1 Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA.
2 Department of Biology, Concordia University, Montreal, QC, Canada.
3 Department of Biology, Concordia University, Montreal, QC, Canada. Electronic address: Christopher.Brett@concordia.ca.
4 Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA; Center for Biophysics & Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA. Electronic address: rfratti@illinois.edu.

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds the last two carbons to VLCFAs that are incorporated into sphingolipids. Cells lacking Elo3 have fragmented vacuoles, which is also seen when WT cells are treated with the sphingolipid synthesis inhibitor Aureobasidin-A. Isolated elo3? vacuoles show acidification defects and increased membrane fluidity, and this correlates with deficient fusion. Fusion arrest occurs at the tethering stage as elo3? vacuoles fail to cluster efficiently in vitro. Unlike HOPS and fusogenic lipids, GFP-Ypt7 does not enrich at elo3? vertex microdomains, a hallmark of vacuole docking prior to fusion. Pulldown assays using bacterially expressed GST-Ypt7 showed that HOPS from elo3? vacuole extracts failed to bind GST-Ypt7 while HOPS from WT extracts interacted strongly with GST-Ypt7. Treatment of WT vacuoles with the fluidizing anesthetic dibucaine recapitulates the elo3? phenotype and shows increased membrane fluidity, mislocalized GFP-Ypt7, inhibited fusion, and attenuated acidification. Together these data suggest that sphingolipids contribute to Rab-mediated tethering and docking required for vacuole fusion.