Jahn Thomas P, Schulz Alexander, Taipalensuu Jan, Palmgren Michael Gjedde
Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
J Biol Chem. 2002 Feb 22;277(8):6353-8. doi: 10.1074/jbc.M109637200. Epub 2001 Dec 13.
Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.
许多在酿酒酵母中表达的异源膜蛋白无法到达其正常的细胞位置，而是在堆叠的内膜中积累。拟南芥质膜H(+)-ATP酶同工型2（AHA2）主要在酵母内膜中表达，并且无法补充缺乏其内源性H(+)-ATP酶Pma1的酵母菌株。我们观察到，异源宿主中AHA2的磷酸化以及随后14-3-3蛋白的结合对于AHA2替代Pma1的能力至关重要。因此，补充pma1的AHA2突变体在倒数第二个残基（Thr(947)）处显示出增加的磷酸化，这为内源性14-3-3蛋白创造了一个结合位点。只有质膜中的一部分ATP酶被磷酸化。另外携带T947A替代突变的双突变体失去了补充pma1的能力。然而，在C末端调节域的两个自抑制区域中受到影响的突变体，无论其在Thr(947)处磷酸化的能力如何，都能补充pma1。这表明，决定H(+)-泵拯救pma1能力的是突变酶的活性状态，而不是运输的重新定向或14-3-3结合本身。
