The regulatory functions of the γ and ε 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 ε (ε(c)) of chloroplast coupling factor (CF1) 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 F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γ(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity og this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in γ(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant ε(c) and ε subunit from TF1 (ε(t)) on α3β3γ(c) were also investigated. ε(c) strongly inhibited the ATP hydrolysis activity of chimeric α3β3γ(c) complex but ε(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of ε(c) or ε(t) to the α3β3γ(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of γ, can be transferred to the bacterial subunit complex; (b) the interaction between the γ(c) subunit and ε(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core.