Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.
Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.
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DOI：
10.1016/s0021-9258(18)48515-8
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发表时间：
1992-01
期刊：
影响因子：
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通讯作者：
Patricia M. KaneS;Margery;Kuehn;Isabelle Howald-Stevenson;T. Stevens
中科院分区：
文献类型：
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作者：
Patricia M. KaneS;Margery;Kuehn;Isabelle Howald-Stevenson;T. Stevens
先前对酵母液泡质子易位atp酶（H(+)- atp酶）的纯化和鉴定表明，它是一个多亚基复合物，由整体和外周膜亚基组成（Uchida， E., Ohsumi， Y., and Anraku， Y. (1985) J. Biol.）。化学，26,1090-1095；（1989） [j] .生物学。化学。264,19236-19244)。我们已经获得了识别42和100 kda多肽的单克隆抗体，这些单克隆抗体是用液泡atp酶活性共纯化的。利用这些抗体，我们提供了进一步的证据，证明42 kda多肽（外周膜蛋白）和100 kda多肽（整体膜蛋白）是酵母液泡H(+)- atp酶的真正亚基。在分别编码69、60和17 kda亚基的TFP1、VAT2或VMA3基因染色体缺失的突变酵母细胞中，研究了三个外周亚基（69、60和42 kda亚基）和两个完整膜亚基（100和17 kda亚基）的合成、组装和靶向性。用Western blotting检测全细胞裂解物和纯化空泡膜中各种亚基的稳态水平，并用免疫荧光显微镜检测60和100 kda亚基的细胞内定位。结果表明，酵母液泡H(+)- atp酶外周亚基的组装和/或液泡靶向性取决于69-，60-和17-kDa亚基的存在。只要17-kDa亚基存在，100-kDa亚基可以独立于外周膜亚基转运到液泡；但在缺乏17-kDa亚基的情况下，100-kDa亚基似乎既不稳定，也无法运输到液泡。
Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.