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Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins

Abstract

The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates—through mechanisms not fully elucidated—with the inner leaflet of the plasma membrane1,2,3. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases4,5,6,7. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP–lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.

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Figure 1: Recombinant Osh6/Osh7 bind and specifically transfer PS in vitro.
Figure 2: In vivo, Osh6/Osh7 transport PS from the ER to the PM.
Figure 3: Structure of the Osh6–PS complex.
Figure 4: Osh6/Osh7 are the first representatives of a PS-binding OSBP subfamily conserved in humans.

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References

  1. Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008)

    Article  CAS  ADS  Google Scholar 

  2. Leventis, P. A. & Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 39, 407–427 (2010)

    Article  CAS  Google Scholar 

  3. Fairn, G. D., Hermansson, M., Somerharju, P. & Grinstein, S. Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity. Nature Cell Biol. 13, 1424–1430 (2011)

    Article  CAS  Google Scholar 

  4. D’Angelo, G., Vicinanza, M. & De Matteis, M. A. Lipid-transfer proteins in biosynthetic pathways. Curr. Opin. Cell Biol. 20, 360–370 (2008)

    Article  Google Scholar 

  5. Lev, S. Non-vesicular lipid transport by lipid-transfer proteins and beyond. Nature Rev. Mol. Cell Biol. 11, 739–750 (2010)

    Article  CAS  Google Scholar 

  6. Holthuis, J. C. & Levine, T. P. Lipid traffic: floppy drives and a superhighway. Nature Rev. Mol. Cell Biol. 6, 209–220 (2005)

    Article  CAS  Google Scholar 

  7. Stefan, C. J. et al. Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites. Cell 144, 389–401 (2011)

    Article  CAS  Google Scholar 

  8. Gavin, A. C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006)

    Article  CAS  ADS  Google Scholar 

  9. Gavin, A. C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002)

    Article  CAS  ADS  Google Scholar 

  10. Raychaudhuri, S., Im, Y. J., Hurley, J. H. & Prinz, W. A. Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides. J. Cell Biol. 173, 107–119 (2006)

    Article  CAS  Google Scholar 

  11. Li, X. et al. Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth. Mol. Biol. Cell 11, 1989–2005 (2000)

    Article  CAS  Google Scholar 

  12. Schulz, T. A. et al. Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues. J. Cell Biol. 187, 889–903 (2009)

    Article  CAS  Google Scholar 

  13. Li, X. et al. Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex. J. Cell Biol. 157, 63–78 (2002)

    Article  CAS  Google Scholar 

  14. Ejsing, C. S. et al. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl Acad. Sci. USA 106, 2136–2141 (2009)

    Article  CAS  ADS  Google Scholar 

  15. Slaughter, B. D. et al. Non-uniform membrane diffusion enables steady-state cell polarization via vesicular trafficking. Nature Commun. 4, 1380 (2013)

    Article  ADS  Google Scholar 

  16. Riekhof, W. R. et al. Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation. J. Biol. Chem. 282, 36853–36861 (2007)

    Article  CAS  Google Scholar 

  17. Spira, F. et al. Patchwork organization of the yeast plasma membrane into numerous coexisting domains. Nature Cell Biol. 14, 640–648 (2012)

    Article  CAS  Google Scholar 

  18. Im, Y. J., Raychaudhuri, S., Prinz, W. A. & Hurley, J. H. Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature 437, 154–158 (2005)

    Article  CAS  ADS  Google Scholar 

  19. de Saint-Jean, M. et al. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers. J. Cell Biol. 195, 965–978 (2011)

    Article  CAS  Google Scholar 

  20. Punta, M. et al. The Pfam protein families database. Nucleic Acids Res. 40, D290–D301 (2012)

    Article  CAS  Google Scholar 

  21. Ngo, M. H., Colbourne, T. R. & Ridgway, N. D. Functional implications of sterol transport by the oxysterol-binding protein gene family. Biochem. J. 429, 13–24 (2010)

    Article  CAS  Google Scholar 

  22. Burgett, A. W. et al. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nature Chem. Biol. 7, 639–647 (2011)

    Article  CAS  Google Scholar 

  23. Beh, C. T., Cool, L., Phillips, J. & Rine, J. Overlapping functions of the yeast oxysterol-binding protein homologues. Genetics 157, 1117–1140 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Fairn, G. D. et al. High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine. J. Cell Biol. 194, 257–275 (2011)

    Article  CAS  Google Scholar 

  25. Pichler, H. et al. A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids. Eur. J. Biochem. 268, 2351–2361 (2001)

    Article  CAS  Google Scholar 

  26. Yu, J. W. & Lemmon, M. A. All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate. J. Biol. Chem. 276, 44179–44184 (2001)

    Article  CAS  Google Scholar 

  27. Yu, J. W. et al. Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell 13, 677–688 (2004)

    Article  CAS  Google Scholar 

  28. Park, W. S. et al. Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging. Mol. Cell 30, 381–392 (2008)

    Article  CAS  Google Scholar 

  29. Gallego, O. et al. A systematic screen for protein–lipid interactions in Saccharomyces cerevisiae. Mol. Syst. Biol. 6, 430 (2010)

    Article  CAS  Google Scholar 

  30. Li, X., Gianoulis, T. A., Yip, K. Y., Gerstein, M. & Snyder, M. Extensive in vivo metabolite-protein interactions revealed by large-scale systematic analyses. Cell 143, 639–650 (2010)

    Article  CAS  Google Scholar 

  31. Weerheim, A. M., Kolb, A. M., Sturk, A. & Nieuwland, R. Phospholipid composition of cell-derived microparticles determined by one-dimensional high-performance thin-layer chromatography. Anal. Biochem. 302, 191–198 (2002)

    Article  CAS  Google Scholar 

  32. Churchward, M. A., Brandman, D. M., Rogasevskaia, T. & Coorssen, J. R. Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles. J. Chem. Biol. 1, 79–87 (2008)

    Article  Google Scholar 

  33. Tong, A. H. & Boone, C. in Yeast Gene Analysis, Methods in Microbiology 2nd edn, Vol. 36 (eds Stansfield, I. and Stark, M. ) 369–386; 706–707 (Elsevier, 2007)

    Google Scholar 

  34. 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, 947–962 (2004)

    Article  CAS  Google Scholar 

  35. Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl Acad. Sci. USA 92, 4947–4951 (1995)

    Article  CAS  ADS  Google Scholar 

  36. Rossanese, O. W. et al. A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae. J. Cell Biol. 153, 47–62 (2001)

    Article  CAS  Google Scholar 

  37. Stefan, C. J., Audhya, A. & Emr, S. D. The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate. Mol. Biol. Cell 13, 542–557 (2002)

    Article  CAS  Google Scholar 

  38. Fischl, A. S. & Carman, G. M. Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase. J. Bacteriol. 154, 304–311 (1983)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Panaretou, B. & Piper, P. Isolation of yeast plasma membranes. Methods Mol. Biol. 313, 27–32 (2006)

    CAS  PubMed  Google Scholar 

  40. Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003)

    Article  CAS  ADS  Google Scholar 

  41. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009)

    Article  CAS  Google Scholar 

  42. Felsenstein, J. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, Washington, USA. (1993)

  43. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23, 127–128 (2007)

    Article  CAS  Google Scholar 

  44. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

    Article  CAS  Google Scholar 

  45. Di Tommaso, P. et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 39, W13–W17 (2011)

    Article  CAS  Google Scholar 

  46. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

  47. Rossmann, M. G. The molecular replacement method. Acta Crystallogr. A 46, 73–82 (1990)

    Article  Google Scholar 

  48. Read, R. J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr. D 57, 1373–1382 (2001)

    Article  CAS  Google Scholar 

  49. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  50. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  51. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  52. Pei, J., Kim, B. H. & Grishin, N. V. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008)

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to C. Schultz and C. Müller for inspiring comments on the manuscript, and to the EMBL Proteomics and the Protein Expression and Purification Core Facilities, E. M. Vilalta, V. Rybin, O. Gallego, M. Skruzny, A. Picco, S. Glatt, F. Voigt and A. Scholz for expert help and the sharing of reagents. The authors thank the beam line staff at the European Synchrotron Radiation Facility (ESRF), beam line ID23–1, Grenoble, France where crystallographic data collection was performed. We also thank C. Müller’s group and other members of M.K.’s and A.-C.G.’s groups for continuous discussions and support. We are grateful to C. Boone, S. Emr and E. Hurt for sharing reagents. This work was partially funded by the Federal Ministry of Education and Research (BMBF; 01GS0865) in the framework of the IG-Cellular System genomics to A.-C.G.; A.K. is supported by the European Molecular Biology Laboratory and the EU Marie Curie Actions Interdisciplinary Postdoctoral Cofunded Programme. K.A. acknowledges support from Marie Curie reintegration grant (ERG). K.M. is supported by the Danish Natural Science Research Council (09-064986/FNU).

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K.M. and A.-C.G. designed the research; K.M., A.C. and A.K. conducted the experiments and performed the analysis; K.A. and K.M. performed X-ray crystallography; M.P. supported the development of the biochemical protocols; M.K. provided technical expertise with instrumentation; and K.M. and A.-C.G. discussed results and wrote the manuscript with support from all the authors.

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Correspondence to Anne-Claude Gavin.

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The authors declare no competing financial interests.

Additional information

The atomic coordinates and structure factors have been deposited in the Protein Data Bank at entry 4B2Z.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 and Supplementary Table 1. The Supplementary Figures show the summary and original data from the screen on lipid transfer protein-lipid interactions, and additional biochemical, structural and cell biological data. Supplementary Table 1 shows the data collection and refinement statistics for the crystal structure of Osh6 in complex with phosphatidylserine. (PDF 3436 kb)

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Maeda, K., Anand, K., Chiapparino, A. et al. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins. Nature 501, 257–261 (2013). https://doi.org/10.1038/nature12430

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