Lengthening the second stalk of F1F0 ATP synthase in Escherichia coli

Paul L. Sorgen, Michael R. Bubb, Brian D. Cain

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58 Scopus citations

Abstract

In Escherichia coli F1F0 ATP synthase, the two b subunits dimerize forming the peripheral second stalk linking the membrane F0 sector to F1. 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 routants with insertions of up to 14 amino acids retained biologically significant levels of activity. Membranes prepared from these strains had readily detectable levels of F1F0-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 FiFo ATP synthase function. Assuming the predicted α- 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 Å. 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.

Original languageEnglish (US)
Pages (from-to)36261-36266
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number51
DOIs
StatePublished - Dec 17 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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