Takeyama M, Noumi T, Maeda M, Futai M
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
J Biol Chem. 1988 Nov 5;263(31):16106-12.
Six chromosomal uncF mutants of Escherichia coli defective in the b subunit of H+-ATPase (156 amino acid residues) were identified (KF92, Met-1----Val; KF164, Gln-64----end; KF61 and KF144, Gln-104----end; KF138, Gln-106----end; and KF79, Gln-123----end). The membranes of all these mutants had low ATPase activities (less than 5% of that of the wild type), and no functional H+ pathway, although the truncated b subunits were integrated into these membranes. These findings suggest that about 30 carboxyl-terminal amino acid residues of the b subunit are essential for formation of the F1-binding site and H+ pathway. For examination of the role(s) of the carboxyl-terminal region(s) or residue(s) of the b subunit, recombinant plasmids carrying truncated uncF genes of various lengths were constructed by in vitro muta-genesis and introduced into a recA1 derivative of strain KF92 (Met-1----Val). Analyses of the membranes from the resulting strains demonstrated that almost the entire carboxyl-terminal region of the b subunit is necessary for formation of functional Fo, since loss of the carboxyl-terminal residue resulted in significant reduction of both F1 binding and H+ translocation, and loss of two or more residues abolished both activities completely.
已鉴定出大肠杆菌中6个H⁺ -ATP酶β亚基（156个氨基酸残基）有缺陷的染色体uncF突变体（KF92，Met-1→Val；KF164，Gln-64→末端；KF61和KF144，Gln-104→末端；KF138，Gln-106→末端；以及KF79，Gln-123→末端）。所有这些突变体的膜都具有低ATP酶活性（不到野生型的5%），并且没有功能性的H⁺途径，尽管截短的β亚基整合到了这些膜中。这些发现表明，β亚基的约30个羧基末端氨基酸残基对于F₁结合位点和H⁺途径的形成至关重要。为了研究β亚基羧基末端区域或残基的作用，通过体外诱变构建了携带不同长度截短uncF基因的重组质粒，并将其导入菌株KF92（Met-1→Val）的recA1衍生物中。对所得菌株的膜进行分析表明，β亚基几乎整个羧基末端区域对于功能性F₀的形成是必需的，因为羧基末端残基的缺失导致F₁结合和H⁺转运都显著减少，而两个或更多残基的缺失则完全消除了这两种活性。
