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.

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.

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.