A Kiss-and-Ride Mechanism for Lipid Exchange at Membrane Contact Sites

Abstract Lipid transfer proteins (LTPs) mediate non-vesicular lipid transport at membrane contact sites, yet how transient membrane tethering contributes to lipid transfer remains poorly understood. Here, we use the single-domain yeast phosphatidylserine transporter Osh6, a member of the oxysterol-binding protein–related protein family, to investigate tethering-dependent lipid transport. We show that Osh6 can access two transport modes depending on membrane composition: conventional shuttling between membranes and a tethering-dependent mode in which the opposing membrane facilitates cargo extraction from the donor membrane. Combining lipid transport and membrane-tethering measurements with kinetic analysis, lipid partitioning, and in vivo complementation, we provide evidence for a transient membrane-bridged intermediate during lipid transfer. Productive phosphatidylinositol 4-phosphate (PI4P) extraction destabilizes this bridged state, allowing cargo-loaded Osh6 to disengage and continue the transport cycle. We term this mechanism kiss-and-ride , in which transient membrane contact facilitates cargo extraction (“kiss”) before the cargo-loaded protein disengages (“ride”). Consistent with this model, PI4P transport is preserved in a lidless Osh6 mutant with impaired membrane dissociation, whereas phosphatidylserine transport is strongly reduced, revealing distinct mechanistic requirements for the two cargoes. Together, our findings reveal a tethering-dependent transport mode in a cup-like LTP and suggest that membrane contact sites can actively shape the mechanism of lipid transport rather than merely bringing donor and acceptor membranes into proximity.