Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli.

The F1F0 H+-ATPase in membranes of Escherichia coli was amplified by heat induction of a lysogenic lambda-unc+ transducing phage. Inverted membrane vesicles were stripped of the F1 sector of the ATPase complex by washing with EDTA. The stripped membranes were treated with dithiobis(succimidylpropionate) to cross-link subunits of the F0 sector of the ATPase complex. After electrophoresis under nonreducing conditions in one dimension, cross-linked subunits were identified by off-diagonal electrophoresis in a second dimension following cleavage of the cross-linked products with beta-mercaptoethanol. A psi-psi dimer was the major cross-linked product identified. In addition, a chi-psi product and chi-psi2 product were identified. These results support the proposed chi-psi2 stoichiometry of subunits in F0. When the F1-stripped membranes were treated with trypsin, the psi subunit was rapidly degraded, whereas psi was protected from degradation when F1 was bound to the membrane. Trypsin-treated, stripped membranes, lacking an intact psi subunit, did not bind the F1 portion of the ATPase with high affinity. However, these trypsin-treated stripped membranes remained as permeable to protons as untreated stripped membranes, and the H+ conductivity was blocked by dicyclohexylcarbodiimide. These results indicate that the portion of the psi subunit exposed on the cytoplasmic face of the inner membrane is involved in the binding of the F1 portion of the ATPase, but is not necessary for H+ conduction mediated by the F0 sector of the complex.