A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
901–908
Year published:
DOI:
doi:10.1038/nsmb.1829
Received
Accepted
Published online

Abstract

The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of ~400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.

At a glance

Figures

  1. Composition of the plasma membrane E-MAP.
    Figure 1: Composition of the plasma membrane E-MAP.

    (a) Genes selected for the plasma membrane E-MAP are classified according to their biological function. (b,c) Genes encoding proteins interacting with each other are more likely to show positive genetic interactions (b) and correlated genetic interaction profiles (c). Green, interaction and correlation scores of gene pairs known to encode interacting proteins; black, the remainder of gene pairs.

  2. Overview of the clustergram of the plasma membrane E-MAP.
    Figure 2: Overview of the clustergram of the plasma membrane E-MAP.

    Top, selected areas are marked in the overview and highlighted as inserts 1–4. Yellow, positive genetic interactions; blue, negative genetic interactions. Bottom, genes with correlating genetic profiles are shared between RTG1 and MKS1. Pairwise correlations between RTG1 and MKS1 and all other genes in the plasma membrane E-MAP were calculated and plotted against each other.

  3. TGMs of the plasma membrane E-MAP.
    Figure 3: TGMs of the plasma membrane E-MAP.

    (a) All four potential TGMs are shown. Nodes in vertical order represent involvement in the same pathway; horizontal orientation indicates possible parallel pathways. (b) Type I TGMs that have PIL1 as a node. Nodes in green represent a gene important for Pil1-GFP localization (YMR031C) or a homolog of such a gene (EMP70)31.

  4. YMR031C/EIS1 encodes an eisosome component.
    Figure 4: YMR031C/EIS1 encodes an eisosome component.

    (a) Affinity purification and MS analysis of heavy labeled cells expressing GFP-tagged Pil1 and untagged control cells. Averaged peptide intensities are plotted against heavy/light SILAC ratios. Significant outliers (P < 0.0001) are colored in orange or light blue (P < 0.05); other identified proteins are shown in dark blue. (b) Pulldown purification from cells expressing tandem affinity-tagged Lsp1, Ymr031c or untagged control cells. Inputs and eluates from the pulldown were blotted and probed with antibodies against Pil1. (c) Colocalization of GFP-tagged Ymr031c with RFPmars-tagged Pil1. Representative confocal midsections are shown. The graph shows the intensity profiles for both channels along the perimeter of the cell. (d) PIL1 is required for normal localization of Ymr031c. Ymr031c-GFP or Lsp1-GFP was expressed and imaged either in WT or pil1Δ cells. Representative confocal midsections are shown. (e,f) Ymr031c is required for normal eisosome formation. Pil1-GFP (e) or Lsp1-GFP (f) was expressed in ymr031cΔ or control cells. Representative midsections are shown. For each experiment, the number of eisosomes per cell, the GFP fluorescence per eisosome and the cytosolic GFP fluorescence were quantified from at least 100 cells and are shown below the images. Scale bars, 2.5 μm.

  5. The eisosome-linked Emp70 is an early endosomal protein.
    Figure 5: The eisosome-linked Emp70 is an early endosomal protein.

    (a) Genes with correlating genetic profiles are shared between PIL1 and EMP70 but not PIL1 and LSP1. Correlation coefficients between the genetic profile of PIL1 and each of the other 373 profiles in the E-MAP are plotted on the x axis against, on the y axis, either the similar set of values for the LSP1 profile with all other profiles (blue) or those for EMP70 with all other profiles (red). Labeled points indicate some genes with profiles that are positively correlated with both the profile of PIL1 and that of EMP70. CC values in blue and red indicate the correlation coefficients for the full set of blue or red points plotted. (b) Emp70 colocalizes with Kex2. Emp70-GFP and Kex2-RFPmars were coexpressed and imaged. Representative confocal midsections are shown. (c) Emp70 localizes to an FM4-64 marked endocytic compartment. Cells expressing Emp70-GFP (green) were pulse labeled with FM4-64 (red) and imaged for 1 h. Images of midsections of cells at selected time are shown as indicated. (d) Emp70 localizes to the class E compartment in SNF7 mutants. GFP-tagged Emp70 was expressed in cells harboring nonfunctional Snf7-RFPmars, resulting in the clustering of endosomal proteins in the class E compartment. Representative confocal midsections are shown. (e) Emp70-GFP foci localize to the cell periphery. Emp70-GFP (green) was expressed in cells harboring the fluorescent eisosomes marker Lsp1-MARS. Representative mid- (left) and top sections (right) are shown. Boxes highlight selected areas of colocalization. (f) PIL1 is required for normal Emp70 localization to the cell periphery. Emp70-GFP was expressed in cells expressing the plasma membrane marker Ylr413w-RFPmars, and foci overlaying this marker were counted in more than 100 WT and pil1Δ cells. Results are shown as a histogram of number of spots opposed to the plasma membrane in each cell. (g) Quantitation of the organelle distribution of Emp70. Emp70-GFP was imaged in live cells and analyzed for colocalization with Kex2-RFPmars (n = 100), vacuolar FM4-64 (n = 91), Snf7-RFPmars (n = 93, diploid strain expressing one tagged Snf7 allele) and Lsp1-Cherry (n = 107). The relative area of overlap between signals was quantified as a percentage of total area occupied by Emp70 signal. Box plots representing maxima, 75th percentile, median, 25th percentile and minima are shown for the colocalization with each marker. Scale bars, 2.5 μm.

  6. Emp70 is required for normal endosome function.
    Figure 6: Emp70 is required for normal endosome function.

    (a) EMP70 is required for normal localization of Kex2-GFP. Kex2-GFP was expressed in either WT or emp70Δ cells, and representative confocal midsections are shown. (b) Emp70 family members are required for late endosomal protein retrieval. Mutants of EMP70, TMN2 or TMN3 were tested alone or in combination for CPY secretion. A representative colony blot is shown. Scale bar, 2.5 μm.

  7. Genetic interactions of sphingolipid metabolism.
    Figure 7: Genetic interactions of sphingolipid metabolism.

    (a) Graphic representation of the sphingolipid synthesis pathway. Blue, negative genetic interactions; yellow, positive interactions. (b) Genes encoding enzymes acting in succession in sphingolipid synthesis show higher correlation than genes further apart in the metabolic network. For each gene pair in sphingolipid synthesis, the pathway distance of genes (that is, the number of metabolic intermediates between the catalyzed reactions) is plotted against the correlation coefficient of the gene pairs. The red line is a best-fit linear regression line fitted for all the data points on the graph.

  8. Rom2 interacts with sphingolipid metabolism.
    Figure 8: Rom2 interacts with sphingolipid metabolism.

    (a) Genes with correlating genetic profiles are shared between SUR4 and ROM2 but not between CSG2 and ROM2. Correlation coefficients between the genetic profile of ROM2 and each of the other 373 profiles in the E-MAP are plotted on the x axis against, on the y axis, either the similar set of values for the SUR4 profile with all other profiles (blue) or those for CSG2 with all other profiles (red). Labeled points indicate genes with profiles that are positively correlated with the profile of ROM2. CC values in blue and red indicate the correlation coefficients for the full set of blue or red points plotted. (b) Lipidome profiling of rom2Δ and selected sphingolipid metabolism mutants. Lipid class abundances were normalized to WT levels. Sterol esters (SE), phosphatidic acid (PA), triacylglycerol (TAG), long chain base (LCB) mannosylinositol phosphoceramide (MIPC), phosphatidylethanolamine (PE), diacylglycerol (DAG), phoshphatidylcholine (PC), phoshphatidylinositol (PI), ceramide (Cer), phosphatidylserine (PS) mannosylinositol-2-phosphoceramide (M(IP)2C) and inositol phosphoceramide (IPC) levels are shown.

References

  1. Grossmann, G., Opekarova, M., Malinsky, J., Weig-Meckl, I. & Tanner, W. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast. EMBO J. 26, 18 (2007).
  2. Walther, T.C. et al. Eisosomes mark static sites of endocytosis. Nature 439, 9981003 (2006).
  3. Grossmann, G. et al. Plasma membrane microdomains regulate turnover of transport proteins in yeast. J. Cell Biol. 183, 10751088 (2008).
  4. deHart, A.K., Schnell, J.D., Allen, D.A. & Hicke, L. The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast. J. Cell Biol. 156, 241248 (2002).
  5. Friant, S., Lombardi, R., Schmelzle, T., Hall, M.N. & Riezman, H. Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast. EMBO J. 20, 67836792 (2001).
  6. Walther, T.C. et al. Pkh-kinases control eisosome assembly and organization. EMBO J. 26, 49464955 (2007).
  7. Aronova, S. et al. Regulation of ceramide biosynthesis by TOR complex 2. Cell Metab. 7, 148158 (2008).
  8. Guan, X.L. et al. Functional interactions between sphingolipids and sterols in biological membranes regulating cell physiology. Mol. Biol. Cell 20, 20832095 (2009).
  9. Tabuchi, M., Audhya, A., Parsons, A.B., Boone, C. & Emr, S.D. The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation. Mol. Cell. Biol. 26, 58615875 (2006).
  10. Tong, A.H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808813 (2004).
  11. Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507519 (2005).
  12. Collins, S.R., Schuldiner, M., Krogan, N.J. & Weissman, J.S. A strategy for extracting and analyzing large-scale quantitative epistatic interaction data. Genome Biol. 7, R63 (2006).
  13. Schuldiner, M., Collins, S.R., Weissman, J.S. & Krogan, N.J. Quantitative genetic analysis in Saccharomyces cerevisiae using epistatic miniarray profiles (E-MAPs) and its application to chromatin functions. Methods 40, 344352 (2006).
  14. Ulitsky, I., Shlomi, T., Kupiec, M. & Shamir, R. From E-MAPs to module maps: dissecting quantitative genetic interactions using physical interactions. Mol. Syst. Biol. 4, 209 (2008).
  15. Bandyopadhyay, S., Kelley, R., Krogan, N.J. & Ideker, T. Functional maps of protein complexes from quantitative genetic interaction data. PLOS Comput. Biol. 4, e1000065 (2008).
  16. Collins, S.R. et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 446, 806810 (2007).
  17. Fiedler, D. et al. Functional organization of the S. cerevisiae phosphorylation network. Cell 136, 952963 (2009).
  18. Wilmes, G.M. et al. A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing. Mol. Cell 32, 735746 (2008).
  19. Collins, S.R. et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae . Mol. Cell. Proteomics 6, 439450 (2007).
  20. Gavin, A.C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631636 (2006).
  21. Krogan, N.J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae . Nature 440, 637643 (2006).
  22. Ren, G., Vajjhala, P., Lee, J.S., Winsor, B. & Munn, A.L. The BAR domain proteins: molding membranes in fission, fusion, and phagy. Microbiol. Mol. Biol. Rev. 70, 37120 (2006).
  23. Revardel, E., Bonneau, M., Durrens, P. & Aigle, M. Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae . Biochim. Biophys. Acta 1263, 261265 (1995).
  24. Breton, A.M. & Aigle, M. Genetic and functional relationship between Rvsp, myosin and actin in Saccharomyces cerevisiae . Curr. Genet. 34, 280286 (1998).
  25. Brizzio, V., Gammie, A.E. & Rose, M.D. Rvs161p interacts with Fus2p to promote cell fusion in Saccharomyces cerevisiae . J. Cell Biol. 141, 567584 (1998).
  26. Friesen, H. et al. Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo . Mol. Biol. Cell 17, 13061321 (2006).
  27. Jonikas, M.C. et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323, 16931697 (2009).
  28. Valdivia, R.H., Baggott, D., Chuang, J.S. & Schekman, R.W. The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins. Dev. Cell 2, 283294 (2002).
  29. Efe, J.A. et al. Yeast Mon2p is a highly conserved protein that functions in the cytoplasm-to-vacuole transport pathway and is required for Golgi homeostasis. J. Cell Sci. 118, 47514764 (2005).
  30. Levin, D.E. Cell wall integrity signaling in Saccharomyces cerevisiae . Microbiol. Mol. Biol. Rev. 69, 262291 (2005).
  31. Frohlich, F. et al. A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling. J. Cell Biol. 185, 12271242 (2009).
  32. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376386 (2002).
  33. Wang, H. et al. A complex-based reconstruction of the Saccharomyces cerevisiae interactome. Mol. Cell. Proteomics 8, 13611381 (2009).
  34. Deng, C., Xiong, X. & Krutchinsky, A.N. Unifying fluorescence microscopy and mass spectrometry for studying protein complexes in cells. Mol. Cell. Proteomics 8, 14131423 (2009).
  35. Schimmoller, F., Diaz, E., Muhlbauer, B. & Pfeffer, S.R. Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution. Gene 216, 311318 (1998).
  36. Vida, T.A. & Emr, S.D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779792 (1995).
  37. Zheng, B., Wu, J.N., Schober, W., Lewis, D.E. & Vida, T. Isolation of yeast mutants defective for localization of vacuolar vital dyes. Proc. Natl. Acad. Sci. USA 95, 1172111726 (1998).
  38. Babst, M., Wendland, B., Estepa, E.J. & Emr, S.D. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17, 29822993 (1998).
  39. Teis, D., Saksena, S. & Emr, S.D. Ordered assembly of the ESCRT-III complex on endosomes is required to sequester cargo during MVB formation. Dev. Cell 15, 578589 (2008).
  40. Brickner, J.H. & Fuller, R.S. SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals. J. Cell Biol. 139, 2336 (1997).
  41. Sipos, G. et al. Soi3p/Rav1p functions at the early endosome to regulate endocytic trafficking to the vacuole and localization of trans-Golgi network transmembrane proteins. Mol. Biol. Cell 15, 31963209 (2004).
  42. Robinson, J.S., Klionsky, D.J., Banta, L.M. & Emr, S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol. 8, 49364948 (1988).
  43. Rothman, J.H., Howald, I. & Stevens, T.H. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae . EMBO J. 8, 20572065 (1989).
  44. Cerantola, V. et al. Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides. Mol. Microbiol. 71, 15231537 (2009).
  45. Ejsing, C.S. et al. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl. Acad. Sci. USA 106, 21362141 (2009).
  46. Schmidt, A., Bickle, M., Beck, T. & Hall, M.N. The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell 88, 531542 (1997).
  47. Kobayashi, T., Takematsu, H., Yamaji, T., Hiramoto, S. & Kozutsumi, Y. Disturbance of sphingolipid biosynthesis abrogates the signaling of Mss4, phosphatidylinositol-4-phosphate 5-kinase, in yeast. J. Biol. Chem. 280, 1808718094 (2005).
  48. Audhya, A. & Emr, S.D. Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade. Dev. Cell 2, 593605 (2002).
  49. Cherry, J.M. et al. SGD: Saccharomyces Genome Database. Nucleic Acids Res. 26, 7379 (1998).
  50. Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 2730 (2000).
  51. Reguly, T. et al. Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae . J. Biol. 5, 11 (2006).
  52. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947962 (2004).
  53. Mullins, C. & Bonifacino, J.S. Structural requirements for function of yeast GGAs in vacuolar protein sorting, α-factor maturation, and interactions with clathrin. Mol. Cell. Biol. 21, 79817994 (2001).
  54. Olsen, J.V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 20102021 (2005).
  55. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 13671372 (2008).
  56. Moore, R.E., Young, M.K. & Lee, T.D. Qscore: an algorithm for evaluating SEQUEST database search results. J. Am. Soc. Mass Spectrom. 13, 378386 (2002).
  57. Peng, J., Elias, J.E., Thoreen, C.C., Licklider, L.J. & Gygi, S.P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. 2, 4350 (2003).
  58. Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 35513567 (1999).

Download references

Author information

  1. These authors contributed equally to this work.

    • Pablo S Aguilar,
    • Florian Fröhlich,
    • Michael Rehman &
    • Mike Shales

Affiliations

  1. Institut Pasteur de Montevideo, Montevideo, Uruguay.

    • Pablo S Aguilar &
    • Agustina Olivera-Couto
  2. Max Planck Institute of Biochemistry, Organelle Architecture and Dynamics, Martinsried, Germany.

    • Florian Fröhlich,
    • Michael Rehman &
    • Tobias C Walther
  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA.

    • Mike Shales,
    • Hannes Braberg &
    • Nevan J Krogan
  4. The Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel.

    • Igor Ulitsky &
    • Ron Shamir
  5. Department of Biochemistry and Biophysics, University of California and Howard Hughes Medical Institute, San Francisco, California, USA.

    • Peter Walter
  6. Max Planck Institute of Biochemistry, Proteomics and Signal Transduction, Martinsried, Germany.

    • Matthias Mann
  7. University of Southern Denmark, Department of Biochemistry and Molecular Biology, Odense, Denmark.

    • Christer S Ejsing
  8. Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Igor Ulitsky

Contributions

All authors contributed to every aspect of this work.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (10M)

    Supplementary Figures 1–5, Supplementary Tables 3–7

Excel files

  1. Supplementary Table 1 (68K)

    Classification of genes used for Figure 1a

  2. Supplementary Table 2 (13M)

    Correlations of genes pairs in the plasma membrane E-MAP used in Figures 1b and 1c.

Movies

  1. Supplementary Video 1 (12M)

    Localization of Emp70-GFP.

  2. Supplementary Video 2 (12M)

    Localization of Emp70-GFP in respect to Lsp1-RFPmars.

Zip files

  1. Supplementary Data (472K)

    Treeview representation for clustergram of the PM E-MAP, composed of 4 files (.atr, .cdt,.gtr.,jtv). These files can be opened by a treeview program such as Java Treeview that can be found at: http://jtreeview.sourceforge.net/.

Additional data