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 .
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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