VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.
VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(18)54027-8
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发表时间：
1993-01
期刊：
影响因子：
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通讯作者：
Ryogo HirataS;N. Umemoto;Margaret N. HolI;Yoshikazu OhyaSS;Tom H. Stevensv;Yasuhiro AnrakuBQ
中科院分区：
文献类型：
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作者：
Ryogo HirataS;N. Umemoto;Margaret N. HolI;Yoshikazu OhyaSS;Tom H. Stevensv;Yasuhiro AnrakuBQ
酿酒酵母的vma12突变体，最初被鉴定为钙敏感(CLS)突变体，也是呼吸缺陷(Pet-)，具有液泡膜H(+)-ATPase活性缺陷(Ohya，Y.，Uemoto，N.，Tanida，I.，Ohta，A.，Iida，H.，Anraku，Y.，J.Biol)。化学。13971-13977)。通过对vma12突变体生长缺陷的互补，克隆了vma12基因。该基因的核苷酸序列预测有215个氨基酸(25.2 kDa)的多肽，有两个假定的跨膜结构域。通过染色体缺失构建的vma12突变体是有活性的，但已经完全失去了液泡膜H(+)-ATPase活性，并表现出与原始vma12突变体相同的生长缺陷。通过全细胞和液泡膜蛋白提取液的Western blotting分析，检测了Delta vma12突变细胞中H(+)-ATPase亚单位的合成和靶向性。我们分析的外周膜亚单位(69 kDa、60 kDa、42 kDa和27 kDa亚基)都没有在液泡膜部分中检测到，尽管这些多肽的细胞水平似乎是正常的。该酶的100 kDa和17 kDa的完整膜亚基在突变的液泡膜组分中缺失或显著降低。抗Vma12p抗体识别一种预期分子质量为25 kDa的空泡蛋白。然而，在用两性离子洗涤剂ZW3-14增溶并通过甘油梯度离心法纯化的液泡膜ATPase复合体中没有检测到Vma12蛋白(Kane，P.M.，Yamashiro，C.T.和Stevens，T.H.(1989)J.Biol)。化学。19236-19244)。这些结果表明，VMA12基因产物不是活性液泡ATPase复合体的组成部分，而是该蛋白在酶复合体组装和/或靶向液泡膜的过程中所必需的。
vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.