Isolation of native plasma membrane H(+)-ATPase (Pma1p) in both the active and basal activation states.
Isolation of native plasma membrane H(+)-ATPase (Pma1p) in both the active and basal activation states.
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DOI：
10.1002/2211-5463.12413
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发表时间：
2018-05
期刊：
影响因子：
2.6
通讯作者：
Palmgren M
中科院分区：
文献类型：
--
作者：
Pedersen JT;Kanashova T;Dittmar G;Palmgren M
酵母质膜H+-ATPase Pma1p是一种为酵母质膜提供能量的P型ATPase。Pma1p以两种激活状态存在：自抑制基础状态和激活状态。在这里，我们展示了功能和稳定的Pma1p可以在不改变其激活状态的情况下以天然形式纯化并在人工脂质体中重组。据报道，乙酰化的微管蛋白可以将Pma1p维持在基础状态，但由于纯化的制剂中没有该蛋白，因此它不是自身抑制机制的重要组成部分。对两种活化态的天然Pma1p的纯化和重组为直接比较这两种活化态的输运性质打开了大门，这使得我们能够证实基态的ATP水解与泵浦的质子之间的耦合比较低，而激活态的耦合比较高。能够在两种激活状态下制备天然的Pma1p将有助于进一步的结构和生物化学研究质膜H+-ATPase被自动抑制的机制。
The yeast plasma membrane H+‐ATPase Pma1p is a P‐type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+‐ATPases are autoinhibited.
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