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Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes

Waugh, MG; Lawson, D; Tan, SK; Hsuan, JJ; (1998) Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes. J BIOL CHEM , 273 (27) 17115 - 17121. Gold open access

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)P-2) 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 C-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.

Type:Article
Title:Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes
Open access status:An open access publication
Publisher version:http://www.jbc.org/content/early/recent/0
Keywords:EPIDERMAL GROWTH-FACTOR, GPI-ANCHORED PROTEINS, KINASE-ACTIVITY, FACTOR RECEPTOR, CELLS, MICRODOMAINS, SECRETION, PIP2, PHOSPHORYLATION, LOCALIZATION

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