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
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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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    • " 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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    ABSTRACT: Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy-a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction.
    Frontiers in Cell and Developmental Biology 09/2015; 3. DOI:10.3389/fcell.2015.00056
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    • "The other human ORPs thus far studied display different affinities for various oxysterols, with K d s in the nM– lM range [14] [15] [16]. Oxysterol binding in vitro by purified ORPs has been demonstrated for OSBP, OSBP2/ORP4, ORP1, and ORP2, and in vitro sterol transfer activity for OSBP, ORP9L, the ORD of ORP5 [17] [18], as well as several of the yeast S. cerevisiae Osh proteins [19] [20]. Of note, unlike OSBP, OSBP2/ORP4 and ORP1, ORP2 binds 25OHC with a low, micromolar affinity [21], and shows high affinity (K d 14 nM) for 22(R)OHC [15]. "
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    ABSTRACT: Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs). We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA). Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP-VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP-VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)-VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2-VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L-VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)-VAPA localized predominantly to the plasma membrane. In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP-VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces.
    Steroids 02/2015; 99(Pt B). DOI:10.1016/j.steroids.2015.01.027 · 2.64 Impact Factor
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    • "The Osh3 β-barrel is too narrow to accommodate a sterol and exclusively binds PI-4P [4]. Similarly, Osh6 and Osh7 bind and transfer phosphatidylserine (PS) [16] but bind sterols weakly compared to other Osh proteins [11]. "
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    ABSTRACT: Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.
    PLoS ONE 09/2014; 9(9):e108368. DOI:10.1371/journal.pone.0108368 · 3.23 Impact Factor
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