Cholesterol plays an essential role in determining the properties of biological membranes. Control of cholesterol levels in organelles depends on sterol transport mechanisms that are poorly understood. Novel methods to study sterol transport among organelles quantitatively have been developed, and this is leading to precise models for transport among organelles. Preliminary data show that a widely expressed, sterol- regulated, soluble sterol transporter, STARD4, plays an important role in nonvesicular transport of cholesterol, and its role and mechanism of transport will be studied. STARD4 is an important component of a complex sterol regulatory network. Recent work showed that STARD4 is responsible for about 25% of sterol transport between two major pools – the plasma membrane and the endocytic recycling compartment - in U2OS cells. The role of STARD4 will also be assessed in macrophages, a cell type in which cholesterol metabolism plays a major role in development of atherosclerosis, and in HepG2 cells, a model for hepatocytes. In preliminary studies it has been found that STARD4 has unique interactions with phosphatidylinositol phosphates (PIPs). STARD4 extraction of sterol from membranes is accelerated nearly 10-fold when the membranes contain PI(4,5)P2, but there is no effect of PI(4,5)P2 in membranes accepting sterol from STARD4. Conversely, PI(3,5)P2 or PI5P in acceptor membranes accelerates transfer ∼10-fold with no effect when these PIPs are in donor membranes. PI3P has a similar but smaller effect. The role of STARD4 in sterol transport to late endosomes, lipid droplets, and autophagosomes (i.e., organelles with good acceptor PIPs) will be analyzed. Molecular dynamics simulations of interactions of STARD4 with membranes is identifying potential sites of interaction with PIPs. The effects of mutations of these sites will be assayed in a liposome to liposome sterol transport assay. Mutant STARD4 molecules with altered PIP sensitivity will be expressed in cells to determine effects on sterol transport and distribution. In preliminary studies, mutants identified by molecular dynamics were confirmed by tests in the liposome assay, and it was found that they do affect transport of sterol among organelles. X-ray crystallography has identified differences between human STARD4 with and without bound sterol. NMR spectroscopy is being used to analyze STARD4 protein dynamics and to determine the sites of interaction with lipids containing various PIPs.