Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum

Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis. A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP. This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively. By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442. This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains. C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization. Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182). OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures. At 40 degrees C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 degrees C. A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis. These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER.     

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.     

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.     

Oxysterol-binding Protein (OSBP)-related Protein 4 (ORP4) Is Essential for Cell Proliferation and Survival

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.     

Efficient trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus requires a VAMP-associated protein-interacting FFAT motif of CERT

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.     

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

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 (HH_488,489AA) 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.     

Structural basis of FFAT motif-mediated ER targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.     

Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.     

Characterization of the sterol-binding domain of oxysterol-binding protein (OSBP)-related protein 4 reveals a novel role in vimentin organization

Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4; also designated OSBP2 and HLM) are implicated in sterol-transport and/or sensing via binding to protein partners. The aggregation of vimentin by an N-terminal-truncated variant of ORP4 (ORP4S), but not full-length ORP4L, suggested a functional interaction with this intermediate filament. Herein, we identify ORP4 domains that interact with vimentin, and determine how sterols and OSBP influence this activity. In CHO cells, ORP4L co-localized with filamentous vimentin but extensive remodeling of vimentin filaments required mutation of a leucine repeat motif (amino acids 361-382) adjacent to the oxysterol-binding domain. Similarly, the absence of the leucine repeat in ORP4S 418-878 resulted in co-localization with aggregated vimentin filaments, suggesting that both the sterol-binding domain and leucine repeat are involved. Transient expression of OSBP leucine repeat mutants also promoted vimentin aggregation by a mechanism involving heterodimerization with ORP4L. Glutathione S-transferase (GST)-ORP4 380-878 bound vimentin, cholesterol and 25-hydroxycholesterol in vitro. However, sterol-binding or a mutation that ablated sterol-binding did not influence the interaction of GST-ORP4 with vimentin. Thus the sterol-binding domain of ORP4 binds vimentin, cholesterol and oxysterols, and interacts with the filamentous vimentin network.     

The targeting of the oxysterol-binding protein ORP3a to the endoplasmic reticulum relies on the plant VAP33 homolog PVA12

In plants, sterols play fundamental roles as membrane constituents in the biosynthesis of steroid hormones, and act as precursors for cell wall deposition. Sterols are synthesized in the endoplasmic reticulum (ER), but mainly accumulate in the plasma membrane. How sterols are trafficked in plant cells is largely unknown. In non-plant systems, oxysterol-binding proteins have been involved in sterol trafficking and homeostasis. There are at least twelve homologs of oxysterol-binding proteins in the Arabidopsis genome, but the biology of these proteins remains for the most part obscure. Here, we report our analysis of the targeting requirements and the sterol-binding properties of a small Arabidopsis oxysterol-binding protein, ORP3a. We have determined that ORP3a is a bona fide sterol-binding protein with sitosterol-binding properties. Live-cell imaging analyses revealed that ORP3a is localized at the ER, and that binding to this organelle depends on a direct interaction with PVA12, a member of the largely uncharacterized VAP33 family of plant proteins. Molecular modeling analyses and site-directed mutagenesis led to the identification of a novel protein domain that is responsible for the PVA12–ORP3a interaction. Disruption of the integrity of this domain caused redistribution of ORP3a to the Golgi apparatus, suggesting that ORP3a may cycle between the ER and the Golgi. These results represent new insights into the biology of sterol-binding proteins in plant cells, and elucidate a hitherto unknown relationship between members of oxysterol-binding protein and VAP33 families of plant proteins in the early plant secretory pathway.     

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.     

Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.     

Dual Targeting of Osh1p, a Yeast Homologue of Oxysterol-binding Protein, to both the Golgi and the Nucleus-Vacuole Junction

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP in Saccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.     

ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism

Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family that has at least 12 members in humans. In the present study we characterize one of the novel OSBP homologs, ORP2, which we show to be expressed ubiquitously in mammalian tissues. The ORP2 cDNA encodes a deduced 55 kDa protein that lacks a pleckstrin homology (PH) domain, a feature found in the other family members. Sucrose gradient centrifugation analysis of Chinese hamster ovary (CHO) cell post-nuclear supernatant demonstrated that ORP2 is distributed in soluble and membrane-bound fractions. Immunofluorescence microscopy of the endogenous and overexpressed ORP2 in CHO cells suggested that the membrane-bound fraction of the protein localizes to the Golgi apparatus. Stably transfected CHO cells that overexpress ORP2 showed an increase in [14C]cholesterol efflux to serum, apolipoprotein A-I (apoA-I), and phosphatidyl choline vesicles. The proportion of cellular [14C]cholesterol that is esterified and the ACAT activity measured as [14C]oleyl-CoA conversion into cholesteryl [14C]oleate by the cellular membranes, were markedly decreased in the ORP2 expressing cells. Transient high level overexpression of ORP2 interfered with the clearance of a secretory pathway protein marker from the Golgi complex. The results implicate ORP2 as a novel regulator of cellular sterol homeostasis and intracellular membrane trafficking.     

Thank ORP9 for FFAT: With endosomal ORP10, it’s fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Most cellular lipids are synthesized in the ER, often undergoing rapid redistribution to other cellular membranes, thereby maintaining low concentrations at the ER. Consequently, lipids exiting the ER may need to be transported against their concentration gradient. Lipid flow along a gradient to the ER can drive countertransport of ER-derived lipid to membranes with a higher lipid concentration. This nonvesicular lipid exchange occurs at membrane contact sites (MCS), where different organelles are closely apposed, providing a platform for lipid transport proteins including oxysterol-binding protein (OSBP)-related proteins (ORPs). Lipid specificity, which varies between ORPs, is defined by the OSBP-related domain (ORD). The ORD of ORP10 shares phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) binding residues with ORP5/8 and can bind and extract PS from liposomes (1), suggesting a potential role in PI4P-PS counter transport, analogous to that of ORP5/8 at ER–plasma membrane MCS (2). ORPs are targeted to specific organelles by interaction between their PH domain and membrane phospholipids. Most ORPs also possess a FFAT motif (two phenylalanines in an acidic tract), which simultaneously targets the ORP to ER-localized VAMP-associated proteins (VAPs) at MCS between the ER and other organelles. ORP10, however, lacks a FFAT motif, yet was found to stabilize ER–Golgi MCSs (Fig. 1 A) for PI4P transport to the ER (2). Kawasaki et al. have now uncovered a novel function for ORP10 in PI4P–PS lipid exchange at the ER–endosome interface (Fig. 1 B), with downstream effects on endosomal fission and retrograde transport (3).Open in a separate windowFigure 1.Regulation of retrograde and secretory traffic by ORP10-mediated lipid exchange. (A) ORP10 interacts with VAP-bound ORP9 at ER–endosome and ER–Golgi MCSs, with downstream effects on retrograde transport of mannose 6-phosphate receptor (M6PR). Boxed region (detailed in B) depicts ORP10 at the ER–endosome interface. (B) ORP10 functions in lipid exchange between the ER and endosomes, transporting endosomal PI4P to the ER in exchange for ER-derived PS. Production of PI4P in endosomes by PI4KIIα-dependent phosphorylation of phosphatidylinositol (PI), coupled with its consumption in the ER by ER-localized Sac1, generates a PI4P concentration gradient from the endosome to the ER. Low membrane PS concentrations in the ER are maintained by PS inhibition of PS synthesis from phosphatidylcholine (PC) by Pss1 or from PE by Pss2, with PS synthesis at ER–endosome contact sites promoting rapid PS export from the ER in yeast (not yet known if a similar mechanism operates in mammalian cells). ORP10 mediates PI4P transport along its gradient to the ER, driving countertransport of PS by ORP10 against its concentration gradient to the endosome. PS enrichment at the endosome leads to recruitment of the ATPase EHD1 to facilitate endosome fission for retrograde transport. (C) Depletion of ORP10 prevents lipid exchange at ER–endosome contact sites, resulting in a loss of retrograde transport of M6PR. Additionally, ER–Golgi MCSs are diminished, and secretion of ApoB-100 is increased.The PH domain of ORP10 selectively binds PI4P and is required for ORP10 recruitment to the TGN (2) and endosomes (3), both home to PI4KIIα, a PI4P-producing kinase. Rapid PI4P degradation at the ER by the phosphatase Sac1 generates a PI4P gradient at the ER–endosome or TGN interface, with PI4P flow to the ER driving countertransport of PS to the endosome (as also predicted for the Golgi). Activity of endosomal PI4P phosphatase Sac2 (4) may hamper formation of an endosome–ER PI4P gradient, but since ORP10 did not colocalise well with Sac2 (3), they likely function at different endosome populations.PS synthesis at MCSs may also contribute to ORP10-mediated lipid exchange. Targeting PS synthase to ER:mitochondria contacts in yeast was found topromote PS transport out of the ER to mitochondria (5). Similarly, ER to endosome PS transport was increased when PS synthase was targeted to ER:endosome MCS. Localized PS gradients from PS synthesis in the ER at MCSs, coupled with rapid decarboxylation of PS to phosphatidylethanolamine (PE) in mitochondria/endosomes by yeast PS decarboxylases Psd1/Psd2, could contribute to lipid exchange. In mammalian cells, though, since no endosomal decarboxylase has been identified, ORP10-mediated lipid exchange is likely to be primarily driven by the PI4P gradient. Whether this process is facilitated by localized activation of PS synthase at MCS has not yet been demonstrated. Since PS synthase activity is negatively regulated by PS, exit from the ER is a key factor in its biogenesis. Recruitment of specific ORPs to endosomes/TGN by PI4P for ER tethering and consequent lipid exchange provides an elegant regulatory pathway for PI4P–PS homeostasis in cellular membranes.ORP10 shares functional similarities with ORP11: both proteins comprise an N-terminal PH domain and a C-terminal ORD, with a linker region in between harboring a coiled-coiled domain. Unlike other ORPs, ORP10 and ORP11 possess neither a FFAT motif nor a membrane spanning domain to enable ER interaction, but heterotypic interaction with ORP9, which does contain a FFAT motif, has been demonstrated for both proteins. Kawasaki et al. identified an ORP9-ORP10 interaction at ER–endosome MCSs that is dependent on the ORP10 linker region. ORP9 was also implicated in ORP10-mediated lipid exchange at the TGN, where it may play a redundant role with OSBP in maintaining ER contact. Similarly, ORP11 is also recruited to the TGN and, to a lesser extent, the endosome, by ORP9, with the interaction depending on the linker region of both proteins (6).The finely tuned regulation of PI4P/PS is emerging as an important determinant of endocytic traffic. Previous studies have shown that endosomal PI4P accumulation inhibits retrograde transport from endosomes to the TGN (7), while endosomal PS regulates endosome to Golgi retrograde traffic. As depicted in Fig. 1 B, Kawasaki et al. have built on this to show that through interaction with VAP-bound ORP9, ORP10 mediates lipid countertransport at ER–endosome MCSs, removing PI4P from, and supplying PS to, the endosome, with consequent recruitment of the membrane scission protein EHD1 to control endosomal fission and retrograde transport (3). Spatial and temporal regulation of endosome fission by ER–endosome MCSs involves recruitment of the ER membrane protein TMCC1 to the budding endosome by the actin regulator Coronin 1C, stabilizing the MCS (7), but the mechanism by which MCS might effect scission has remained elusive. The findings of Kawaski et al. present an explanation: by providing a platform for lipid exchange, MCS promote the recruitment of EHD1, which belongs to a conserved class of ATPases that can oligomerise in ring-like structures around tubules to mediate fission (8). VAP interaction with OSBP at ER–endosome MCSs is also required for retrograde transport (7), but potential redundancy between ORP9/OSBP in ORP10-mediated lipid exchange, or if ORP10 functions at Coronin 1C/TMCC1-regulated MCS is not yet established.Interestingly, ORP10 function at the TGN has been implicated in regulating ApoB-100 secretion (Fig. 1 C), with hypersecretion reported in ORP10-depleted cells (9). FFAP1, which promotes PI4P consumption by Sac1 at ER:TGN contacts, also negatively regulates ApoB-100 exit from the TGN in a PI4KIIIβ-dependent manner, suggesting direct regulation of ApoB-100 secretion by PI4P at the TGN (9). Could PI4P coordinate nutrient sensing with cargo sorting and secretion at the TGN? PI4P has been described as lipid biosensor of cytosolic pH, with protonation of its head group regulating protein interactions (10). The influence of cytosolic pH on ORP10-PI4P interaction may provide an additional layer of regulation of lipoprotein secretion in response to changes in cellular energy/pH.How ORP10 function is coordinated at Golgi and endosomal membranes and the significance of potential redundancy with ORP11 remains unclear. The regulation of Sac2 activity and how it relates to ER-endosome lipid exchange is also intriguing. While questions still remain, an important role for ORP10 is emerging in maintaining homoeostasis between endosome maturation, retrograde traffic and secretory transport.     

Promotion of Neurite Extension by Protrudin Requires Its Interaction with Vesicle-associated Membrane Protein-associated Protein

Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth.The molecular mechanisms that underlie neurite formation include both cytoskeletal remodeling and membrane trafficking. Membrane components are transported in a directional manner within the cell by a membrane recycling system, resulting in expansion of the surface area of the neurite. The small GTPase Rab11 regulates membrane recycling and constitutive exocytosis (1), and it is thought to contribute to neurite formation through regulation of directional membrane transport.We have recently identified protrudin as a key regulator of Rab11-dependent membrane trafficking during neurite extension. Protrudin interacts with FKBP38 (also known as FKBP8) (2), which is a member of the immunophilin family of proteins that bind the immunosuppressant drug FK506 (3). FKBPs are multifunctional proteins that regulate the folding or export of other proteins as a result of their peptidyl-prolyl cis-trans-isomerase activity (4). Protrudin was found to interact with FKBP38, but not with other FKBP proteins such as FKBP12 or FKBP52 (5). Protrudin is hyperphosphorylated in Fkbp38^-/- mice, which manifest abnormal extension of nerve fibers (5).Protrudin contains a Rab11-binding domain (RBD11), two transmembrane domains (TM1 and TM2),² an FFAT (two phenylalanines in an acidic tract) motif (6), a coiled-coil domain, and a FYVE domain (7). These structural characteristics suggested that protrudin might function in membrane trafficking, particularly in membrane recycling. The gene encoding ZFYVE27 (a synonym of human protrudin) was recently found to be mutated in a German family with an autosomal dominant form of hereditary spastic paraplegia (AD-HSP), which is characterized by selective degeneration of axons (8). The phenotype of the affected individuals is similar to that of patients with AD-HSP caused by mutation of spastin, a protein implicated in neuronal vesicular trafficking (9), and protrudin was shown to interact with spastin (8). These findings support the notion that protrudin plays a key role in Rab11-mediated directional membrane transport during neurite formation.The subcellular localization of protrudin is dynamic. Whereas it is localized to the endoplasmic reticulum (ER) under basal conditions, nerve growth factor (NGF) triggers the translocation of protrudin from the ER, via recycling endosomes, to the tip of membrane protrusions in neuronal cells. Given that the FFAT motif is thought to serve as an ER targeting signal (6), this motif might be expected to contribute both to the localization of protrudin to the ER and to the regulation of neurite formation by this protein. The FFAT motif (consensus amino acid sequence of EFFDAXE, where X is any amino acid) is present in several lipid-binding proteins that are implicated in the transfer of lipids between the ER and other organelles such as the Golgi apparatus (10, 11). Vesicle-associated membrane protein-associated protein (VAP) interacts with these lipid-binding proteins through their FFAT motifs (6, 11, 12). The VAP-A and VAP-B isoforms of mammalian VAP are ER-resident type II membrane proteins (13) that are encoded by different genes (14); VAP-C is a splicing variant of VAP-B that lacks the membrane-spanning domain. VAP-A and VAP-B share ∼60% amino acid sequence identity, form homo- or heterodimers, and are expressed in many tissues (14-16). In addition to their localization to the ER (16), VAP-A and VAP-B are present in a wide range of intracellular membranes or membrane structures, including the Golgi, the ER-Golgi intermediate compartment (17), tight junctions (18), neuromuscular junctions (19), recycling endosomes, and the plasma membrane (20).We have now identified VAP-A and VAP-B as proteins that interact with protrudin. Protrudin preferentially interacts with VAP-A via its FFAT motif, and this motif was found to be required for the protrudin-dependent formation of membrane protrusions in HeLa cells. In addition, depletion of VAP-A by RNA interference resulted in inhibition of NGF-induced neurite outgrowth in the PC12 rat pheochromocytoma cell line. This inhibition of neurite outgrowth was reversed by expression of human VAP-A but not by that of VAP-A mutants that have lost the ability to bind to protrudin. These results suggest that interaction of protrudin with VAP-A is important both for its ER retention and for its ability to stimulate neurite formation.     

A highly conserved binding site in vesicle-associated membrane protein-associated protein (VAP) for the FFAT motif of lipid-binding proteins

A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associated-membrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.     

Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components

BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting. RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p. CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.     

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.     

The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.