Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes.
J BIOL CHEM
17115 - 17121.
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.
|Title:||Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes|
|Open access status:||An open access publication|
|Keywords:||EPIDERMAL GROWTH-FACTOR, GPI-ANCHORED PROTEINS, KINASE-ACTIVITY, FACTOR RECEPTOR, CELLS, MICRODOMAINS, SECRETION, PIP2, PHOSPHORYLATION, LOCALIZATION|
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