Role of Very Long-Chain Fatty Acyl-Ceramides in Endocytic Uptake

Specialized endoplasmic reticulum (ER) subdomains termed ER sterol exit sites (ERSES), transiently contact endocytic sites to locally supply sterols, which in turn initiate actin polymerization and drive membrane invagination. Osh2, an oxysterol-binding protein (OSBP)-related protein (ORP), mediates the non-vesicular sterol transfer at ER-endocytic contact sites (ER-ECS). In search for additional ERSES components, we identified Fat1, a fatty acid (FA) transporter and acyl-CoA synthetase with specificity for very long-chain (VLC) FAs (C20-C26). Analysis of VLC-FA anabolic and catabolic pathways revealed a specific requirement for VLC fatty acyl-ceramides (VLCA-Cers) in the constriction of endocytic invaginations. Strikingly, the Cer synthase physically interacted with the endocytic machinery, pointing to a direct role of VLCFA-Cers in membrane remodelling. Consistent with this view, molecular dynamics and live-cell imaging of a Cer probe indicated that the ORPs Osh1 and Osh3 contribute to Cer export from at ER-ECS and and that their depletion recapitulates the endocytic defects observed in the absence of VLCFA-Cer. Our findings establish that local transport of VLCA-Cers is required for endocytic budding and reveal ERSES as hubs where Cer and sterol biosynthesis and transport converge