Topological characterization of the c, c′, and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae
Topological characterization of the c, c′, and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m406767200
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发表时间：
2004-09-17
影响因子：
4.8
通讯作者：
Stevens, TH
中科院分区：
文献类型：
--
作者：
Flannery, AR;Graham, LA;Stevens, TH
液泡型ATPase(V-ATPase)是真核生物细胞内细胞器酸化的多亚单位酶。与F型ATP合成酶(F-ATPase)类似，V-ATPase由V-1和V-0两个亚复合体组成。V-1亚基中ATP的水解与V-0亚基完成的质子转运紧密相连，V-0亚基由五个独特的亚基(a，d，c，c‘和c“)组成，其中三个亚基c(Vma3p)，c’(Vma11p)和c”(Vma16p)是称为“蛋白脂”的小分子疏水性完整膜蛋白，其序列与F-ATPase亚基c有相似的序列。而来自F-ATPase的c亚基两次跨越膜双层，而V-ATPase蛋白脂被模拟为至少有四个跨膜螺旋。用表位标记的蛋白质脂拷贝进行的有限蛋白质分解实验表明，c(Vma3p)和c‘(Vma11p)的N和C末端位于液泡的管腔中。对表位标记的c“(Vma16p)进行有限的蛋白分解，结果表明，Vma16p的N端位于液泡的细胞质表面，而C端位于液泡内。此外，Vma16p与Vma3p的嵌合融合Vma16-Vma3p可组装成一个功能齐全的V-ATPase复合体，进一步支持Vma16p的C端位于液泡腔内的结论。这些结果表明，c和c‘亚基有四个跨膜片段，它们的N和C端位于液泡内，c”有五个跨膜片段，N端暴露在细胞质和C端的腔内。
The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (F-ATPase), the V-ATPase is composed of two subcomplexes, V-1 and V-0. Hydrolysis of ATP in the V-1 subcomplex is tightly coupled to proton translocation accomplished by the V-0 subcomplex, which is composed of five unique subunits (a, d, c, c', and c"). Three of the subunits, subunit c (Vma3p), c' (Vma11p), and c" (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c' (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c" ( Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c' have four transmembrane segments with their N and C termini in the lumen and that c" has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.