Oligomerization of the Saccharomyces cerevisiae Na+/H+ antiporter Nha1p: implications for its antiporter activity.
Oligomerization of the Saccharomyces cerevisiae Na+/H+ antiporter Nha1p: implications for its antiporter activity.
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DOI：
10.1016/j.bbamem.2005.11.005
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发表时间：
2005-12
期刊：
影响因子：
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通讯作者：
Keiji Mitsui;H. Yasui;N. Nakamura;H. Kanazawa
中科院分区：
文献类型：
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作者：
Keiji Mitsui;H. Yasui;N. Nakamura;H. Kanazawa
芽殖酵母（Saccharomycescerevisiae）的Na+/H+逆向转运蛋白（Nha 1 p）在细胞内pH和Na+稳态中起重要作用。在这里，我们通过在同一细胞中表达的不同标记的Nha 1 p蛋白的共沉淀表明酵母Nha 1 p l形成了寡聚体。然后，体外交联实验揭示Nha 1 p-FLAG作为二聚体存在于膜中。标记的Nha 1 p蛋白也从sec 18 -1突变细胞中共沉淀，其中ER到高尔基体的交通在非允许温度下被阻断，这表明Nha 1 p可能已经在ER膜中二聚化。当我们在同样表达野生型Nha 1 p-EGFP融合蛋白的细胞中过度表达具有缺陷反向转运蛋白活性的突变体Nha 1 p时，我们发现在高盐条件下细胞生长受损，即使野生型蛋白被适当表达并正确定位。免疫共沉淀试验表明，失活的Nha 1 p-FLAG突变体与野生型Nha 1 p-EGFP蛋白相互作用。这些结果支持的概念，Nha 1 p存在于膜作为二聚体，其单体的相互作用是重要的，其反向转运活性。
The Na+/H+antiporter (Nha1p) from the budding yeast Saccharomyces cerevisiae plays an important role in intracellular pH and Na+homeostasis. Here, we show by co-precipitation of differently tagged Nha1p proteins expressed in the same cell that the yeast Nha1p l forms an oligomer. In vitro cross-linking experiments then revealed that Nha1p-FLAG is present in the membranes as a dimer. Differently tagged Nha1p proteins were also co-precipitated from sec18-1 mutant cells in which ER-to-Golgi traffic is blocked under non-permissive temperatures, suggesting that Nha1p may already dimerize in the ER membrane. When we over-expressed a mutant Nha1p with defective antiporter activity in cells that also express the wild-type Nha1p-EGFP fusion protein, we found impaired cell growth in highly saline conditions, even though the wild-type protein was appropriately expressed and localized correctly. Co-immunoprecipitation assays then showed the inactive Nha1p-FLAG mutant interacted with the wild-type Nha1p-EGFP protein. These results support the notion that Nha1p exists in membranes as a dimer and that the interaction of its monomers is important for its antiporter activity.