酵母液泡由v型H+-ATPase(V-ATPase)酸化,所述V型H+-ATPase由膜包埋的VO复合物和可溶性细胞质V1复合物组成。V1-VO全酶在液泡上的组装部分是通过VOa亚基直向同源物Vph1与脂质磷脂酰肌醇3,5-双磷酸酯(PI(3,5)P2)之间的相互作用而稳定的。PI(3,5)P2还影响通过通道Yvc1的液泡Ca2+释放和通过Ca2+泵Pmc1的摄取。这里,我们询问H+和Ca2+转运活动是否通过PI(3,5)P2连接。我们发现,过度活跃的fab1T2250A突变体过度生产PI(3,5)P2会增加液泡酸化,而激酶失活的fab1EEE突变体减弱了H梯度的形成。分别,我们测试了过量Ca2对液泡酸化的影响。添加微摩尔Ca2+阻断液泡酸化,而螯合Ca2+加速酸化。当Ca2/H反转运蛋白Vcx1不存在时,添加Ca2对酸化的影响被消除,表明空泡H+梯度可以通过Vcx1活性在Ca2+胁迫期间崩溃。这个,然而,与PI(3,5)P2无关,表明PI(3,5)P2在亚微摩尔Ca2通量中起作用,但在Ca2冲击下不发挥作用。看看Ca2+和H+传输之间的联系是否是双向的,当液泡酸化受到抑制时,我们检查了Ca2的运输。我们发现,通过用巴弗洛霉素停止V-ATPase活性或用氯喹中和液泡pH来抑制Ca2转运。一起,这些数据显示Ca2+转运和V-ATP酶功效是连接的,但不一定通过PI(3,5)P2。
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.