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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 elo3Δ vacuoles show acidification defects and increased membrane fluidity, and this correlates with deficient fusion. Fusion arrest occurs at the tethering stage as elo3Δ vacuoles fail to cluster efficiently in vitro. Unlike HOPS and fusogenic lipids, GFP-Ypt7 does not enrich at elo3Δ vertex microdomains, a hallmark of vacuole docking prior to fusion. Pulldown assays using bacterially expressed GST-Ypt7 showed that HOPS from elo3Δ 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 elo3Δ 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. 

Abstract The transport of Ca²⁺across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca²⁺stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P₂pools. Ca²⁺is also released during vacuole fusion upontrans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P₂on Ca²⁺flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P₂were required for Ca²⁺uptake into the vacuole, increased concentrations abolished Ca²⁺efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P₂or increased endogenous production of by the hyperactivefab1^T2250Amutant. In contrast, the lack of PI(3,5)P₂on vacuoles from the kinase deadfab1^EEEmutant showed delayed and decreased Ca²⁺uptake. The effects of PI(3,5)P₂were linked to the Ca²⁺pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P₂. Experiments with Verapamil inhibited Ca²⁺uptake when added at the start of the assay, while adding it after Ca²⁺had been taken up resulted in the rapid expulsion of Ca²⁺. Vacuoles lacking both Pmc1 and the H⁺/Ca²⁺exchanger Vcx1 lacked the ability to take up Ca²⁺and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P₂can modulate which path predominates.