Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote robust homotypic vacuolar fusion in Saccharomyces cerevisiae . The elongase Elo3 adds two carbons to 24-carbon (C24) acyl chains to make C26 VLCFAs that are incorporated into sphingolipids. Vacuoles isolated from elo3 Δ cells had increased fluidity relative to the wild-type and were attenuated for fusion. Upon further testing we found that vesicle the tethering stage was affected as elo3 Δ vacuole clusters contained fewer vesicles versus the WT. Vacuole tethering requires the interactions of late endosomal Rab GTPase Ypt7 and the HOPS tethering complex. Pulldown assays using GST-Ypt7 showed that HOPS from elo3 Δ vacuole extracts failed to bind Ypt7 while HOPS from WT extracts interacted with GST-Ypt7. Furthermore GFP-Ypt7 failed to localize at vertex microdomains of elo3 Δ vacuoles relative to the WT, whereas HOPS and regulatory lipids did accumulate at vertices. Finally, we found that elo3 Δ vacuoles had reduced V-ATPase. Together these data show that C26-VLCFA containing sphingolipids are important for Ytp7 function and vacuole homeostasis.