inactivation of SCFD1. This epistatic relationship is used to argue for a sequential model of recruitment with SCFD1 and perhaps client SNARE proteins operating upstream of ACBD3-Golgin interaction. This argument is not persuasive because we do not know whether SCFD1 and its downstream activities increase the rate of ACBD3-Golgin complex asssembly, or alternatively stabilizes ACBD3-Golgin complexes, decreasing the rate of their dissociation.

    In general the methods are fairly clear but that there is room for improvement. The "high throughput" imaging pipeline is not clearly described. Each figure legend should specify the microscopy methods used, and for each result the number of biological replicates and cells analyzed should be specified. The statistical methods (Student, Tukey, etc.) used for each experiment should be specified. Saying that statistics were calculated using Python 3.7 is useless without additional details. e.g. at least the libraries and codebase used should be indicated or deposited. Many figure labels (e.g. Fig. 2) use absurdly small fonts. The mass spec hits obtained should be provided both with and without exclusion of non-Golgi-localized proteins.

    Significance

    In general I think this is a useful and well controlled set of experiments producing useful insights. However, the interpretations need to be more carefully considered, and alternative interpretations must laid out as clearly as possible. Specifying the limitations of the study will make it more, not less, useful to the field. If the authors want to make the case more robustly that the interactions described are mediated through direct binding, or that the operation of SCFD1 and Golgins operate sequentially to recruit ACBD3, additional wet bench work will be required which will of course take time to complete.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This is a very interesting and potentially important paper for the field of membrane biology and membrane trafficking, in which the authors have studied the molecular mechanisms by which ACBD3 (and consequently PI4KIIIb) is recruited to the cis-Golgi membranes. The authors suggest that this recruitment is based on a two-step process, mediated by interactions to, on the one hand, SCFD-1 (SLY1) and, on the other hand, two redundant golgins (golgin-45 and giantin).

    Comments:

    • Pg.1 : arfaptins, as far as I know, have not been shown to be involved in intra-golgi trafficking but rather in Golgi export (see e.g. ref. 12)
    • Pg. 1: reigon --> region
    • Arf1 also recruits PI4KIIIb right?
    • Fig. S1: the information about the number of cells per experiment is missing. Also, please add the information about what exactly is represented in the box plots (is it the distribution of the mean value of R per experiment? or the total distribution on a cell-by-cell basis of a representative experiment?)
    • The definition of Avg. Golgi int/avg. cell int. (a.u.) in Fig 1E,F is a bit difficult to understand to me. If I understand correctly, the total fl. int in the Golgi mask was computed and divided by the area of the Golgi mask (this is the av. Golgi intensity). A similar computation is done for the entire cell (including the Golgi), i.e., total fl. intensity in the cell mask is computed and divided by the area of the cell mask. Then the two av. intensities are divided (ratio = av. Golgi int / av. cell int.). This ratio, for a protein that is enriched in the Golgi area, should be larger than 1. For a protein that is equally distributed all over the cell, it should be 1, and for a protein that is excluded from the Golgi area, smaller than 1. Then to this value, the authors subtract the value of the ratio found for an inert construct (GFP of Halo alone), which I imagine should have an original ratio value of the order of 1, and hence, after this subtraction, norm. ratio values larger than 0 mean that they are more enriched at the Golgi area than GFP/HaloTag themselves. Is this correct? In principle, I don't see anything entirely wrong with this way of thought, but I just found it a bit difficult to understand, and in general one has to be careful when computing rations (quotients) and then subtract another ratio. Also, the units are not a.u., the value is dimensionless, what is "arbitrary" is the definition of 0 value and the based on this definition, also the actual value. I think it would probably be much clearer for the readers to compute somthing like the relative enrichment in the Golgi area as compared to the rest of the cell (excluding the Golgi area). That is, a value r'=(Int. Golgi mask / Area Golgi mask) / [(Int. Cell mask - Int. Golgi mask)/(Area cell mask - Area Golgi mask)]. This can be computed directly or defining a mask that is the cell mask - the Golgi mask. Also, some maths (unless I made a mistake) give that this r'= r (1-aG)/(1-r aG); where r is the ratio (before subtraction) defined by the authors, and aG=Area cell mask/Area Golgi mask. In any case, I'd suggest the authors to either adopt this other quantitation (without subtraction of the GFP/HAloTAG), which gives directly the fold-enrichment in the intensity density in the Golgi area with respect to the rest of the cell; or explain in more detail the maths of the value they are plotting now.
    • Fig. 1C&F: Besides the MWT mutant, the FKE mutant also seems to have a somewhat compromised Golgi localization. Have the authors followed on that, or what is the reason that they have just focused on the MWT mutant?
    • Very minor point, and without wanting to sound pedant at all, but I think (I might be wrong of course, so apologies if I am) that the plural of apparatus in latin is not apparati, but apparatus (fourth declination). So, I'd change the word in page 2 (or just rephrase the sentence: e.g. "resulting in Golgi fragmentation"). But of course, I'd leave this to the authors' discretion.
    • Fig. 3A: have the authors tried or been able to perform IF of the endogenous SCFD1 protein?
    • Similarly to what has been done for other panels, could you quantify Fig. 3C? Are PI4KIIIb protein levels affected upon the different KOs?
    • The last paragraph of the "SCFD1 and ACBD3 interact upstream of PI4KIIIβ recruitment
      to the Golgi apparatus" section reads a bit odd placed there. I think it is more appropriate for the discussion or for the intro part on SCFD1.
    • I am confused on Fig. 5A/B. The labels in the blots show that 390-528 (without UR) does not bind sec22 or scfd1, but the 368-529 does? Or I guess, judging by the MW seen in the middle blots, that there's some error in the labelling? also, the IP efficiency of the MWT mutant in the panel A blot is quite low, still sec22 seems to be very efficiently pulled down. Can the authors comment on that please? Would co-IPing against endogenous sec22 and scfd1 would work (so you don't need to rely on HaloTag+ligand?)
    • I really like the experiment 6B. Have the authors tested whether SEC22 is also recruited to mitochondria in those conditions? But not SCFD1?
    • The results shown in Fig 7 might show a partial depletion in the interactions, but to be fully trusted they would need to be quantified and a statistical test used to compare the values. I think this part is important to show very clearly, because even with low binding to golgins (remember, single knockouts do not prevent Golgi localization of ACBD3), one could expect that ACBD3 still localized to the Golgi but it does not in the absence of SCFD1 as shown in this paper.
    • A prediction of the proposed model is that in cells depleted of the two Golgins, SCFD1 and ACBD3 should still bind to one another, right? Did the authors test this?
    • The model presented here (fig 8) seems to suggest that only the conformational variation of ACBD3 that binds Golgins is able to recruit (bind) PI4KIIIb. Is this known, or is there any experimental evidence for that?
    • Have the authors thought about testing the FKE mutant in the experiemnts shown in Fig. 5?
    • In general, I think the title might be a bit misleading because of the use of PI4Kiiib. I understand what the authors mean, but because they have not thoroughly tested PI4Kiiib recruitment in their experiments, I think they should focuse rather on the mechanism of recruitment of ACBD3 the authors have found.

    Significance

    I think, as said above, that this is potentially an important paper for the field of membrane trafficking and membrane biology. Most of the experiments are in general well performed and well controlled, and the paper is clearly written and follows a logical line.

  9. 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

    Summary

    This manuscript describes molecular mechanisms by which ACBD3 is recruited to the Golgi complex. ACBD3 recruits PI4KIII which is required to generate PI4P, a phosphoinositide which is key for the recruitment of essential Golgi proteins and hence is key to Golgi identity. The authors have used a combination of mass spectrometry, high quality fluorescence imaging, transient CRISPR knockdowns, and biochemical approaches such as IPs to identify the key determinant for recruitment of ACBD3 to the Golgi complex. They map the interaction between ACBD3 and the Golgi as a unique region (UR) upstream of its GOLD domain, identifying, in particular, an MWT motif as key for this recruitment. Using mass spectrometry they identify several novel interactors of ACBD3 as well as some established binding partners. Knockdown of these interactors reveal a key role for the SNARE, SCFD1, where reduced levels lead to complete loss of ACBD3 localisation to the Golgi without apparent disruption of Golgi structure. They further validate this interaction and that of another SNARE (Sec22b), which is part of the same SNARE complex as SCFD1, mapping the interaction to the longin domain of Sec22b. Surprisingly however they demonstrate that the UR domain does not mediate the interaction between ACBD3 and these SNAREs suggesting an alternative mechanism of recruitment. Previously identified ACBD3 interactors, Golgi proteins giantin and golgin-45 were also identified in the mass spectrometry screen and the authors demonstrate that these two proteins can recruit ACBD2 to the Golgi and this is dependent on the MWT motif identified in the UR domain. By knocking down SCFD1, they show reduced recruitment of ACBD3 leading them to propose a model of sequential recruitment of ACBD3 by SCFD1 followed by interactions with the golgins.

    Major points:

    This study is a well-executed and rigorous study of the molecular requirements for the recruitment of ACBD3 to the Golgi. The experimental approaches are state-of-the-art and the data are clean and convincing. The only caveat, raised by the authors themselves, is their interpretation that there are two sequential steps for Golgi recruitment of ACBD3. While they show that loss of SCFD1 reduces the interaction of ACBD3 with giantin and golgin 45, their model depends on doing the reverse experiment, i.e. assessing the effects of knocking down either giantin or golgin-45. This is especially relevant given the demonstration that golgin-45 is sufficient to recruit ACBD3 to mitochondria. It may well be that recruitment involves a tripartite complex, which is not uncommon in vesicular transport mechanisms Giantin is not an essential protein do it should be feasible to perform this experiment. The authors are equipped in the quantitative fluorescence microscopy which would be required and which would help resolve whether sequential or redundant mechanisms are required for ACBD3 recruitment.

    Significance

    PI4P is a phosphoinositide that is important for the recruitment of Golgi proteins. As with most PIs it is likely to act by coincidence detection in that Golgi associated proteins will recognise PI4P as well as other factors on Golgi membranes. This results in different local membrane environments which will be specific for particular functions. PI4KIII is key for PI4P production although the absolute levels of PI4P are likely to be determined by a balance of lipid kinases and phosphatases. However, since ACBD3 is key for the recruitment of PI4KIII it is important to understand the molecular mechanisms by which it is recruited. The manuscript thus makes a significant contribution to understanding one of the underlying mechanisms for PI4KIII recruitment although, as indicated above, stops short of establishing a clear model for the roles SCDF1 and Sec22b versus golgin 45 and giantin. For the future it will be of interest to determine why either a sequential or a redundant mechanism is required for the recruitment of ACBD3 as a scaffold protein.

  10. Version published to 10.1101/2023.04.13.536018v2 on bioRxiv
  11. Version published to 10.1101/2023.04.13.536018v1 on bioRxiv