RESUMO
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.
RESUMO
Yeast vacuoles are acidified by the v-type H+-ATPase (V-ATPase) that is comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 also affects vacuolar Ca2+ release through the channel Yvc1 and uptake through the Ca2+ pump Pmc1. Here, we asked if H+ and Ca2+ transport activities were connected through PI(3,5)P2. We found that overproduction of PI(3,5)P2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P2, suggesting that PI(3,5)P2 plays a role in submicromolar Ca2+ flux but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional, we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P2.