The interdependent transport of yeast vacuole Ca2+ and H+ and the role of phosphatidylinositol 3,5-bisphosphate

Chi Zhang, Yilin Feng, Adam Balutowski, Gregory E. Miner, David A. Rivera-Kohr, Michael R. Hrabak, Katherine D. Sullivan, Annie Guo, Jorge D. Calderin, Rutilio A. Fratti

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number102672
JournalJournal of Biological Chemistry
Issue number12
StatePublished - Dec 2022


  • Fab1
  • PIKfyve
  • Pmc1
  • Stv1
  • V-ATPase
  • Vcx1
  • Vph1
  • calcium ATPase
  • calcium transport
  • lysosome
  • organellar pH homeostasis
  • phosphoinositide

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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