Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
J Proteome Res. 2010 Feb 5;9(2):833-42. doi: 10.1021/pr900743b.
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.
高通量分析蛋白质-蛋白质相互作用可以提供前所未有的见解，了解细胞过程如何在分子水平上整合。然而，由于膜蛋白的疏水性和低丰度，它们往往在这些研究中被忽视。在这里，我们使用了一种基于蛋白质组学的策略，专门用于鉴定膜相关的蛋白质复合物。我们方法的一个重要方面是使用化学交联来捕获在细胞裂解之前发生在活细胞中的瞬时和低亲和力蛋白质相互作用。我们应用这种方法来鉴定酵母高尔基体 P(4)-ATP 酶 Drs2p 的结合伙伴，Drs2p 是假定的氨基磷脂转运体保守家族的成员。Drs2p 被内源性地标记上多组氨酸和生物素化肽，允许在高度严格的条件下进行包含 Drs2p 的蛋白质复合物的串联亲和纯化。分离的复合物的质谱分析产生了一个已知的和九个新的 Drs2p 结合伙伴。通过正交的体内膜蛋白相互作用测定验证了结合特异性，证实了我们方法的有效性。引人注目的是，三个新的 Drs2p 相互作用因子参与了磷酸肌醇代谢。其中之一，磷脂酰肌醇-4-磷酸酶 Sac1p，也与 Drs2p 显示出遗传相互作用。这些发现表明，氨基磷脂转运和磷酸肌醇代谢在高尔基体中相互关联。
