Functional complementation of yeast vma1Δ cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+-ATPase activity
Functional complementation of yeast vma1Δ cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+-ATPase activity
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DOI：
10.1046/j.1365-313x.1999.00402.x
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发表时间：
1999-03-01
期刊：
影响因子：
7.2
通讯作者：
Wilkins, TA
中科院分区：
文献类型：
--
作者：
Kim, W;Wan, CY;Wilkins, TA
研究了植物液泡H+- atp酶（v - atp酶）亚基同源物（亚基a）拯救酵母vma突变表型的能力，这是研究植物亚基分子细节结构和功能的第一步。在vma突变型酵母细胞中，异源表达编码A亚基的棉花cdna成功地挽救了vma突变体的表型，这表明植物和酵母的A亚基在进化过程中保留了V-ATPases必需的元件。虽然液泡酸化，但植物-酵母杂交全酶只能部分恢复突变酵母细胞中的v - atp酶活性（约60%）。不同的N或c末端的结构域替换只略微增强了v -ATP酶的活性，而交换两个结构域则起协同作用，相对于原生植物亚基，增加了偶联ATP水解和质子易位约22%。免疫印迹分析表明，类似数量的酵母、植物或植物-酵母嵌合亚基是膜结合的。这些结果表明亚基A末端结构域包含影响V-ATPase结构和功能的结构信息。
The ability of a vacuolar H+-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma id yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.