Jackson D D, Stevens T H
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
J Biol Chem. 1997 Oct 10;272(41):25928-34. doi: 10.1074/jbc.272.41.25928.
The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment.
酿酒酵母液泡膜质子转运ATP酶（V-ATP酶）可分为一个外周膜复合物（V1）和一个整合膜复合物（V0），V1包含至少8种多肽，分子量分别为69、60、54、42、32、27、14和13 kDa，V0包含至少5种多肽，分子量分别为100、36、23、17和16 kDa。已鉴定出其他酵母基因，它们是V-ATP酶组装所必需的，但它们的蛋白质产物不能与该酶复合物共纯化。其中一个这样的基因VMA12编码一种25 kDa的蛋白质（Vma12p），预计该蛋白质含有两个跨膜结构域。生化分析表明，Vma12p表现为一种整合膜蛋白，其N端和C端均朝向胞质溶胶，并且该蛋白免疫定位在内质网（ER）。在缺乏Vma12p的细胞（vma12Δ）中，V0复合物的100 kDa亚基（包含6至8个推定的跨膜结构域）迅速降解（半衰期约30分钟）。蛋白酶保护试验表明，100 kDa亚基正确插入/转运到vma12Δ细胞的ER膜中。这些数据表明，Vma12p在V0亚基插入ER膜后在内质网中发挥作用。我们提出，Vma12p直接在内质网中发挥作用，将V0亚基组装成复合物，并且这种组装对于V0亚基的稳定性及其作为复合物从该隔室转运出去是必需的。
