Abstract
In Escherichia coli F(1)F(0) ATP synthase, the two b subunits dimerize forming the peripheral second stalk linking the membrane F(0) sector to F(1). Previously, we have demonstrated that the enzyme could accommodate relatively large deletions in the b subunits while retaining function (Sorgen, P. L., Caviston, T. L., Perry, R. C., and Cain, B. D. (1998) J. Biol. Chem. 273, 27873-27878). The manipulations of b subunit length have been extended by construction of insertion mutations into the uncF(b) gene adding amino acids to the second stalk. Mutants with insertions of seven amino acids were essentially identical to wild type strains, and mutants with insertions of up to 14 amino acids retained biologically significant levels of activity. Membranes prepared from these strains had readily detectable levels of F(1)F(0)-ATPase activity and proton pumping activity. However, the larger insertions resulted in decreasing levels of activity, and immunoblot analysis indicated that these reductions in activity correlated with reduced levels of b subunit in the membranes. Addition of 18 amino acids was sufficient to result in the loss of F(1)F(0) ATP synthase function. Assuming the predicted alpha-helical structure for this area of the b subunit, the 14-amino acid insertion would result in the addition of enough material to lengthen the b subunit by as much as 20 A. The results of both insertion and deletion experiments support a model in which the second stalk is a flexible feature of the enzyme rather than a rigid rod-like structure.
Highlights
F1F0 ATP synthases are multimeric enzymes that function through a complex mechanism similar to a rotary motor [1,2,3]
Construction and Growth Characteristics of Mutants—To determine the maximum length of a b subunit that can be incorporated into a functional F1F0 ATP synthase, a collection of insertion mutations were generated within the stalk region of the b subunit by site-directed mutagenesis
The segment of the b subunit thought to form the portion of the F1F0 ATP synthase visualized as the second stalk in electron micrographs extends from about Ala-32 to at least Ser-85
Summary
Strains and Media—The E. coli uncF(b) deletion strain KM2 [29] and the wild type b subunit expression plasmid pKAM14 have been described previously [30]. Two base substitutions were generated by using the Stratagene QuikChange kit on plasmid pSD59 (bsol) at uncF gene nucleotides 96 and 99; this resulted in silent mutations in the codons for bsol Ala-32,Ile while generating the unique MunI site in plasmid pAUL45 (bsol). Preparative Procedures—Inverted membrane vesicles were prepared from 500-ml cultures of strain KM2 (⌬b) carrying the recombinant uncF(b) gene expression plasmids according to methods described previously [25]. E. coli strain 1100 was transformed with bsol expression plasmids pAUL49 and pAUL50 and inoculated into 500 mL of LB supplemented with glucose medium. Chemical cross-linking of bsol peptides with bis(sulfosuccinimidyl) suberate and analytic ultracentrifugation experiments were conducted according to procedures published in earlier work [27]
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