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

Abstract

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.

• Authors: 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
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 298 Issue 12 Pg. 102672 (12 2022) ISSN: 1083-351X [Electronic] United States
• PMID: 36334632 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Chemical References

• phosphatidylinositol 3,5-diphosphate
• Saccharomyces cerevisiae Proteins
• Phosphatidylinositols
• Vacuolar Proton-Translocating ATPases
• PMC1 protein, S cerevisiae
• Plasma Membrane Calcium-Transporting ATPases
• FAB1 protein, S cerevisiae
• Phosphotransferases (Alcohol Group Acceptor)

Topics

• Saccharomyces cerevisiae (metabolism)
• Saccharomyces cerevisiae Proteins (genetics, metabolism)
• Phosphatidylinositols
• Vacuoles (metabolism)
• Vacuolar Proton-Translocating ATPases (genetics, metabolism)
• Plasma Membrane Calcium-Transporting ATPases
• Phosphotransferases (Alcohol Group Acceptor) (metabolism)