Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Reconstitution of ORP-mediated lipid exchange process coupled to PI(4)P metabolism.

Lipid distribution in the eukaryotic cells depends on tight couplings between lipid transfer and lipid metabolism. Yet these couplings remain poorly described. Notably, it is unclear to what extent lipid exchangers of the OSBP-related proteins (ORPs) family, coupled to PI(4)P metabolism, contribute to the formation of sterol and phosphatidylserine gradient between the endoplasmic reticulum (ER) and other cell regions. To address this question, we have examined in vitro the activity of Osh4p, a representative ORP, between Golgi mimetic membranes in which PI(4)P is produced by a PI 4-kinase and ER mimetic membranes in which PI(4)P is hydrolyzed by the phosphatase Sac1p. Using quantitative, real-time assays, we demonstrate that Osh4p creates a sterol gradient between the two membranes by sterol/PI(4)P exchange as soon as a PI(4)P gradient is generated at this interface following ATP addition, and define how much PI(4)P must be synthesized for this process. Then, using a kinetic model supported by our in vitro data, we estimate to what extent PI(4)P metabolism can drive lipid transfer in cells. Finally, we show that Sec14p, by transferring phosphatidylinositol between membranes, can support the synthesis of PI(4)P and the creation of a sterol gradient by Osh4p. These results indicate to what extent ORPs, under the control of PI(4)P metabolism, can distribute lipids in the cell.

Creating and sensing asymmetric lipid distributions throughout the cell.

A key feature of eukaryotic cells is the asymmetric distribution of lipids along their secretory pathway. Because of the biological significance of these asymmetries, it is crucial to define the mechanisms which create them. Extensive studies have led to the identification of lipid transfer proteins (LTPs) that work with lipid-synthesizing enzymes to carry lipids between two distinct membranes in a directional manner, and are thus able to create asymmetries in lipid distribution throughout the cell. These networks are often in contact sites where two organelle membranes are in close proximity for reasons we have only recently started to understand. A question is whether these networks transfer lipids en masse within the cells or adjust the lipid composition of organelle membranes. Finally, recent data have confirmed that some networks organized around LTPs do not generate lipid asymmetries between membranes but sense them and rectify the lipid content of the cell.

MOSPD2 is an endoplasmic reticulum-lipid droplet tether functioning in LD homeostasis.

Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER-anchored proteins, such as MOSPD2, that function as major ER-organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER-lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein-membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.

In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities.

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum-resident vesicle-associated membrane protein-associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

Functional analyses of phosphatidylserine/PI(4)P exchangers with diverse lipid species and membrane contexts reveal unanticipated rules on lipid transfer.

BACKGROUND: Lipid species are accurately distributed in the eukaryotic cell so that organelle and plasma membranes have an adequate lipid composition to support numerous cellular functions. In the plasma membrane, a precise regulation of the level of lipids such as phosphatidylserine, PI(4)P, and PI(4,5)P2, is critical for maintaining the signaling competence of the cell. Several lipid transfer proteins of the ORP/Osh family contribute to this fine-tuning by delivering PS, synthesized in the endoplasmic reticulum, to the plasma membrane in exchange for PI(4)P. To get insights into the role of these PS/PI(4)P exchangers in regulating plasma membrane features, we question how they selectively recognize and transfer lipid ligands with different acyl chains, whether these proteins exchange PS exclusively for PI(4)P or additionally for PI(4,5)P2, and how sterol abundance in the plasma membrane impacts their activity. RESULTS: We measured in vitro how the yeast Osh6p and human ORP8 transported PS and PI(4)P subspecies of diverse length and unsaturation degree between membranes by fluorescence-based assays. We established that the exchange activity of Osh6p and ORP8 strongly depends on whether these ligands are saturated or not, and is high with representative cellular PS and PI(4)P subspecies. Unexpectedly, we found that the speed at which these proteins individually transfer lipid ligands between membranes is inversely related to their affinity for them and that high-affinity ligands must be exchanged to be transferred more rapidly. Next we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for exchange processes, because it is a low-affinity ligand, and do not transfer more PS into sterol-rich membranes. CONCLUSIONS: Our study provides new insights into PS/PI(4)P exchangers by indicating the degree to which they can regulate the acyl chain composition of the PM, and how they control PM phosphoinositide levels. Moreover, we establish general rules on how the activity of lipid transfer proteins relates to their affinity for ligands.

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism.

Lipids are amphiphilic molecules that self-assemble to form biological membranes. Thousands of lipid species coexist in the cell and, once combined, define organelle identity. Due to recent progress in lipidomic analysis, we now know how lipid composition is finely tuned in different subcellular regions. Along with lipid synthesis, remodeling and flip-flop, lipid transfer is one of the active processes that regulates this intracellular lipid distribution. It is mediated by Lipid Transfer Proteins (LTPs) that precisely move certain lipid species across the cytosol and between the organelles. A particular subset of LTPs from three families (Sec14, PITP, OSBP/ORP/Osh) act as lipid exchangers. A striking feature of these exchangers is that they use phosphatidylinositol or phosphoinositides (PIPs) as a lipid ligand and thereby have specific links with PIP metabolism and are thus able to both control the lipid composition of cellular membranes and their signaling capacity. As a result, they play pivotal roles in cellular processes such as vesicular trafficking and signal transduction at the plasma membrane. Recent data have shown that some PIPs are used as energy by lipid exchangers to generate lipid gradients between organelles. Here we describe the importance of lipid counter-exchange in the cell, its structural basis, and presumed links with pathologies.

Osh6 requires Ist2 for localization to ER-PM contacts and efficient phosphatidylserine transport in budding yeast.

Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS levels at the PM are key for many cellular functions. Intriguingly, Osh6 and Osh7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homolog of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy osh6Δ osh7Δ deletion: they decrease cellular PS levels and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.This article has an associated First Person interview with the first author of the paper.

MapPIng PI inside cells brings new light to polyphosphoinositide biology.

It is unclear how phosphatidylinositol (PI), the precursor of polyphosphoinositides, is distributed within cell membranes. Pemberton et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.201906130) and Zewe et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.201906127) describe new approaches to map the subcellular PI abundance and clarify how polyphosphoinositide metabolism relates to PI distribution.

An electrostatic switching mechanism to control the lipid transfer activity of Osh6p.

A central assumption is that lipid transfer proteins (LTPs) bind transiently to organelle membranes to distribute lipids in the eukaryotic cell. Osh6p and Osh7p are yeast LTPs that transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol-4-phosphate (PI4P) exchange cycles. It is unknown how, at each cycle, they escape from the electrostatic attraction of the PM, highly anionic, to return to the ER. Using cellular and in vitro approaches, we show that Osh6p reduces its avidity for anionic membranes once it captures PS or PI4P, due to a molecular lid closing its lipid-binding pocket. Thus, Osh6p maintains its transport activity between ER- and PM-like membranes. Further investigations reveal that the lid governs the membrane docking and activity of Osh6p because it is anionic. Our study unveils how an LTP self-limits its residency time on membranes, via an electrostatic switching mechanism, to transfer lipids efficiently.

In Vitro Strategy to Measure Sterol/Phosphatidylinositol-4-Phosphate Exchange Between Membranes.

Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for phosphatidylinositol-4-phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificial membranes using Förster resonance energy transfer (FRET)-based assays and a standard spectrofluorometer. Such an approach can serve to better characterize the activity of known sterol/PI4P exchangers, but also to reveal whether ill-defined ORP/Osh proteins or LTPs belonging to other families have such an exchange activity. Besides, this protocol is amenable to test whether molecules can act as Orphilins, which have been found to inhibit the sterol/PI4P exchange activity of certain ORPs. Last, our strategy to measure in real-time PI4P transport using a known lipid-binding domain can serve as a basis for the design of novel in vitro protocols aiming to detect other lipid species.

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites.

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP-A and VAP-B, interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain-containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro MOSPD2 is an ER-anchored protein, and it interacts with several FFAT-containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle-bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems.

Kes1/Osh4 is a member of the conserved, but functionally enigmatic, oxysterol binding protein-related protein (ORP) superfamily that inhibits phosphatidylinositol transfer protein (Sec14)-dependent membrane trafficking through the trans-Golgi (TGN)/endosomal network. We now report that Kes1, and select other ORPs, execute cell-cycle control activities as functionally non-redundant inhibitors of the G1/S transition when cells confront nutrient-poor environments and promote replicative aging. Kes1-dependent cell-cycle regulation requires the Greatwall/MASTL kinase ortholog Rim15, and is opposed by Sec14 activity in a mechanism independent of Kes1/Sec14 bulk membrane-trafficking functions. Moreover, the data identify Kes1 as a non-histone target for NuA4 through which this lysine acetyltransferase co-modulates membrane-trafficking and cell-cycle activities. We propose the Sec14/Kes1 lipid-exchange protein pair constitutes part of the mechanism for integrating TGN/endosomal lipid signaling with cell-cycle progression and hypothesize that ORPs define a family of stage-specific cell-cycle control factors that execute tumor-suppressor-like functions.

Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth.

STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites.

StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER-anchored partner, Vesicle-associated membrane protein-associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER-endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.

Running up that hill: How to create cellular lipid gradients by lipid counter-flows.

Lipids are unevenly distributed within eukaryotic cells, allowing various processes to work in an optimized membrane environment. Rather than following vesicular trafficking pathways, certain lipids are transported by lipid transfer proteins (LTPs), some at sites of close apposition of two organelles called membrane contact sites (MCSs). An important question is whether or not LTPs are able to convey lipids to create and maintain the lipid gradients observed between organelles. Recent data shows that LTPs from the ORP/Osh family transport sterols and phosphatidylserine (PS) to the Golgi and plasma membrane, respectively, through counter-exchange for the phosphoinositide phosphatidylinositol 4-phosphate (PI4P). Coupling PI4P-driven exchange to PI4P metabolism allows for an accumulation of these lipids in specific organelles and for a regulation of ORP/Osh proteins in MCSs. Additionally, data indicate that PI4P/sterol exchanges are hijacked by various virus strains to generate replication organelles. Compounds called ORPhilins block PI4P/sterol exchange and have thereby powerful antiviral activities, indicating in turn that some ORP/Osh proteins might be relevant pharmaceutical targets.

New molecular mechanisms of inter-organelle lipid transport.

Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated.

INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.

In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.