title: A P4-ATPase protein interaction network reveals a link between aminophospholipid transport and phosphoinositide metabolism.
abstract: High-throughput analysis of protein-protein interactions can provide unprecedented insight into how cellular processes are integrated at the molecular level. Yet membrane proteins are often overlooked in these studies owing to their hydrophobic nature and low abundance. Here we used a proteomics-based strategy with the specific intention of identifying membrane-associated protein complexes. One important aspect of our approach is the use of chemical cross-linking to capture transient and low-affinity protein interactions that occur in living cells prior to cell lysis. We applied this method to identify binding partners of the yeast Golgi P(4)-ATPase Drs2p, a member of a conserved family of putative aminophospholipid transporters. Drs2p was endogeneously tagged with both a polyhistidine and a biotinylation peptide, allowing tandem-affinity purification of Drs2p-containing protein complexes under highly stringent conditions. Mass-spectrometric analysis of isolated complexes yielded one known and nine novel Drs2p binding partners. Binding specificity was verified by an orthogonal in vivo membrane protein interaction assay, confirming the efficacy of our method. Strikingly, three of the novel Drs2p interactors are involved in phosphoinositide metabolism. One of these, the phosphatidylinositol-4-phosphatase Sac1p, also displays genetic interactions with Drs2p. Together, these findings suggest that aminophospholipid transport and phosphoinositide metabolism are interconnected at the Golgi.
authors.affiliation: Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Catheleyne F
authors.initials: CF
authors.lastname: Puts
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lenoir
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
authors.forename: Patrick
authors.initials: P
authors.lastname: Williamson
authors.forename: Joost C M
authors.initials: JC
authors.lastname: Holthuis
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002846:Chromatography, Affinity; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D053719:Tandem Mass Spectrometry
chemical_list: D010716:Phosphatidylinositols; D010743:Phospholipids; D000251:Adenosine Triphosphatases