The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man 1, 2 and are implicated in regulation of sterol pathways 3,4 and in signal transduction 5 . The structure of the full-length yeast ORP Osh4 was determined at 1.5-1.9 Å 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 Å 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 propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.OSBP was first discovered 6,7 as a cytosolic receptor for oxysterols that downregulate cholesterol synthesis 8 . The cloning of OSBP 1 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 2 . 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 3,4 , and OSBP is essential for embryonic development in mice (M. Brown, personal communication). All ORPs contain a core OSBPrelated domain (ORD), and many also contain pleckstrin homology (PH) domains, transmembrane regions, endoplasmic reticulum (ER)-targeting FFAT motifs, GOLD domains, and/or ankyrin repeats 2 . These additional domains localize ORPs by binding to phosphoinositides 9 , the ER protein VAP 10 , 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 9,11 . ORPs can bind lipids other than oxysterols, including phosphoinositides and phosphatidic acid 12,13 . OSBP is a cholesterol sensing regulator of two protein phosphatases, a PTPPBS family member, and Ser/Thr phosphatase PP2A 5 .The structure of full-length Osh4 was determined by multiple isomorphous replacement and refined to a free R-factor of 23 % at 1.5 Å resolution (Supplementary Fig. 1 Table 1). Osh4 is built around a central antiparallel β-sheet of 19 strands (residues 115-293) which form a nearly complete β-barrel (Fig. 1a, Supplementary Fig. 2). The strands of the barrel are pitched at ~45 degrees to its axis. The barrel has structural similarity to the large β-barrels of various bacterial outer membrane transporters 14 as scored by Dali 15 ( Supplementary Fig. 3). There is no PH domain within the ORD 13 . A tunnel with largely hydrophobic walls...

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)–related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.