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.

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 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 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 homologue 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.

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.

Osh6 and Osh7 are lipid transfer proteins (LTPs) that transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 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 homologue 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 osh6D osh7D 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.

Several members of the oxysterol-binding protein-related proteins (ORPs)/oxysterol-binding homology (Osh) family exchange phosphatidylserine (PS) and phosphatidylinositol 4-phosphate (PI(4)P) at the endoplasmic reticulum/plasma membrane (PM) interface. It is unclear whether they preferentially exchange PS and PI(4)P with specific acyl chains to tune the features of the PM, whether they use phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) instead of PI(4)P for exchange processes and whether their activity is influenced by the association of PS with sterol in the PM. Here, we measured in vitro how the yeast Osh6p and human ORP8 transported diverse PS and PI(4)P subspecies, including major cellular species, between membranes. We established how their activity is impacted by the length and unsaturation degree of these lipids. Surprisingly, the speed at which they individually transfer these ligands inversely depends on their affinity for them. To be fast, the transfer of high-affinity ligands requires an exchange with a counterligand of equivalent affinity. Besides, we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for intracellular lipid exchange, as they have a low affinity for this lipid compared to PI(4)P, and do not transfer more PS into sterol-rich membranes. This study provides insights into PS/PI(4)P exchangers and sets unanticipated rules on how the activity of lipid transfer proteins relates to their affinity for ligands.

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