title: Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha.
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.
authors.affiliation: Department of Cell Biology, Emory University, 615 Michael Street Room 446, Atlanta, GA, 30322, USA.
authors.forename: Avanti
authors.initials: A
authors.lastname: Gokhale
authors.affiliation: Department of Cell Biology, Emory University, 615 Michael Street Room 446, Atlanta, GA, 30322, USA.
authors.forename: Pearl V
authors.initials: PV
authors.lastname: Ryder
authors.affiliation: Department of Cell Biology, Emory University, 615 Michael Street Room 446, Atlanta, GA, 30322, USA.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Zlatic
authors.affiliation: Department of Cell Biology, Emory University, 615 Michael Street Room 446, Atlanta, GA, 30322, USA. vfaunde@emory.edu.|Center for Social Translational Neuroscience, Emory University, Atlanta, GA, 30322, USA. vfaunde@emory.edu.
authors.forename: Victor
authors.initials: V
authors.lastname: Faundez
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D002352:Carrier Proteins; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase
keywords: Cross-linking; Endosome; Interactome; Mass spectrometry; Phosphatidylinositol; Phosphatidylinositol kinase; Phosphoinositide; Phospholipid