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.
Highlights
Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase
Through the use of a variety of cell fractionation, immunoisolation, and immunofluorescence imaging techniques, we demonstrated that a pool of CDS activity localizes to calnexin-containing endoplasmic reticulum (ER) membrane domains that cofractionate with low-buoyant density caveolae but with biochemical properties that are different to those ascribed to plasma membrane rafts
The novel phospholipid-synthesizing membrane domains we describe here are detergent-soluble and are not enriched for either cholesterol or glycosphingolipids. This sets them apart from the detergent-resistant ER domains implicated in the synthesis of mammalian glycosylphosphatidylinositol anchor intermediates [54], the organization of export to the Golgi [55], and from the detergent-insoluble membrane domains to which erlin-1/2 [7] and sigma-1 chaperones [6] are targeted. Another novel finding from our coimmunoprecipitation studies is the origin of the CDP-DAG-synthesizing domains in the calnexin and type III inositol 1 (IP3R)-containing region of ER
Summary
Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. There are reports that MAMs contain detergent-insoluble membrane microdomains [6, 7] analogous to plasma membrane rafts, and that MAMs are enriched for molecular chaperones such as calnexin [8] as well as signaling molecules such as type III inositol [1,4,5]-trisphosphate receptors (IP3R) [9, 10] This has led to the proposal that MAMs represent important sites for the compartmentation and regulation of intracellular Ca2+ release [10], a key event in phosphatidylinositol (PI)-dependent, phospholipase C (PLC) signaling.
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