V-type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus -: Subunit structure and operon
V-type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus -: Subunit structure and operon
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DOI：
10.1074/jbc.275.18.13955
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Yoshida, M
中科院分区：
文献类型：
--
作者：
Yokoyama, K;Ohkuma, S;Yoshida, M
从嗜热真细菌Thermus thermophilus中分离到一种V型ATP酶（VoV 1），该酶具有ATP驱动的H+泵和H+梯度驱动的ATP合成功能。当在十二烷基硫酸钠存在下通过凝胶电泳分析该酶时，它显示出八条多肽带，其中四条是V-1的亚基。我们还分离了VoV 1操纵子，其包含9个基因，顺序为atpG-I-L-E-X-F-A-B-D），其编码的蛋白质的分子大小分别为13、43、10、20、35、11、64、53和25 kDa。最后四个基因被鉴定为V-1亚基的基因; atpA、B、D和F分别编码A、B、γ和δ亚基。前五个基因atpG-atpX被鉴定为V-o亚基的基因。atpL的蛋白脂质亚基的产物缺乏19个氨基酸的前序列，并且与V型ATP酶不同，其包含两个跨膜结构域而不是四个。atpI的疏水性43-kDa产物是迄今为止发现的真核生物100-kDa亚基家族中最小的成员。其电泳带与A亚基的电泳带重叠。因此，在我们纯化的VoV 1中发现了所有的基因产物。我们从分离的亚基中分离出A（3）B（3）亚复合物，并从表达亚基的大肠杆菌中分离出A（3）B（3）γ亚复合物。电镜观察表明，V-1的γ亚基填充在A、B的中心空腔中，可能是中心亚基，与F-1-ATPase的γ亚基相似。
V-type ATPase (VoV1) capable of ATP-driven H+ pumping and of H+ gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V-1. We also isolated the VoV1 operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D), which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V-1 subunits; atpA, B, D, and F encoded the A, B, gamma, and delta subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V-o subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified VoV1. We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V-1 filled the central cavity of A,B, and might be central subunit, similar to the gamma subunit of F-1-ATPase.