Marzuki S, Watkins L C, Choo W M
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Biochim Biophys Acta. 1989 Jul 13;975(2):222-30. doi: 10.1016/s0005-2728(89)80252-x.
Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.
酿酒酵母的线粒体aap1基因发生特定损伤的突变体已被研究，该基因编码H⁺-ATP酶的膜亚基8，以检测这些突变对酶复合体功能和组装的影响。这些突变体包括三个mit-突变体，由于它们无法合成全长的亚基8，所以不能通过氧化代谢生长，以及其中一个突变体的三个部分回复突变体。这些菌株中的突变先前已通过DNA测序进行了表征。使用针对H⁺-ATP酶β亚基的单克隆抗体作为组装缺陷的探针，结果表明亚基8的存在对于亚基6组装到酶复合体中至关重要。从mit-突变体中分离出的线粒体具有可忽略不计的[³²Pi]ATP交换活性，并且它们表现出对寡霉素抑制不敏感的ATP酶活性，这表明膜F0区段存在缺陷。在回复突变体菌株中观察到亚基8（和亚基6）的正常组装，尽管其中两个菌株的H⁺-ATP酶亚基8的跨膜区域有8-9个氨基酸替换。然而，组装后的复合体表现出降低的[³²Pi]ATP交换活性和对寡霉素的低敏感性，这表明aap1基因的产物是线粒体H⁺-ATP酶的一个功能亚基。
