Lipid exchange at ER-trans-Golgi contact sites governs polarized cargo sorting.

Oxysterol binding protein (OSBP) extracts cholesterol from the ER to deliver it to the TGN via counter exchange and subsequent hydrolysis of the phosphoinositide PI(4)P. Here, we show that this pathway is essential in polarized epithelial cells where it contributes not only to the proper subcellular distribution of cholesterol but also to the trans-Golgi sorting and trafficking of numerous plasma membrane cargo proteins with apical or basolateral localization. Reducing the expression of OSBP, blocking its activity, or inhibiting a PI4Kinase that fuels OSBP with PI(4)P abolishes the epithelial phenotype. Waves of cargo enrichment in the TGN in phase with OSBP and PI(4)P dynamics suggest that OSBP promotes the formation of lipid gradients along the TGN, which helps cargo sorting. During their transient passage through the trans-Golgi, polarized plasma membrane proteins get close to OSBP but fail to be sorted when OSBP is silenced. Thus, OSBP lipid exchange activity is decisive for polarized cargo sorting and distribution in epithelial cells.

Structure-Based Design of a Lead Compound Derived from Natural Schweinfurthins with Antitumor Properties That Target Oxysterol-Binding Protein.

Schweinfurthins (SWs) are naturally occurring prenylated stilbenes with promising anticancer properties. They act through a novel mechanism of action similar to that of other families of natural compounds. Their known target, oxysterol-binding protein (OSBP), plays a crucial role in controlling the intracellular distribution of cholesterol. We synthesized 15 analogues of SWs and demonstrated for the first time that their cytotoxicity as well as that of natural derivatives correlates with their affinity for OSBP. Through this extensive SAR study, we selected one synthetic analogue obtained in one step from SW-G. Using its fluorescence properties, we showed that this compound recapitulates the effect of natural SW-G in cells and confirmed that it leads to cell death via the same mechanism. Finally, after pilot PK experiments, we provided the first evidence of its in vivo efficacy in combination with temozolomide in a patient-derived glioblastoma xenograft model.

Stranger Twins: A Tale of Resemblance and Contrast Between VAP Proteins.

When considering the vesicle-associated membrane protein-associated protein (VAP) family, major receptors at the surface of the endoplasmic reticulum (ER), it appears that VAP-A and VAP-B paralogs largely overlap in structure and function, and that specific features to distinguish these two proteins hardly exist or are poorly documented. Here, we question the degree of redundancy between VAP-A and VAP-B: is one simply a backup plan, in case of loss of function of one of the two genes, or are there molecular and functional divergences that would explain their maintenance during evolution?

New insights into the OSBP‒VAP cycle.

VAP-A is a major endoplasmic reticulum (ER) receptor that allows this organelle to engage numerous membrane contact sites with other organelles. One highly studied example is the formation of contact sites through VAP-A interaction with Oxysterol-binding protein (OSBP). This lipid transfer protein transports cholesterol from the ER to the trans-Golgi network owing to the counter-exchange of the phosphoinositide PI(4)P. In this review, we highlight recent studies that advance our understanding of the OSBP cycle and extend the model of lipid exchange to other cellular contexts and other physiological and pathological conditions.

VAP-A intrinsically disordered regions enable versatile tethering at membrane contact sites.

Membrane contact sites (MCSs) are heterogeneous in shape, composition, and dynamics. Despite this diversity, VAP proteins act as receptors for multiple FFAT motif-containing proteins and drive the formation of most MCSs that involve the endoplasmic reticulum (ER). Although the VAP-FFAT interaction is well characterized, no model explains how VAP adapts to its partners in various MCSs. We report that VAP-A localization to different MCSs depends on its intrinsically disordered regions (IDRs) in human cells. VAP-A interaction with PTPIP51 and VPS13A at ER-mitochondria MCS conditions mitochondria fusion by promoting lipid transfer and cardiolipin buildup. VAP-A also enables lipid exchange at ER-Golgi MCS by interacting with oxysterol-binding protein (OSBP) and CERT. However, removing IDRs from VAP-A restricts its distribution and function to ER-mitochondria MCS. Our data suggest that IDRs do not modulate VAP-A preference toward specific partners but do adjust their geometry to MCS organization and lifetime constraints. Thus, IDR-mediated VAP-A conformational flexibility ensures membrane tethering plasticity and efficiency.

Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites.

Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.

[A lipid exchange market : vectorial cholesterol transport by the protein OSBP]. / Un marché d'échange de lipides - Transport vectoriel du cholestérol par la protéine OSBP.

Cholesterol is synthesized in the endoplasmic reticulum (RE) and then transported to cellular compartments whose functions require high cholesterol levels. Here, we describe the mechanism by which cholesterol is transported from the RE to the trans-Golgi network (TGN) by the protein OSBP (Oxysterol-Binding Protein). OSBP has two complementary activities. First, it tethers the RE to the TGN by forming a contact site where the two membranes are about twenty nanometers away. Then, it exchanges RE cholesterol for a TGN lipid, phosphatidylinositol 4-phosphate (PI4P). Eventually, PI4P is hydrolyzed at the RE, making the exchange cycle irreversible. Thus, OSBP is at the center of a lipid exchange market where a transported cholesterol "costs" a PI4P. Antiviral or anti-cancer molecules target OSBP, suggesting the importance of the OSBP cycle in different physiopathological contexts. The general principles of this cycle are shared by other lipid-transfer proteins.

TITLE: Un marché d'échange de lipides - Transport vectoriel du cholestérol par la protéine OSBP. ABSTRACT: Le cholestérol est synthétisé dans le réticulum endoplasmique (RE) puis transporté vers les compartiments cellulaires dont la fonction en nécessite un taux élevé. Nous décrivons ici le mécanisme de transport du cholestérol du RE vers le réseau trans golgien (TGN) par la protéine OSBP (oxysterol binding protein). Celle-ci présente deux activités complémentaires : elle arrime les deux compartiments, RE et TGN, en formant un site de contact où les deux membranes sont à une vingtaine de nanomètres de distance ; puis elle échange le cholestérol du RE avec un lipide présent dans le TGN, le phosphatidylinositol 4-phosphate (PI4P). Dans le RE, le PI4P est hydrolysé, rendant le cycle d'échange irréversible. OSBP est donc au cœur d'un marché d'échange de lipides dans lequel un cholestérol transporté « coûte ¼ un PI4P. Des molécules à activités antivirales ou anticancéreuses ont pour cible OSBP, suggérant une importance dans différents contextes physiopathologiques du cycle d'OSBP, dont les bases générales sont partagées par d'autres protéines transporteurs de lipides.

Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor.

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.

An Intrinsically Disordered Region in OSBP Acts as an Entropic Barrier to Control Protein Dynamics and Orientation at Membrane Contact Sites.

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of oxysterol-binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density, and facilitates protein mobility in the narrow and crowded MCS environment.

Lipid exchange and signaling at ER-Golgi contact sites.

Meticulous observations of the cell perinuclear region where the Golgi and the endoplasmic reticulum (ER) networks intermingle have revealed close contact sites of barely 20 nm between these two organelles. Recent studies demonstrate that molecular machineries, including lipid-transfer proteins, enriched in membrane contact sites between ER and trans-Golgi are capable of bridging membranes and exchanging key lipids such as sphingolipid precursors and cholesterol while bypassing the early secretory compartments. This occurs at the cost of an intense phosphoinositide turnover in order to prepare a membrane environment conducive to the signaling and trafficking functions of the trans-Golgi network. A tight control operates in the contact zone to adjust lipid transport and metabolism to the cellular needs.

The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol.

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP.

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.

Mitochondrial lipid transport and biosynthesis: A complex balance.

Little is known about how mitochondrial lipids reach inner membrane-localized metabolic enzymes for phosphatidylethanolamine synthesis. Aaltonen et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602007) and Miyata et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601082) now report roles for two mitochondrial complexes, Ups2-Mdm35 and mitochondrial contact site and cristae organizing system, in the biosynthesis and transport of mitochondrial lipids.

The counterflow transport of sterols and PI4P.

Cholesterol levels in intracellular membranes are constantly adjusted to match with specific organelle functions. Cholesterol is kept high in the plasma membrane (PM) because it is essential for its barrier function, while low levels are found in the endoplasmic reticulum (ER) where cholesterol mediates feedback control of its own synthesis by sterol-sensor proteins. The ER→Golgi→PM concentration gradient of cholesterol in mammalian cells, and ergosterol in yeast, appears to be sustained by specific intracellular transport processes, which are mostly mediated by lipid transfer proteins (LTPs). Here we review a recently described function of two LTPs, OSBP and its yeast homolog Osh4p, which consists in creating a sterol gradient between membranes by vectorial transport. OSBP also contributes to the formation of ER/Golgi membrane contact sites, which are important hubs for the transfer of several lipid species. OSBP and Osh4p organize a counterflow transport of lipids whereby sterols are exchanged for the phosphoinositide PI4P, which is used as a fuel to drive sterol transport. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes.

Lipids are unevenly distributed within eukaryotic cells, thus defining organelle identity. How non-vesicular transport mechanisms generate these lipid gradients between membranes remains a central question. Here using quantitative, real-time lipid transport assays, we demonstrate that Osh4p, a sterol/phosphatidylinositol-4-phosphate (PI(4)P) exchanger of the ORP/Osh family, transports sterol against its gradient between two membranes by dissipating the energy of a PI(4)P gradient. Sterol transport is sustained through the maintenance of this PI(4)P gradient by the PI(4)P-phosphatase Sac1p. Differences in lipid packing between membranes can stabilize sterol gradients generated by Osh4p and modulate its lipid exchange capacity. The ability of Osh4p to recognize sterol and PI(4)P via distinct modalities and the dynamics of its N-terminal lid govern its activity. We thus demonstrate that an intracellular lipid transfer protein actively functions to create a lipid gradient between membranes.

Building lipid 'PIPelines' throughout the cell by ORP/Osh proteins.

In eukaryotic cells, a sterol gradient exists between the early and late regions of the secretory pathway. This gradient seems to rely on non-vesicular transport mechanisms mediated by specialized carriers. The oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) family has been assumed initially to exclusively include proteins acting as sterol sensors/transporters and many efforts have been made to determine their mode of action. Our recent studies have demonstrated that some ORP/Osh proteins are not mere sterol transporters, but sterol/phosphatidylinositol 4-phosphate [PI(4)P] exchangers. They exploit the PI(4)P gradient at the endoplasmic reticulum (ER)/Golgi interface, or at membrane-contact sites between these compartments, to actively create a sterol gradient. Other recent reports have suggested that all ORP/Osh proteins bind PI(4)P and recognize a second lipid that is not necessary sterol. We have thus proposed that ORP/Osh proteins use PI(4)P gradients between organelles to convey various lipid species.

A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP.

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

Insights into the mechanisms of sterol transport between organelles.

In cells, the levels of sterol vary greatly among organelles. This uneven distribution depends largely on non-vesicular routes of transfer, which are mediated by soluble carriers called lipid-transfer proteins (LTPs). These proteins have a domain with a hydrophobic cavity that accommodates one sterol molecule. However, a demonstration of their role in sterol transport in cells remains difficult. Numerous LTPs also contain membrane-binding elements, but it is not clear how these LTPs couple their ability to target organelles with lipid transport activity. This issue appears critical, since many sterol transporters are thought to act at contact sites between two membrane-bound compartments. Here, we emphasize that biochemical and structural studies provide precious insights into the mode of action of sterol-binding proteins. Recent studies on START, Osh/ORP and NPC proteins suggest models on how these proteins could transport sterol between organelles and, thereby, influence cellular functions.