Abstract
Trace amounts ( approximately 5%) of the chloroplast alpha subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores of Rhodospirillum rubrum with the chloroplast beta subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the alpha subunit in the rebinding of beta, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast alpha and beta subunits. The treated chromatophores were found to have lost, in addition to most of their beta subunits, approximately a third of the alpha subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum alpha and chloroplast beta subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast alpha and beta subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum alpha subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast alpha subunit is required together with the chloroplast beta subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the beta subunit on sensitivity to tentoxin.
Highlights
The photosynthetic F0F1 ATP synthases found in the thylakoids of chloroplasts and in the cytoplasmic membranes of photosynthetic bacteria couple the movement of protons down an electrochemical proton gradient to the synthesis of ATP during photophosphorylation
More recently it was shown that the presence of small amounts of the ␣ subunit was a requirement for the reconstitution of a hybrid ATP synthase with the CF1  subunits [15, 16] or a native enzyme with RrF1  [17, 18], suggesting the possibility that the LiCl treatment removed some of the ␣ subunit from the
LiCl-treated chromatophores could be restored by their reconstitution either with CF1  containing trace amounts (ϳ5%) of CF1 ␣ [14], or with a preparation of CF1 ␣ containing an equimolar ratio of both subunits but not with a highly purified preparation of CF1  [15]
Summary
Materials—CF1 ␣, was isolated as described previously [27]. Recombinant RrF1 ␣ [28] and RrF1  [17] were expressed as inclusion bodies in unc operon-deleted E. coli DK8 or LM3115 strains, respectively, grown to steady state at 37 °C. The pET3a-NE3 plasmid used only for the  mutations contained a modified  subunit gene in which the codon at position 378 (AGG) was modified to UGG, which replaced the arginine with tryptophan This construct was found to increase the yield of folded monomeric  subunits without affecting any of the catalytic properties of the protein.. Washed chromatophores equivalent to 1–3 g of BChl were incubated for 10 min at 35 °C in a 0.5-ml reaction mixture containing 50 mM Tricine-NaOH (pH 8.0), 50 mM NaCl, and either 2 mM MgCl2 and 4 mM ATP, or 5 mM CaCl2 and 5 mM ATP. The residual activity of 80 mol ATP hydrolyzed/h per mg of BChl of the LiCl-treated chromatophores was subtracted from the values shown
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