Identification of the interactome of a palmitoylated membrane protein, phosphatidylinositol 4-Kinase type II Alpha

• Type of Material: Article
• Authors: Avanti S Gokhale, Emory University; Pearl V. Ryder, Emory University; Stephanie Zlatic, Emory University; Victor Faundez, Emory University
• Language: English
• Date: 2016-01-01
• Publisher: Humana Press
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © Springer Science+Business Media New York 2016.
• Final Published Version (URL): http://dx.doi.org/10.1007/978-1-4939-3170-5_4
• Title of Journal or Parent Work: Methods in Molecular Biology
• ISSN: 1064-3745
• Volume: 1376
• Start Page: 35
• End Page: 42
• Grant/Funding Information: This work was supported by grants from the National Institutes of Health (GM077569 and NS42599) and CHOA Children’s Center for Neuroscience to V.F. P.V.R. was supported by National Research Service Award Fellowship F31NS0765.
• Keywords: Phospholipid; Minor Histocompatibility Antigens; Mass spectrometry; Animals; Phosphatidylinositol; Phosphatidylinositol kinase; Protein Interaction Mapping; Immunoprecipitation; Protein Binding; Interactome; Humans; Phosphoinositide; Phosphotransferases (Alcohol Group Acceptor); Cross-linking; Carrier Proteins; Chromatography, Affinity; Endosome
• Research Categories: Biology, Neuroscience; Biology, Cell

Abstract

Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KIIα participates in transient, low-affinity interactions that are stabilized by the use of DSP. Using this optimized protocol we have successfully identified actin cytoskeleton regulators—the WASH complex and RhoGEF1, as major novel interactors of PI4KIIα. While this chapter focuses on the PI4KIIα interactome, this protocol can and has been used to generate other membrane interactome networks.