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bioRxiv · 10.1101/2020.05.22.111153

Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate

Miner, G. E.·Rivera-Kohr, D. A.·Zhang, C.·Sullivan, K. D.·Guo, A.·Fratti, R. A.
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Abstract

Yeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H+ into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H+ and Ca2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H+ pumping is reduced. Conversely, chelating free Ca2+ with EGTA stimulated vacuole acidification. Not only did Ca2+ levels affect H+ translocation, we also show that blocking V-ATPase activity inhibited Ca2+ transport into the vacuole lumen. Together, these data illustrate that Ca2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles. Summary StatementHere we show that Ca2+ and H+ transport across the vacuole membrane is reciprocally regulated and that it is linked to the production of Phosphatidylinositol 3,5-bisphoshpate.

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Miner, G. E., Rivera-Kohr, D. A., Zhang, C., Sullivan, K. D., Guo, A., Fratti, R. A.. 2020-05-25. Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate. https://doi.org/10.1101/2020.05.22.111153

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