Molecular Dissection of the E Subunit of the Chloroplast ATP Synthase of Spinach ’ Jeffrey

The gene encoding the E subunit (atpf?) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia COK The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain E that i s as biologically active as E purified from chloroplastcoupling factor 1 (CF,). Recombinant E folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF, deficient in e and restores proton impermeability to thylakoid membranes reconstituted with CF, deficient i n E . Site-directed mutagenesis was used to generate truncations and single amino acid substitutions i n the primary structure of E . I n the five mutants tested, alterations that weaken ATPase inhibition by recombinant E affect i ts ability t o restore proton impermeability to a similar extent, wi th one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeabilty. As in the case of E. coli, it appears that N-terminal truncations of the 4 subunit have more profound effects than C-terminal deletions on the function of E. Recombinant E with six amino acids deleted from the C terminus, which i s the only region of significant mismatch between the E of spinach and the E of Pisum sativum, inhibits ATPase activity with a reduced potency similar t o that of purified pea E . Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in E-CF, interactions.