The objective of the proposed research is to undertake a detailed analysis of a long-recognized, but remarkably underinvestigated, class of proteins: the phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs). These ubiquitous proteins catalyze the transport of either phosphatidylinositol or phosphatidylcholine, as monomers, between membrane bilayers in vitro. The goal of this project is to elucidate the in vivo function and mechanism of action of the phosphatidylinositol/ phosphatidylcholine transfer protein of yeast (SEC14p), a prototypical member of a novel class of regulatory/signalling molecules in cells. All available evidence indicates that SEC14p is a negative effector of choline-phosphate cytidylyltransferase (CCTase), the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine (PC) biosynthesis, in yeast Golgi membranes. The proposed studies are designed to thoroughly test three basic hypotheses: (i) that the functional form of SEC14p in vivo is the PC-bound form and not the phosphatidylinositol (PI)-bound form, (ii) that the essential in vivo role of SEC14p is a PL-modulated negative effector function, not a PL-transfer function, and (iii) that SEC14p does not function to maintain reduced Golgi membrane PC per se, but functions to reduce flux through the CDP-choline pathway so that a metabolic intermediate required for Golgi secretory function is not inappropriately consumed by CDP-choline pathway activity. These hypotheses will be tested by comprehensive sets of structural studies coupled with functional analyses of SEC14p derivatives that are either chemically or genetically modified at predetermined positions, and by molecular and cell biological analyses of gene products whose altered function effects a bypass of the SEC14p requirement for Golgi secretory function and cell viability. The available evidence further suggests that PI-TPs play central, and previously unrecognized roles in phospholipid-mediated signal transduction processes that interface with such diverse cellular processes as protein secretion, phototransduction, and receptor-mediated signalling. As all eukaryotic cells are likely to employ PI-TPs in various ways to potentiate, or otherwise regulate, phospholipid-mediated signal transduction pathways, these studies will provide new and fundamental advances on ubiquitous issues that bear directly on receptor-mediated control of cell behavior (relevant to function of neuronal cells, cellular development, and cancer), vision, and phospholipid metabolism (relevant to metabolic disorders).
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