Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol 4-phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to the presence of charged lipids and to membrane fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.

• MeSH
• Biological Transport MeSH
• Cell Membrane * metabolism   MeSH
• Membrane Fluidity MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Phosphatidylserines metabolism   MeSH
• Oxysterol Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Receptors, Steroid metabolism   MeSH
• Unilamellar Liposomes metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphatidylinositol Phosphates MeSH
• Phosphatidylserines MeSH
• phosphatidylinositol 4-phosphate MeSH Browser
• Oxysterol Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Receptors, Steroid MeSH
• Unilamellar Liposomes MeSH

ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.

• Keywords
• ER, ORD, ORP8, PI4P, PS, lipid transport, plasma membrane,
• MeSH
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Lipids * chemistry   MeSH
• Carrier Proteins * metabolism   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipids * MeSH
• Carrier Proteins * MeSH