Preview

PDF, English Download (5MB) | Terms of use

Abstract

Sphingolipid transport between organelles has been increasingly studied during the last years, and a large body of evidence points to protein-mediated sphingolipid transfer at organelle contact sites. This is well characterized for sphingolipid transfer along the biosynthetic pathway from the endoplasmic reticulum (ER) towards the plasma membrane. However, the catabolic pathway is far less well studied. Here, a crucial but unexplored step is the recycling of the sphingosine backbone upon lysosomal sphingolipid degradation and its reintegration into the biosynthetic pathway at the ER. In this study, I propose that the lysosomal cholesterol transporter STARD3 acts as a sphingosine transfer protein at lysosome-ER contact sites to facilitate sphingosine entry into the recycling pathway. STARD3 has been previously described to tether lysosomes to the ER via its FFAT motif that binds to ER-resident proteins. At this contact, its START-domain transfers cholesterol from the ER to lysosomes to support endosome maturation and to maintain cholesterol levels at the ER. I employed functionalized, photocrosslinkable sphingosine in intact cells to show that STARD3 also binds to sphingosine. Functionally, I could show that overexpression of STARD3 drives the sphingosine metabolism towards biogenesis of higher sphingolipid species, such as sphingomyelin, while depleting cellular STARD3 levels results in a delayed sphingosine metabolism, which indicates STARD3 as a lysosomal sphingosine exporter. This transfer is dependent on a functional FFAT motif, consistent with sphingosine transfer taking place at the lysosome-ER contact site. STARD3 FFAT mutation or drug-induced prevention of sphingosine entry into the recycling pathway switches the sphingolipid metabolism towards the degradation pathway. Finally, molecular simulations, mutational analyses, and cholesterol competition studies revealed the START domain as a sphingosine transfer domain that can either transfer cholesterol or sphingosine at lysosome-ER contact sites. Overall, I hypothesize that STARD3 transfers sphingosine from the lysosome towards the ER along a subcellular lipid gradient, potentially in exchange for the transfer of cholesterol. As such, STARD3 is the first protein able to facilitate sphingosine exit from lysosomes towards other organelles. This represents an important connection between the catabolic and anabolic sphingolipid pathways and has wide-ranging implications for future studies relating to lysosomal lipid efflux and sphingolipid-mediated processes in health and disease.