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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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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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