ABSTRACT
Yeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H+ into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H+ and Ca2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H+ pumping is reduced. Conversely, chelating free Ca2+ with EGTA stimulated vacuole acidification. Not only did Ca2+ levels affect H+ translocation, we also show that blocking V-ATPase activity inhibited Ca2+ transport into the vacuole lumen. Together, these data illustrate that Ca2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles.
Summary Statement Here we show that Ca2+ and H+ transport across the vacuole membrane is reciprocally regulated and that it is linked to the production of Phosphatidylinositol 3,5-bisphoshpate.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AO
- acridine orange
- DAG
- diacylglycerol
- FCCP
- Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone
- PA
- phosphatidic acid
- PI
- phosphatidylinositol
- PI3P
- phosphatidylinositol 3-phosphate
- PI(3,5)P2
- phosphatidylinositol 3,5-bisphosphate
- PKC
- protein kinase C
- PLC
- phospholipase C
- SNARE
- soluble N-ethylmaleimide-sensitive factor attachment protein receptor
- TRP
- transient receptor potential
- YPD
- yeast extract, peptone, dextrose