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 proteomicsbased 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 lowaffinity 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.

P4 ATPases: Flippases in Health and Disease

“…Genetic associations with drs2 have been described for ADP-ribosylation factors (recruitment of coat protein complexes) and Rab proteins [54,90–92]. Using tandem-affinity purification and mass spectrometric analyses, Puts et al have identified additional Drs2p-interacting proteins with a role in vesicular trafficking [91]. Amongst others, three proteins involved in phosphoinositide metabolism were identified, including a PI4P phosphatase.…”

“…In addition, the different subcellular distributions of the newly identified Drs2p-interacting proteins ( i.e. , Golgi apparatus (GA), ER, plasma membrane, and vacuole) suggest that Drs2p resides in different protein complexes within distinct trafficking pathways [91]. …”

Bacterial Electron Transfer Chains Primed by Proteomics

“…For instance, amino acids required for cross-linking are rather rare in hydrophobic domains. Yet, cross-linking may stabilise the interaction between soluble and membrane-embedded subunits, supporting their combined isolation (Puts, Lenoir, Krijgsveld, Williamson, & Holthuis, 2010). Anyway, an examination of at least the hydrophilic stretches of membrane subunits and of the associated solventexposed subunits should be feasible (Fig.…”

P4-ATPases: lipid flippases in cell membranes

“…The yeast P4-ATPases Drs2p and Neo1p were found to interact with cytosolic proteins such as guanine nucleotide exchange factors and small GTPases that are crucial for the recruitment of coat proteins during membrane budding [7, 11, 15, 31, 92, 102]. Furthermore, a systematic search for Drs2p-binding partners yielded proteins involved in phosphoinositide metabolism [70]. Phosphoinositides are important signaling molecules in membrane traffic that help establish organelle identity through recruitment of effector proteins.…”