Reconstitution of photophosphorylation with coupling factor 1 ATPases from the thermophilic bacterium PS3 and lettuce chloroplasts

(1) EDTA-resolved particles (partially depleted of CF₁) from lettuce chloroplast thylakoids were reconstituted with CF₁ from lettuce and with TF₁ from the thermophilic bacterium PS3. The binding affinity of both F₁ proteins for the membranal CF₀ is similar. (2) The phosphorylation activities of the EDTA-resolved particles after reconstitution with either the tentoxin-sensitive CF₁ or the tentoxin-resistant TF₁ are equally inhibited by tentoxin. The same degree of reconstitution of photophosphorylation is observed either with the native TF₁, the DCCD-modified TF₁ or the DCCD-modified CF₁. Particles reconstituted with native CF₁ always catalyzed a higher rate of ATP formation. (3) NaBr-resolved particles (CF₁-depleted) bind TF₁ with the reconstitution of the light-dependent H⁺ uptake. However, no significant enhancement of ATP formation accompanies the binding of TF₁ to the membranal CF₀. (4) These findings suggest a close relationship between the F₀-F₁ complex of the prokaryotic thermophilic bacterium PS3 and that from lettuce, a eukaryotic organism. It is also apparent that the rebinding of added TF₁ or CF₁ to CF₁-depleted particles reforms ATP synthetase complexes that are not fully catalytically functional. In particular, the TF₁ incorporated into the membrane appears to have only a structural role of sealing the membrane and preventing protons from leaking out but does not participate in the catalytic process itself. The endogenous CF₁ that is present in the native undissociated ATP synthetase complex and still remains in these CF₁-partially depleted membranes is the catalytically active enzyme species.