Deletions in the second stalk of F1F0-ATP synthase in Escherichia coli.
In Escherichia coli F1F0-ATP synthase, the two b subunits form the second stalk spanning the distance between the membrane F0 sector and the bulk of F1. Current models predict that the stator should be relatively rigid and engaged in contact with F1 at fixed points. To test this hypothesis, we constructed a series of deletion mutations in the uncF(b) gene to remove segments from the middle of the second stalk of the subunit. Mutants with deletions of 7 amino acids were essentially normal, and those with deletions of up to 11 amino acids retained considerable activity. Membranes prepared from these strains had readily detectable levels of F1-ATPase activity and proton pumping activity. Removal of 12 or more amino acids resulted in loss of oxidative phosphorylation. Levels of membrane-associated F1-ATPase dropped precipitously for the longer deletions, and immunoblot analysis indicated that reductions in activity correlated with reduced levels of b subunit in the membranes. Assuming the likely alpha-helical conformation for this area of the b subunit, the 11-amino acid deletion would result in shortening the subunit by approximately 16 A. Since these deletions did not prevent the b subunit from participating in productive interactions with F1, we suggest that the b subunit is not a rigid rodlike structure, but has an inherent flexibility compatible with a dynamic role in coupling.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
P L
PL
Sorgen
T L
TL
Caviston
R C
RC
Perry
B D
BD
Cain
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D008566:Membranes / Q000201:enzymology; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010539:Permeability; D017494:Proton Pumps; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D017494:Proton Pumps; C074430:uncF protein, E coli; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases