The regulatory functions of the gamma and epsilon subunits from chloroplast CF1 are transferred to the core complex, alpha3beta3, from thermophilic bacterial F1.
The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(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 gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(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 CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated. Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(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 epsilon(c) or epsilon(t) to the alpha3beta3gamma(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 gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.
Hisabori
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan. thisabor@res.titech.ac.jp
Kato
Y
Y
Motohashi
K
K
Kroth-Pancic
P
P
Strotmann
H
H
Amano
T
T
D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D004789:Enzyme Activation; D000432:Methanol / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018724:Spinacia oleracea / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases; D000432:Methanol