The Regulatory Functions of the γ and ɛ Subunits from Chloroplast CF₁ are Transferred to the Core Complex, α₃β₃, from Thermophilic Bacterial F₁

Abstract

The expression plasmids for the subunit γ (γ c ) and the subunit ɛ (ɛ c ) of chloroplast coupling factor (CF 1 ) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γ c was mixed with recombinant α and β subunits of F 1 from thermophilic Bacillus PS3 (TF 1 ), a chimeric subunit complex (α 3 β 3 γ c ) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCI 2 , which induces formation of a disulfide bond between two cysteine residues in γ c . Hence, this complex has similar modulation characteristics as CF 1 . The effects of recombinant ɛ c , and ɛ subunit from TF 1 (ɛ 1 ) on α 3 β 3 γ c were also investigated. ɛ c , strongly inhibited the ATP hydrolysis activity of chimeric α 3 β 3 γ c complex but ɛ 1 did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of ɛ c , or ɛ 1 , to the α 3 β 3 γ t complex, which is the authentic subunit complex from TF 1 , resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of γ c can be transferred to the bacterial subunit complex; (b) the interaction between the γ c subunit and ɛ c , strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α 3 β 3 core.