Localization of a conformational energy-coupling determinant near the C terminus of the beta subunit of the F1F0-ATPase.
Escherichia coli mutants in the beta subunit of the F1F0-ATPase can be complemented with the beta subunit from the obligate aerobe Bacillus megaterium. It has been shown that cells carrying such hybrid ATPases have an unusual energy-coupling phenotype. Although they are able to grow on minimal succinate medium, and therefore carry a functional ATP synthase, they are defective in the ability to grow anaerobically, indicating some defect in ATP-driven proton pumping (Scarpetta, M., Hawthorne, C. A., and Brusilow, W. S. A. (1991) J. Biol. Chem. 266, 18567-18572). In this study, chimeric beta subunits were constructed consisting of the E. coli or the B. megaterium beta subunit carrying the C-terminal 18% of the other's beta subunit. The phenotypes of an E. coli beta mutant complemented with these chimeric subunits showed that the energy-coupling defect was located in this C-terminal region. The E. coli beta subunit carrying the B. megaterium C-terminal region displayed the energy-coupling defect, while the B. megaterium beta subunit carrying the E. coli C-terminal region did not. In ATP-dependent fluorescence quenching assays, membranes isolated from cells displaying the energy-coupling defect also pumped protons less well than membranes isolated from cells that were able to grow anaerobically. These results demonstrate that the C terminus of the beta subunit is involved in the conformational coupling pathway, which, through the polypeptide backbone of the beta subunit, physically links ATP synthesis or hydrolysis to the energy of proton translocation.
Schemidt
R A
RA
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. wbrusil@cms.cc.wayne.edu
Brauning
C K
CK
Bouvier
A
A
Brusilow
W S
WS
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001410:Bacillus megaterium; D004926:Escherichia coli; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010641:Phenotype; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases