Ho M N, Hirata R, Umemoto N, Ohya Y, Takatsuki A, Stevens T H, Anraku Y
Department of Biology, Faculty of Science, University of Tokyo, Japan.
J Biol Chem. 1993 Aug 25;268(24):18286-92.
Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.
先前对酿酒酵母液泡质子转运ATP酶（V-ATP酶）的纯化和特性研究表明，该酶是一种多亚基复合物，由至少8个亚基组成，分子量分别为100、69、60、42、36、32、27和17千道尔顿（凯恩，P.M.，山代，C.T.，和史蒂文斯，T.H.（1989年）《生物化学杂志》264卷，19236 - 19244页）。我们报告了另一个V-ATP酶亚基，即54千道尔顿亚基的克隆和特性研究，它由VMA13基因编码。VMA13是通过互补与vma13突变相关的生长表型而分离得到的，vma13突变最初被描述为cls11（大矢，Y.，梅本，N.，谷田，I.，太田，A.，饭田，H.，和荒乐，Y.（1991年）《生物化学杂志》266卷，13971 - 13977页）。VMA13基因的核苷酸序列预测了一种亲水性多肽，计算分子量为54415道尔顿。VMA13的54千道尔顿基因产物位于液泡膜上，并与活性V-ATP酶复合物共纯化。对vma13基因缺失突变体（Δvma13）的特性研究表明，Vma13多肽对V-ATP酶活性至关重要。然而，如从Δvma13细胞分离的液泡膜上这些亚基的结合情况所示，Vma13多肽对于其他V-ATP酶亚基（100、69、60、42、27或17千道尔顿亚基）靶向液泡膜并非必需。尽管如此，Δvma13突变体中V-ATP酶“复合物”的性质与野生型酶有根本不同。这一点由以下事实证明：来自Δvma13细胞的无活性V-ATP酶复合物比野生型酶更不稳定。综上所述，这些结果表明VMA13编码V-ATP酶的54千道尔顿亚基，并且该亚基对酶复合物的活性至关重要，但对其组装并非必需。
