Deletions in the Second Stalk of F1F0-ATP Synthase in Escherichia coli

• Published in: The Journal of biological chemistry 1998-10, Vol.273 (43), p.27873-27878
• Main Authors: Sorgen, Paul L., Caviston, Tamara L., Perry, Regina C., Cain, Brian D.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proton-Translocating ATPases; Base Sequence; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins; Membranes - enzymology; Models, Theoretical; Molecular Sequence Data; Mutagenesis; Permeability; Proton Pumps; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Sequence Deletion
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.43.27873

In Escherichia coliF1F0-ATP synthase, the two bsubunits 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 theuncF(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 α-helical conformation for this area of the b subunit, the 11-amino acid deletion would result in shortening the subunit by approximately 16 Å. Since these deletions did not prevent the b subunit from participating in productive interactions with F1, we suggest that theb subunit is not a rigid rodlike structure, but has an inherent flexibility compatible with a dynamic role in coupling.