Essential aspartate in subunit c of F1F0 ATP synthase. Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-1.
Subunit c of the F1F0 type, H(+)-transporting ATP synthase contains an essential Asp that is thought to function in H+ transport. Subunit c folds as a helical hairpin of two transmembrane helices with the essential Asp centered at residue 61 in transmembrane helix-2. Miller et al. (Miller, M. J., Olderburg, M., and Fillingame, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4900-4904) have described a functional subunit c variant in which the essential Asp was moved from helix-2 to residue 24 on helix-1 with replacement of Asp61 by Gly. The function of the A24D/D61G subunit c variant is not optimal. In this study, 11 position 61 variants of an A24D subunit c were generated by site-directed mutagenesis in order to test the importance of the position 61 residue. Three functional combinations were found with activities in the order:A24D/D61N > A24D/D61G > or = A24D/D61S. Other substitutions at position 61, including Ala and Cys, did not support function in the A24D protein. Although the A24D/D61N variant showed the highest rates of ATPase-coupled H+ transport, its F0 was inactive in passive H+ transport when F1 was stripped from the membrane. On the other hand, passive H+ transport by A24D/D61G and A24D/D61S stripped membranes approached that of wild type. The defect in function in these two mutants must be ascribed to events related to coupling ATPase and H+ transport. An A24D subunit c (with Asp at both position 24 and 61) was also generated. Its function proved to be pH-dependent. Activity approaching that of wild type was observed at pH 7.0, but function was almost completely lost at pH 7.8. The pH-dependent loss of ATP synthase function led to a slowing of growth on succinate as carbon source on raising the pH from 7.0 to 7.8. In the A24D mutant, with a second Asp at position 61, we postulate that 1 Asp must be protonated before the other can function in H+ transport.
Zhang
Y
Y
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706.
Fillingame
R H
RH
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism
D017931:DNA Primers; D046911:Macromolecular Substances; D001224:Aspartic Acid; D006180:Proton-Translocating ATPases