Fraga D, Hermolin J, Oldenburg M, Miller M J, Fillingame R H
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706.
J Biol Chem. 1994 Mar 11;269(10):7532-7.
Two substitutions were made for Arg41 in the polar loop of subunit c of the Escherichia coli F1F0 H(+)-transporting ATP synthase. The R41K and R41H mutants were initially studied by use of a plasmid carrying the complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon. The extent of F0 incorporation into membranes of these cells was quite variable, and the system was concluded to be unsuitable for biochemical characterization. Ultimately, the mutant genes were recombined into the chromosome using a novel method for the unc system. The biochemical phenotype of the chromosomally expressed mutants proved to be reproducible. The c R41H mutation causes a specific defect in assembly of F0, i.e. subunit a was not incorporated into the membrane despite near normal incorporation of subunits b and c. On the other hand, c R41K mutant F0 assembled normally in one of two background strains studied. (In the second genetic background, subunit a was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F0, R41K F0 was found to bind F1 with near normal affinity and to transport H+ at near normal rates. Although R41K F0 binds F1, F1-ATPase activity and H+ transport remained uncoupled. The uncoupling was indicated by a lack of ATP-driven H+ translocation and by the high proton permeability of membranes with F1 bound to F0. The uncoupled phenotype of the R41K mutant closely resembles that previously reported for the c Q42E mutant.
对大肠杆菌F1F0 H⁺转运ATP合酶c亚基极性环中的Arg41进行了两处替换。最初通过使用携带完整的c R41K或c R41H unc（F1F0）操纵子的质粒，在缺失unc操纵子的染色体菌株中研究R41K和R41H突变体。这些细胞中F0整合到膜中的程度变化很大，并且得出结论认为该系统不适合进行生化表征。最终，使用一种针对unc系统的新方法将突变基因重组到染色体中。染色体表达突变体的生化表型被证明是可重复的。c R41H突变导致F0组装出现特定缺陷，即尽管亚基b和c的整合接近正常，但亚基a未整合到膜中。另一方面，c R41K突变体F0在研究的两种背景菌株之一中正常组装。（在第二种遗传背景中，亚基a低效整合到c R41K膜中。）在由组装完整F0的c R41K菌株制备的膜中，发现R41K F0以接近正常的亲和力结合F1，并以接近正常的速率转运H⁺。尽管R41K F0结合F1，但F1 - ATP酶活性和H⁺转运仍未偶联。未偶联通过缺乏ATP驱动的H⁺转运以及F1与F0结合的膜的高质子渗透性来表明。R⁴¹K突变体的未偶联表型与先前报道的c Q42E突变体非常相似。
