The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate

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).