Glycosphingolipids regulate phosphatidylserine transport machinery that operates at ER-PM contact sites.

Plasma membrane (PM) localization of KRAS requires specific glycosphingolipids in the outer leaflet and phosphatidylserine (PS) in the inner leaflet. PM PS content is controlled by lipid transport proteins ORP5 and ORP8, which operate at ER-PM membrane contact sites (MCSs). Using high-resolution imaging, we now show that GSLs, including GM3 and SM4, are required to maintain ORP5 and ORP8 localization to MCSs. Genetic deletion or pharmacologic inhibition of enzymes required for the biosynthesis of GM3 or SM4 displaces PI4-kinase Type IIIα (PI4KIIIα) and its adaptor EFR3A from the PM, thereby reducing PM phosphatidylinositol 4-phosphate (PI4P) content. PM interactions of ORP5 and ORP8 are also disrupted. Since ORP5 and ORP8 transport PS to the PM by counter-transporting PI4P to the ER, PM PS content is substantially reduced. We further show that GM3 and GM2 regulate the assembly of ER-PM-MCSs, such that the function of other MCS-localized macromolecular machineries including calcium release-activated calcium channels is abrogated when glycosphingolipid biosynthesis is blocked. Together, this study establishes glycosphingolipids as organizers of PS transport and ER-PM MCSs, expanding the regulators of MCSs beyond protein tethers to include glycosylated lipids and revealing how glycosphingolipids control KRAS function.