Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs
Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs
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DOI：
10.1074/jbc.ra118.006192
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发表时间：
2019-05-17
影响因子：
4.8
通讯作者：
Toshima, Jiro
中科院分区：
文献类型：
--
作者：
Abe, Michiko;Saito, Mayu;Toshima, Jiro
胞浆型H+-ATP酶（V-ATP酶）是一种高度保守的质子泵，负责细胞内细胞器的酸化和潜在的药物靶点。它是一种多亚基复合物，包含负责ATP水解的细胞质V-1结构域和有助于质子跨膜转运的膜包埋V-0结构域。酿酒酵母V-ATP酶由14个亚基组成，其中任何一个亚基的缺失都会导致明确的生长缺陷。由于V-ATP酶的结构和各亚基的功能已在酵母中得到很好的表征，该生物体已被认为是研究V-ATP酶的优选模型。在本研究中，为了评估酵母和人类V-ATP酶亚基的功能相关性，我们研究了人类V-ATP酶亚基是否可以补充钙或pH敏感性生长、液泡腔酸化、V-ATP酶复合物的组装以及蛋白质分选缺乏同等酵母基因的酵母突变体。这些评估显示，13个人V-ATP酶亚基中的9个可以部分或完全补充相应酵母亚基的功能。重要的是，序列相似性不一定与功能互补相关。我们还发现，除了所有的V-O结构域亚基，V-1F亚基是必需的V-O结构域在内质网的适当装配。此外，人类H亚基完全恢复了液泡酸化的水平，但只有部分挽救钙敏感的增长，这表明在V-ATP酶活性的H亚基的特定作用。这些发现为酵母和人类V-ATP酶之间的功能同源性提供了重要的见解。
Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V-1 domain responsible for ATP hydrolysis and a membrane-embedded V-o domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all V-o domain subunits, the V-1 F subunit is required for proper assembly of the V-o domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.