Escherichia coliF1-ATPase Subunit Interactions: β and γ Subunit Peptides Inhibitin VitroReconstitution of the Active αβγ Complex

For biochemical analysis of subunit interactions in the proton-translocating ATPase, a new approach within vitroreconstitution of theEscherichia coliαβγ complex and the peptides derived from the subunits was established. Various portions of the β or γ subunits were used forin vitroreconstitution of the αβγ complex from the purified subunits. For the β subunits, peptides corresponding to residues 226–459, 254–459, and 226–365 inhibited reconstitution, while those corresponding to residues 1–105, 1–146, and 295–459 did not. For the γ subunits, peptides corresponding to residues 1–192 and 74–286 exhibited inhibitory effect on reconstitution, but the peptide containing residues 191–286 did not. Only inhibitory peptides blocked the assembly of the αβγ complex which was detected by nondenaturing polyacrylamide gel electrophoresis. These inhibitory peptides bound to the α or β subunit on the filter, but the noninhibitory peptides did not. These results suggested that regions β 254–294 and γ 74–190 have sequences important for subunit interactions which interfered with those in the reconstitution mixtures. Based on comparison between X-ray crystallographic data of bovine αβγ complex and the present results, we discussed here the significance of the biochemical approach adopted in this study.