Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes
| Autor: | J. Justin Hsuan, Mark G. Waugh, Siow Khoon Tan, Durward Lawson |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Phosphatidylinositol 4-phosphate
Caveolin 1 Biology Biochemistry Caveolins Cell Line Substrate Specificity chemistry.chemical_compound Phosphatidylinositol Phosphates Caveolae Caveolin Phosphatidylinositol Kinase activity Molecular Biology 1-Phosphatidylinositol 4-Kinase Phosphatidylinositol transfer protein Vesicle Cell Membrane Membrane Proteins Cell Biology Precipitin Tests Cell biology Microscopy Electron chemistry Cell fractionation Subcellular Fractions |
| Zdroj: | The Journal of biological chemistry. 273(27) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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