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A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids


Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo–assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30–50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography–mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.

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Figure 1: Overview of the procedure to capture protein-lipid complexes assembled in vivo in Saccharomyces cerevisiae.
Figure 2: Purification of the Osh4/Kes1-ergosterol complex.
Figure 3: Performance of the protocol for lipid-transfer proteins (LTPs).

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  1. Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).

    Article  CAS  Google Scholar 

  2. 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 

  3. 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 

  4. Li, X.Y., 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 

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

    Article  CAS  Google Scholar 

  6. Haberkant, P. et al. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids. Angew. Chem. Int. Ed. Engl. 52, 4033–4038 (2013).

    Article  CAS  Google Scholar 

  7. Saliba, A.E. et al. A quantitative liposome microarray to systematically characterize protein-lipid interactions. Nat. Methods 11, 47–50 (2014).

    Article  CAS  Google Scholar 

  8. Lee, J.M. et al. A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects. Nature 474, 506–510 (2011).

    Article  CAS  Google Scholar 

  9. Hait, N.C. et al. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 325, 1254–1257 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Maeda, K. et al. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins. Nature 501, 257–261 (2013).

    Article  CAS  Google Scholar 

  12. van Meer, G., Voelker, D.R. & Feigenson, G.W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Krogan, N.J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643 (2006).

    Article  CAS  Google Scholar 

  15. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Sha, B.D., Phillips, S.E., Bankaitis, V.A. & Luo, M. Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol-transfer protein. Nature 391, 506–510 (1998).

    Article  CAS  Google Scholar 

  18. 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 

  19. 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 

  20. Brugger, B., Erben, G., Sandhoff, R., Wieland, F.T. & Lehmann, W.D. Quantitative analysis of biological membrane lipids at the low-picomole level by nano-electrospray ionization tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 94, 2339–2344 (1997).

    Article  CAS  Google Scholar 

  21. Griffiths, W.J. & Wang, Y.Q. Mass spectrometry: from proteomics to metabolomics and lipidomics. Chem. Soc. Rev. 38, 1882–1896 (2009).

    Article  CAS  Google Scholar 

  22. Fuchs, B., Suss, R., Teuber, K., Eibisch, M. & Schiller, J. Lipid analysis by thin-layer chromatography: a review of the current state. J. Chromatogr. A 1218, 2754–2774 (2011).

    Article  CAS  Google Scholar 

  23. Welti, S., Fraterman, S., D'Angelo, I., Wilm, M. & Scheffzek, K. The sec14 homology module of neurofibromin binds cellular glycerophospholipids: mass spectrometry and structure of a lipid complex. J. Mol. Biol. 366, 551–562 (2007).

    Article  CAS  Google Scholar 

  24. Gavin, A.C., Maeda, K. & Kuhner, S. Recent advances in charting protein-protein interaction: mass spectrometry–based approaches. Curr. Opin. Biotech. 22, 42–49 (2011).

    Article  CAS  Google Scholar 

  25. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  Google Scholar 

  26. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  Google Scholar 

  27. Hsu, F.F. & Turk, J. Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: assignment of fatty acyl groups on the glycerol backbone and location of double bonds. J. Am. Soc. Mass Spectrom. 21, 657–669 (2010).

    Article  CAS  Google Scholar 

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We are grateful to E.M. Vilalta, F. O'Reilly, A. Tarafder and the Protein Expression and Purification Core Facility (V. Rybin) for expert help. We also thank other members of A.-C.G.'s groups for continuous discussions and support. K.M. is supported by the Danish Natural Science Research Council (09-064986/FNU). This work is partially funded by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the Cluster of Excellence, CellNetworks Initiative of the University of Heidelberg.

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K.M. and A.-C.G. designed the protocol. K.M. and M.P. were responsible for the development and optimization of the protocol. A.C. supported the protocol optimization and A.-C.G. provided valuable advice.

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Correspondence to Anne-Claude Gavin.

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The authors declare no competing financial interests.

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Maeda, K., Poletto, M., Chiapparino, A. et al. A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids. Nat Protoc 9, 2256–2266 (2014).

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