Cross‐reconstitution of the FOF1‐ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit δ

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Abstract
FoF1-ATP synthases catalyse ATP formation from ADP And P1 by using the free energy supplied by the transmembrane electrochemical potential of the proton. The .delta. subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0. We wanted to know whether or not .delta. of one species was effective between F0 and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1 (-.delta.), and .delta. were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-.delta.) + chloroplast.delta., EF1, EF1 (-.delta.) + E. coli .delta. were also effective but to lesser extent. CF1 (-.delta.) + E. coli .delta. and EF1(-.delta.) + chloroplast .delta. restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1 (-.delta.) + chloroplast .delta. and CF1 (-.delta.) + E. coli .delta. gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1 (-.delta.) + E. coli .delta. by energization of the vesicles with NADH, while EF1 (-.delta.) + chloroplast .delta. was ineffective. All ''mixed'' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed Fo (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning a view of the weak sequence similarity between chloroplast .delta. and E. coli .delta.. It favors a role of .delta. as a conformational transducer rathern than as a proton conductor between F0 and F1.

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