A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin
A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin
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DOI：
10.1074/jbc.m909690199
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Morsomme, P
中科院分区：
文献类型：
--
作者：
Maudoux, O;Batoko, H;Morsomme, P
带有 His 标签的烟草质膜 H+ ATP 酶亚型 PMA2 在酿酒酵母中表达并纯化。出乎意料的是，纯化的标记 PMA2 的一部分与两种酵母 14-3-3 调节蛋白 BMH1 和 BMH2 相关。这种复合物是在体内形成的，无需用梭菌素处理，梭菌素是一种真菌毒素，已知可稳定植物中的等效复合物。当使用凝胶过滤色谱法将游离 ATP 酶与 14-3-3.H+-ATP 酶复合物分离时，复合 ATP 酶的活性是游离形式的两倍。该复合物经胰蛋白酶处理后释放出一个较小的复合物，该复合物由 14-3-3 二聚体和 PMA2 C 末端区域的片段组成。后者通过 Edman 降解和质谱鉴定为 PMA2 C 端 57 个残基，其倒数第二个残基 (Thr-955) 被磷酸化。该 C 端片段的体外去磷酸化阻止了 14-3-3 蛋白的结合，即使在存在梭菌素的情况下也是如此。Thr-955 突变为丙氨酸、天冬氨酸或终止密码子可阻止 PMA2 与酵母互补H+-ATPase，这些突变也被引入到激活的 PMA2 突变体（Gln-14 --> Asp）中，其特征是具有更高的 H+ 泵活性。每个直接修饰 Thr-955 的突变都会阻止 14-3-3 结合、降低 ATP 酶比活性并减少酵母生长。我们得出的结论是，14-3-3 结合需要 Thr-955 的磷酸化，并且复合物的形成会激活该酶。
The Nicotiana plumbaginifolia plasma membrane H+ ATPase isoform PMA2, equipped with a His, tag, was expressed in Saccharomyces cerevisiae and purified, Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H+-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated, In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin, Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H+-ATPase, These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H+ pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.