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.     

Phosphatidylethanolamine Biosynthesis in Mitochondria: PHOSPHATIDYLSERINE (PS) TRAFFICKING IS INDEPENDENT OF A PS DECARBOXYLASE AND INTERMEMBRANE SPACE PROTEINS UPS1P AND UPS2P*

Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1Δ mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.     

氧化固醇结合蛋白结构、功能与应用

氧化固醇结合蛋白(oxysterol binding protein,OSBP)是存在于真核细胞内的一类参与脂质代谢的非囊泡运输蛋白质,在哺乳动物中被称为氧化固醇结合蛋白相关蛋白质(oxysterol binding protein-related proteins,ORPs),而在酵母中被称为氧化固醇结合蛋白同源物质(oxysterol-binding protein homologues,OSH）。近年来人们对氧化固醇结合蛋白的研究不断深入,特别是对其同源蛋白质（例如,ORP5/8、Osh3/4、ORP4L等）的结构功能差异和其在信号转导中的作用的相关研究,以及在生物医药方面的应用更成为了本领域的热点。本文综述了关于OSBP及其同源蛋白质结构和功能的相关研究,指出了该领域存在的一些关键问题。与此同时,对OSH和ORPs在细胞内的膜接触位点（membrane contact sites,MCS）进行对比,以及对今后OSBP的研究方向做了展望。     

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.     

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.     

Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport,signaling and metabolism

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation.The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

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.     

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly,allowing small headgroup lipids to support the terminal steps of membrane fusion

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.     

A Conserved, Lipid-Mediated Sorting Mechanism of Yeast Ist2 and Mammalian STIM Proteins to the Peripheral ER

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS^Ist2) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS^Ist2 targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P₂ at the PM created ER–PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca²⁺ entry (SOCE). The CSS^Ist2 and the C-terminus of the ER-located Ca²⁺ sensor STIM2 were sufficient to bind PI(4,5)P₂ and PI(3,4,5)P₃ at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P₂, suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.     

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.     

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.     

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

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.     

Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides

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.     

Role of Flippases,Scramblases and Transfer Proteins in Phosphatidylserine Subcellular Distribution

It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then distributed to different cellular locations in order to create the distinct membrane compositions observed in eukaryotes. However, the mechanisms by which specific lipid species are trafficked to and maintained in specific areas of the cell are poorly understood and constitute an active area of research. Of particular interest is the distribution of phosphatidylserine (PS), an anionic lipid that is enriched in the cytosolic leaflet of the plasma membrane. PS transport occurs by both vesicular and non‐vesicular routes, with members of the oxysterol‐binding protein family (Osh6 and Osh7) recently implicated in the latter route. In addition, the flippase activity of P4‐ATPases helps build PS membrane asymmetry by preferentially translocating PS to the cytosolic leaflet. This asymmetric PS distribution can be used as a signaling device by the regulated activation of scramblases, which rapidly expose PS on the extracellular leaflet and play important roles in blood clotting and apoptosis. This review will discuss recent advances made in the study of phospholipid flippases, scramblases and PS‐specific lipid transfer proteins, as well as how these proteins contribute to subcellular PS distribution.      

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p-PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers.     

Dynamics of cholesterol exchange in the oxysterol binding protein family

The oxysterol-binding protein-related protein (ORP) family is essential to sterol transfer and sterol-dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol-bound states. In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.     

Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E‐Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E‐Syt1 tethers and transports lipids in a Ca²⁺‐dependent manner, but the role of Ca²⁺ in this regulation is unclear. Of the five C2 domains of E‐Syt1, only C2A and C2C contain Ca²⁺‐binding sites. Using liposome‐based assays, we show that Ca²⁺ binding to C2C promotes E‐Syt1‐mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P₂‐rich membranes, as previously suggested by studies in semi‐permeabilized cells. Importantly, Ca²⁺ binding to C2A enables lipid transport by releasing a charge‐based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E‐Syt1 constructs defective in Ca²⁺ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E‐Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca²⁺‐triggered phosphatidylserine scrambling. Thus, a main effect of Ca²⁺ on E‐Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.     

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.