Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These ...processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient.
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.
Numerous data have been collected on lipid-binding amphipathic helices involved in membrane-remodeling machineries and vesicular transport. Here we describe how, with regard to lipid composition, the ...physicochemical features of some amphipathic helices explain their ability to recognize membrane curvature or to participate in membrane remodeling. We propose that sensing highly-curved membranes requires that the polar and hydrophobic faces of the helix do not cooperate in lipid binding. A more detailed description of the interaction between amphipathic helices and lipids is however needed; notably to explain how new helices contribute to detection of modest changes in curvature or even negative curvature.
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.
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.
Synopsis
The cholesterol transfer protein STARD3, which scaffolds ER–endosome contact sites, controls cellular cholesterol distribution by efficiently delivering ER cholesterol to endosomes.
STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane.
STARD3‐mediated cholesterol routing depends on both its START domain‐dependent lipid transfer activity and its ability to create ER–endosome contacts with the ER‐anchored VAP proteins.
In vitro reconstitution assays indicate that STARD3 and VAP assemble into a highly efficient cholesterol transport machine.
The cholesterol transfer protein STARD3, which scaffolds ER–endosome contact sites, controls cellular cholesterol distribution by efficiently delivering ER cholesterol to endosomes.
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.
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.
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.
Synopsis
Membrane contact sites (MCS) are subcellular domains formed by close apposition between the membranes of two organelles, which regulate lipid transport and organelle dynamics. Here, phosphorylation of a non‐conventional FFAT motif on Endoplasmic Reticulum (ER) receptor partner proteins is shown to mediate MCS formation and lipid transport.
The ER receptors VAP‐A, VAP‐B and MOSPD2 bind a non‐conventional FFAT motif via their MSP domain.
The alternative FFAT motif (Phospho‐FFAT) is characterized by a key phosphorylated serine/threonine residue.
Structural analyses show that conventional FFATs and alternative phospho‐FFATs bind to VAP differently.
An algorithm to predict Phospho‐FFAT motifs reveals that Phospho‐FFATs are as common as conventional FFATs in the human proteome.
Phosphorylation acts as a switch that activates the ER–endosome MCS formation and sterol exchange.
Phosphorylation of a non‐conventional FFAT motif promotes ER‐endosome membrane contact sites and sterol exchange.
Lipids are precisely distributed in the eukaryotic cell where they help to define organelle identity and function, in addition to their structural role. Once synthesized, many lipids must be ...delivered to other compartments by non-vesicular routes, a process that is undertaken by proteins called Lipid Transfer Proteins (LTPs). OSBP and the closely-related ORP and Osh proteins constitute a major, evolutionarily conserved family of LTPs in eukaryotes. Most of these target one or more subcellular regions, and membrane contact sites in particular, where two organelle membranes are in close proximity. It was initially thought that such proteins were strictly dedicated to sterol sensing or transport. However, over the last decade, numerous studies have revealed that these proteins have many more functions, and we have expanded our understanding of their mechanisms. In particular, many of them are lipid exchangers that exploit PI(4)P or possibly other phosphoinositide gradients to directionally transfer sterol or PS between two compartments. Importantly, these transfer activities are tightly coupled to processes such as lipid metabolism, cellular signalling and vesicular trafficking. This review describes the molecular architecture of OSBP/ORP/Osh proteins, showing how their specific structural features and internal configurations impart unique cellular functions.
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
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.
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