Schulz, T. A. et al. Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues. J. Cell Biol. 187, 889-903

Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 12/2009; 187(6):889-903. DOI: 10.1083/jcb.200905007
Source: PubMed


Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein-related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

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    • "Yeast contain extensive contacts between the PM and underlying cortical ER that involve the ER-resident proteins Tcb1p1/2/3, Ist1p, Scs2p and Scs21p[187]. PM-cortical ER contacts are also sites for PI 4-phosphate metabolism and sterol trafficking/ signaling mediated by Sac1p and oxysterol binding proteins, respective- ly[188,189](Fig. 1). "

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    • " Mitochondria - associated Membrane of the ER ( MAM ) ] , the PAM is involved in the transport of sterol compounds between the ER and the plasma membrane ( Toulmay and Prinz , 2011 ) ( Figure 1 ) . In yeast , oxysterol - binding protein ( OSBP ) - related proteins ( ORPs ) have been proposed as shuttles between apposed ER / plasma membrane sites ( Schulz et al . , 2009 ) and deletion of all ORPs in a yeast strain has been shown to decrease sterol exchange significantly ( Beh et al . , 2001 ) . A subset of yeast ORPs possess Pleckstrin homology domains and a motif containing two phenylalanine residues in an acidic tract ( FFAT ) , which bind PIPs of the plasma membrane and Vamp - associated proteins of"
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    • "To test whether Ltc1 facilitates lipid transfer, we used an established in vitro lipid transfer assay (Schulz et al., 2009), in which dense sucrose-loaded donor liposomes containing one of three different types of radiolabeled lipids, phospholipids, triolein (a triglyceride), or cholesterol, were incubated with lighter, unlabeled acceptor liposomes in the presence and absence of Ltc1(TM) and subsequently separated by centrifugation (Fig. 2 B). Radioactive cholesterol, but not triolein or major classes of phospholipids, was transferred to acceptor liposomes in an Ltc1-dependent manner (Fig. 2 B), and transport efficiency was dependent on Ltc1(TM) concentration and time (Fig. 2, C and D). "
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    ABSTRACT: Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER-mitochondria and ER-vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER-mitochondria and ER-vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER-mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER-mitochondria and ER-vacuole contact sites. © 2015 Murley et al.
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