Role of Vma21p in assembly and transport of the yeast vacuolar ATPase
Role of Vma21p in assembly and transport of the yeast vacuolar ATPase
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DOI：
10.1091/mbc.e04-06-0514
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发表时间：
2004-11-01
影响因子：
3.3
通讯作者：
Schekman, R
中科院分区：
文献类型：
--
作者：
Malkus, P;Graham, LA;Schekman, R
酿酒酵母（Saccharomycescerevisiae）液泡H ~+-ATPase（V-ATPase）是一个多亚基复合体，由负责ATP水解的外周膜部分（V-1）和质子转运所需的完整膜部分（V-0）组成。V-0的生物发生需要内质网（ER）定位的辅助因子，Vma 21 p。我们发现，在vma 21 Delta细胞中，V-0的主要蛋白脂质亚基未能与100 kDa的V-0亚基Vph 1 p相互作用，表明Vma 21 p是V-0组装所必需的。来自野生型膜的Vma 21 p的免疫沉淀导致所有五个V-0亚基的共免疫沉淀。对vmaDelta菌株的分析表明，V-0亚基与Vma 21 p的结合是由蛋白脂质亚基Vma 21 p介导的。虽然Vma 21 p/蛋白脂质相互作用独立于Vph 1 p，Vma 21 p/Vph 1 p协会是依赖于所有其他V-0亚基;表明组装V-0发生在一个定义的序列，与Vph 1 p招聘到Vma 21 p/蛋白脂质/Vma 6p复合物代表的最后一步。ER输出的体外测定用于证明完全组装的Vma 21 p/蛋白脂质/Vma 6 p/Vph 1 p复合物优先包装到COPII包被的运输囊泡中。脉冲追踪实验表明Vma 21 p与V-0之间的相互作用是短暂的，Vma 21 p/V-0的解离伴随着V-0/V-1的组装。在体内阻断ER输出稳定了Vma 21 p和V-0之间的相互作用，并消除了V-0/V-1的组装。虽然Vma 21 p突变体缺乏ER检索信号仍然与V，在液泡中，这种相互作用并不影响液泡V-0/V-1复合物的组装。我们的结论是，Vma 21 p是不参与调节V-0和V-1部门之间的相互作用，但它有一个至关重要的作用，在协调组装的V-0亚基和护送组装的V-0复合物到ER衍生的运输囊泡。
The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V-1) responsible for ATP hydrolysis and an integral membrane sector (V-0) required for proton translocation. Biogenesis of V-0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Delta cells, the major proteolipid subunit of V-0 failed to interact with the 100-kDa V-0 subunit, Vph1p, indicating that Vma21p is necessary for V-0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V-0 subunits. Analysis of vmaDelta strains showed that binding of V-0 subunits to Vma21p was mediated by the proteolipid subunit Vma21p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V-0 subunits; indicating that assembly of V-0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/ Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V-0 was transient and that Vma21p/V-0 dissociation was concomitant with V-0/V-1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V-0 and abrogated assembly of V-0/V-1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V, in the vacuole, this interaction did not affect the assembly of vacuolar V-0/V-1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V-0 and V-1 sectors, but that it has a crucial role in coordinating the assembly of V-0 subunits and in escorting the assembled V-0 complex into ER-derived transport vesicles.