Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution
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eLife Assessment:
In this manuscript, the authors perform an extensive systematic analysis of membrane contacts sites to uncover novel proteins required for tethering organelles and modulation of membrane contacts. The authors identify over 100 new potential contact site proteins and effectors including proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. Further, the authors identify and characterize novel lipid transport proteins associated with the pClip as well as Lec1, an ER-Lipid droplet contact site associated protein which contains a novel putative lipid binding domain and may facilitate ergosterol transport between the plasma membrane and lipid droplets.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 ( L ipid-droplet E rgosterol C ortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.
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Author Response
Reviewer #1 (Public Review):
Main concerns:
- Validation of the MCS reporters is not shown. This is particularly important for pCLIP and GoPo, which have not been reported before. Fluorescence complementation between two proteins that normally localize to different organelles is far from demonstrating the existence of a MCS between those organelles. It would be important to demonstrate using marker proteins and ideally electron microscopy/CLEM the existence of the mentioned MCS and the suitability of the fluorescent reporter.
We thank the reviewer for pointing this out and have now added supplementary characterization of the pCLIP and GoPo contact sites. The pCLIP has been previously described by us (Shai et al. 2018 Nat Commun 9, 1761. doi:10.1038/s41467-018-03957-8) and so we have only added one new figure (Figure …
Author Response
Reviewer #1 (Public Review):
Main concerns:
- Validation of the MCS reporters is not shown. This is particularly important for pCLIP and GoPo, which have not been reported before. Fluorescence complementation between two proteins that normally localize to different organelles is far from demonstrating the existence of a MCS between those organelles. It would be important to demonstrate using marker proteins and ideally electron microscopy/CLEM the existence of the mentioned MCS and the suitability of the fluorescent reporter.
We thank the reviewer for pointing this out and have now added supplementary characterization of the pCLIP and GoPo contact sites. The pCLIP has been previously described by us (Shai et al. 2018 Nat Commun 9, 1761. doi:10.1038/s41467-018-03957-8) and so we have only added one new figure (Figure 2 S1A) which shows the co-localization of the contact site reporter with a LD marker (MDH) and a cell periphery marker (TRITC-ConA). For the GoPo, since this is the first demonstration of a reporter for this contact site, we have rigorously characterized it by looking at the frequency of co-localization between peroxisomes and the Golgi in the absence of the reporter (Figure 1 S1B), the co-localization of the contact site reporter with a peroxisome marker (CFP-SKL) and a Golgi marker (Sec7-mCherry) (Figure 1 S1C), and by identifying a condition where this contact site is increased (Figure 1 S1D).
Since all supported their function as bone-fide reporters and since performing electron microscopy experiments on these reporters was not possible for us at this time and has not been the standard in the field for other reporters, we hope that this is satisfactory.
- As pointed out above, the identification of a phenotype in ergosterol distribution for Ypr097W/Lec1 is very interesting. However, it is unclear how this observation relates with the localization of Lec1 to LDs, which is observed only upon over-expression.
We would like to clarify that at endogenous levels Lec1 also localizes to LDs. However, this localization is less pronounced. To clarify this in the text and show this experimentally we have now added an example of the endogenous GFP-tagged protein with the LD marker Faa4-mCherry (Figure 3 S1B), and added a section in the text.
Instead, further characterization of Ypr097w phenotype (via mutagenesis, modulation of ergosterol biosynthetic pathway, test ability to bind ergosterol, etc) in ergosterol distribution would be a plus.
To further characterize the Lec1 phenotype, we looked at changes in ADHpr-GFP-Lec1 localization in cells treated with 40 µg/ml of fluconazole for 3h (Figure 5 S2B-C). Fluconazole is a known inhibitor of Erg11 and treatment with this drug strongly reduces the overall levels of cellular ergosterol, which can be clearly observed by the loss of binding of mCherry-D4H to the plasma membrane (cytosolic signal) (Figure 5 S2B lower right panels). After 3h of treatment with fluconazole, there is a small increase in the number of cells with bud/ bud neck localization for GFP-Lec1. The GFP-Lec1 signal in these cells generally appears brighter than in untreated cells, suggesting that loss of ergosterol potentiates Lec1 accumulation at the bud / bud neck. This result suggests that Lec1 cellular localization is affected by the levels of ergosterol. However, since treatment with high concentration of fluconazole leads to growth arrest (Zhang et al. 2010. PLOS Pathogens 6:e1000939. doi:10.1371/journal.ppat.1000939), it is also possible that this signal increase is the result of Lec1 accumulation at the bud due to a stalling in budding. We now discuss this in the text.
We have also extensively mutagenized Lec1 as requested in an attempt to find a mutant that is still localized to LDs and stable yet not causing sterol redistribution. However, despite great efforts this has proven to be challenging (See below in detailed response to this request from reviewer #2).
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eLife Assessment:
In this manuscript, the authors perform an extensive systematic analysis of membrane contacts sites to uncover novel proteins required for tethering organelles and modulation of membrane contacts. The authors identify over 100 new potential contact site proteins and effectors including proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. Further, the authors identify and characterize novel lipid transport proteins associated with the pClip as well as Lec1, an ER-Lipid droplet contact site associated protein which contains a novel putative lipid binding domain and may facilitate ergosterol transport between the plasma membrane and lipid droplets.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors …
eLife Assessment:
In this manuscript, the authors perform an extensive systematic analysis of membrane contacts sites to uncover novel proteins required for tethering organelles and modulation of membrane contacts. The authors identify over 100 new potential contact site proteins and effectors including proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. Further, the authors identify and characterize novel lipid transport proteins associated with the pClip as well as Lec1, an ER-Lipid droplet contact site associated protein which contains a novel putative lipid binding domain and may facilitate ergosterol transport between the plasma membrane and lipid droplets.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Exploiting a previously established fluorescence complementation microscopy-based approach, Castro and colleagues screen a large library of fluorescently tagged proteins for their potential localization and regulation of MCS sites. This approach pinpointed known as well as previously unidentified components of MCS. Based on the homology models and structural prediction in silico, new classes of potential lipid transfer proteins were identified. While more work will be necessary, these are important results to the field. In a screen for components of the ER-LD MCS the authors identified ypr097W (re-named Lec1). This protein, which distributes to the cytosol, plasma membrane and upon overexpression also to LDs, appears to affect the distribution of ergosterol between mother and daughter cells. While these are …
Reviewer #1 (Public Review):
Exploiting a previously established fluorescence complementation microscopy-based approach, Castro and colleagues screen a large library of fluorescently tagged proteins for their potential localization and regulation of MCS sites. This approach pinpointed known as well as previously unidentified components of MCS. Based on the homology models and structural prediction in silico, new classes of potential lipid transfer proteins were identified. While more work will be necessary, these are important results to the field. In a screen for components of the ER-LD MCS the authors identified ypr097W (re-named Lec1). This protein, which distributes to the cytosol, plasma membrane and upon overexpression also to LDs, appears to affect the distribution of ergosterol between mother and daughter cells. While these are all interesting results, in its current form, the manuscript presents some shortcomings, as described below.
Main concerns:
Validation of the MCS reporters is not shown. This is particularly important for pCLIP and GoPo, which have not been reported before. Fluorescence complementation between two proteins that normally localize to different organelles is far from demonstrating the existence of a MCS between those organelles. It would be important to demonstrate using marker proteins and ideally electron microscopy/CLEM the existence of the mentioned MCS and the suitability of the fluorescent reporter.
As pointed out above, the identification of a phenotype in ergosterol distribution for Ypr097W/Lec1 is very interesting. However, it is unclear how this observation relates with the localization of Lec1 to LDs, which is observed only upon over-expression. Instead, further characterization of Ypr097w phenotype (via mutagenesis, modulation of ergosterol biosynthetic pathway, test ability to bind ergosterol, etc) in ergosterol distribution would be a plus.
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Reviewer #2 (Public Review):
In this manuscript, the authors used powerful yeast genetics tools to systemically identify proteins that colocalize with 6 contact site markers and/or impact the size and distribution of contact site markers. The major strength is the non-biased, high content screening utilizing the power of yeast. The identified lists of proteins will be very useful to colleagues studying membrane contact sites. The discovery of new members of the VPS13 family, and the characterization of a potential lipid binding fold are both interesting and valuable.
There are however some weaknesses. For instance, no biochemical characterization was provided for Csf1, Hob1 and 2. Further, the study on Ypr097w, a key finding of this paper, remains preliminary.
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Reviewer #3 (Public Review):
In this manuscript, the authors Castro, Shortill, Dziurdzik, Cadou and colleagues perform an extensive systematic analysis of six membrane contacts sites (MCSs) to uncover novel proteins required for organelle tethering and modulation of membrane contacts. This work is critical as few if any proteins have been identified to regulate the formation and/or function of these contact sites. The authors identify over 100 new potential contact site proteins and effectors, including identification of proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. This data set alone represents a huge contribution to the MCS field. The authors go on to identify and characterize novel proteins associated with the pClip homologous to known lipid binding proteins …
Reviewer #3 (Public Review):
In this manuscript, the authors Castro, Shortill, Dziurdzik, Cadou and colleagues perform an extensive systematic analysis of six membrane contacts sites (MCSs) to uncover novel proteins required for organelle tethering and modulation of membrane contacts. This work is critical as few if any proteins have been identified to regulate the formation and/or function of these contact sites. The authors identify over 100 new potential contact site proteins and effectors, including identification of proteins associated with the recently discovered plasma membrane-LD (pClip) and Golgi-peroxisome (GoPo) contact sites. This data set alone represents a huge contribution to the MCS field. The authors go on to identify and characterize novel proteins associated with the pClip homologous to known lipid binding proteins which may facilitate the transfer of lipids at membrane contact sites. Finally, the authors investigate the lipid droplet-ER (LiDER) contact site associated protein Lec1, which contains a novel putative lipid binding domain and may facilitate ergosterol transport between the plasma membrane and lipid droplets. The screening approach and experiments characterizing the function of Lec1 are for the most part straight forward, well-controlled, and easy to interpret. However, the authors' conclusions regarding the identified family of VPS13 like proteins role at the pCLIP is not strongly supported. Overall, these findings greatly expand our knowledge of proteins that regulate MCSs and will serve as the foundation for the identification of MCS tethers.
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