title: Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes.
abstract: This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.
authors.affiliation: Ludwig Institute for Cancer Research, University College London Medical School, Courtauld Building, 91 Riding House Street, London W1P 8BT, United Kingdom.
authors.forename: M G
authors.initials: MG
authors.lastname: Waugh
authors.forename: D
authors.initials: D
authors.lastname: Lawson
authors.forename: S K
authors.initials: SK
authors.lastname: Tan
authors.forename: J J
authors.initials: JJ
authors.lastname: Hsuan
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis* / Q000378:metabolism; D011233:Precipitin Tests; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D019870:1-Phosphatidylinositol 4-Kinase