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 V₁ complex. The assembly of the V₁-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)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 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 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.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Zhang C, Feng Y, Balutowski A, Miner GE, Rivera-Kohr DA, Hrabak MR, Sullivan KD, Guo A, Calderin JD, Fratti RA

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