The interdependent transport of yeast vacuole Ca2+ 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 VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO 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 fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE 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.

High throughput analysis of vacuolar acidification.

Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is lumenal pH. In the endocytic and secretory pathways, lumenal pH is controlled by isoforms and concentration of the vacuolar-type H+-ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes.