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
Abstract
Yeast vacuoles are acidified by the v-type H + -ATPase (V-ATPase) that is comprised of the membrane embedded V O complex and the soluble cytoplasmic V 1 complex. The assembly of the V 1 -V O holoenzyme on the vacuole is stabilized in part through interactions between the V O a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ). PI(3,5)P 2 also affects vacuolar Ca 2+ release through the channel Yvc1 and uptake through the Ca 2+ pump Pmc1. Here, we asked if H + and Ca 2+ transport activities were connected through PI(3,5)P 2 . We found that overproduction of PI(3,5)P 2 by the hyperactive fab1 T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1 EEE mutant attenuated the formation of a H + gradient. Separately, we tested the effects of excess Ca 2+ on vacuole acidification. Adding micromolar Ca 2+ blocked vacuole acidification, whereas chelating Ca 2+ accelerated acidification. The effect of adding Ca 2+ on acidification was eliminated when the Ca 2+ /H + antiporter Vcx1 was absent, indicating that the vacuolar H + gradient can collapse during Ca 2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P 2 , suggesting that PI(3,5)P 2 plays a role in submicromolar Ca 2+ flux but not under Ca 2+ shock. To see if the link between Ca 2+ and H + transport was bidirectional, we examined Ca 2+ transport when vacuole acidification was inhibited. We found that Ca 2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca 2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P 2 .