The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man and are implicated in regulation of sterol pathways and in signal transduction. OSBP was first discovered as a cytosolic receptor for oxysterols that downregulate cholesterol synthesis. The cloning of OSBP led to the identification of a large family of OSBP-related proteins, the ORPs, with 7 members in S. cerevisiase and 12 in H. sapiens. ORPs are essential for life in eukaryotes. The deletion of all 7 ORPs leads to severe defects in sterol and lipid distribution and endocytosis in yeast, and OSBP is essential for embryonic development in mice. All ORPs contain a core OSBP-related domain (ORD), and many also contain pleckstrin homology (PH) domains, transmembrane regions, endoplasmic reticulum (ER)-targeting FFAT motifs, GOLD domains, andor ankyrin repeats. These additional domains localize ORPs by binding to phosphoinositides, the ER protein VAP, and other targeting signals. The localization of ORPs is dynamic. Oxysterol binding changes the subcellular localization of certain ORPs from the cytosol to the Golgi or ER. ORPs can bind lipids other than oxysterols, including phosphoinositides and phosphatidic acid. OSBP is a cholesterol sensing regulator of two protein phosphatases, a PTPPBS family member, and SerThr phosphatase PP2A. In previous work, we determined the structure of the full-length yeast ORP Osh4 at 1.5-1.9 Angstrom resolution in complexes with ergosterol, cholesterol, and 7-, 20-, and 25-hydroxycholesterol. A single sterol molecule binds in a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible N-terminal lid and surrounded by functionally critical basic residues. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 Angstrom resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favoring transport across aqueous barriers and transmitting signals. The unliganded structure exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. Based on these observations we proposep a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.[unreadable] [unreadable] On the basis of the structural data, a series of mutants were designed to test determinants for binding and transfer. As part of an ongoing collaboration, these reagents were used in Will Prinzs lab (NIDDK) to determine the roles of the binding and transfer processes in ergosterol (the physiological counterpart of cholesterol in yeast) transport in S. cerevisiase. These studies determined that Osh4 specifically facilitates the non-vesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4 transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. This finding was confirmed by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. These findings argue that ORPs move sterols among cellular compartments and that transport is regulated by PIPs.