title: CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum.
abstract: Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.
authors.affiliation: Centre for Molecular Cell Biology, Division of Medicine, University College London, Royal Free Campus, London NW3 2PF, United Kingdom. Electronic address: m.waugh@ucl.ac.uk.
authors.forename: Mark G
authors.initials: MG
authors.lastname: Waugh
authors.affiliation: Centre for Molecular Cell Biology, Division of Medicine, University College London, Royal Free Campus, London NW3 2PF, United Kingdom.
authors.forename: Shane
authors.initials: S
authors.lastname: Minogue
authors.affiliation: Centre for Molecular Cell Biology, Division of Medicine, University College London, Royal Free Campus, London NW3 2PF, United Kingdom.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Clayton
authors.affiliation: Centre for Molecular Cell Biology, Division of Medicine, University College London, Royal Free Campus, London NW3 2PF, United Kingdom.
authors.forename: J Justin
authors.initials: JJ
authors.lastname: Hsuan
mesh_terms: D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D022461:Caveolins / Q000378:metabolism; D045744:Cell Line, Tumor; D003567:Cytidine Diphosphate Diglycerides / Q000096:biosynthesis*; D003902:Detergents / Q000737:chemistry*; D002798:Diacylglycerol Cholinephosphotransferase / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D057054:Molecular Imaging; D010743:Phospholipids / Q000096:biosynthesis*; D021381:Protein Transport; D012995:Solubility
chemical_list: D022461:Caveolins; D003567:Cytidine Diphosphate Diglycerides; D003902:Detergents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010743:Phospholipids; D037281:Calnexin; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002798:Diacylglycerol Cholinephosphotransferase