The objective of the proposed research is to undertake a detailed structure/function analysis of a long-recognized, but underinvestigated, class of proteins: the phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs). 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. Our previous data indicate that Sec14p is an essential regulatory factor that operates at the interface of phospholipid metabolism and Golgi secretory function in yeast, and that Sec14p independently employs its PI- and PC-binding activities to promote yeast Golgi secretory function. The proposed studies are designed to test three basic hypotheses: (i) that a particular hydrophobic surface helix of Sec 14p promotes phospholipid exchange by physically inserting into the lipid bilayer, (ii) that Sec14p is an internally redundant protein that employs its PI- and PC- binding/transfer activities in independent, yet complementary, ways to execute biological function, and (iii) that the essential in vivo role of Sec 14p is to maintain a Golgi pool of diacylglycerol that is required for Golgi secretory function. These hypotheses will be tested by comprehensive sets of structural, biochemical, and biophysical analyses. These approaches will be coupled with functional analyses of suitably modified Sec 14p derivatives, and by molecular and cell biological analyses of gene products whose altered function effects a bypass of the Sec14p requirement for Golgi function and cell viability. The available evidence suggests that PITPs 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 at least two cases of inherited PITP insufficiency in higher eukaryotes result in neurodegeneration, the proposed studies will provide new and fundamental information that will bear directly on the molecular mechanisms by which PITPs protect the mammalian nervous system from neurodegenerative disease.
Showing the most recent 10 out of 44 publications
