SUBUNIT-III OF THE CHLOROPLAST ATP-SYNTHASE CAN FORM A CA2-BINDING SITE ON THE LUMENAL SIDE OF THE THYLAKOID MEMBRANE()

Subunit III, the 8 kDa component of the chloroplast CF, H+ channel, wa s isolated and purified from pea thylakoids for the purpose of studyin g its Ca2+-binding properties. After n-butanol extraction and ether pr ecipitation, HPLC purification was accomplished using a poly(styrene-d ivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic prot eins of near 4 kDa molecular mass, the psaI and psbK gene products ass ociated with PSI and PSII reaction centers, respectively. Purified sub unit III as well as the unfractionated organic-solvent soluble prepara tion were used in a Ca-45(2+)-ligand blot assay known to detect high a ffinity Ca2+-binding sites in proteins. Polypeptides were separated wi th SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with (CaCl2)-Ca-45, in the presence of 10-fold excess of MgC l2, and 200-fold excess KCl led to the labeling of only the 8 kDa poly peptide. The Ca2+ binding was inhibited after derivatizing aqueously e xposed carboxyl groups with a water soluble carbodiimide plus a nucleo phile, after de-formylation of the N-terminal methionine, or with a su bsequent treatment with La3+ Ca2+ binding was maximum at pH 7.5-8.5 an d was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatme nt (no nucleophile was added) of thylakoid membranes, which derivativi zes the hydrophobically located Glu-61, decreased the electrophoretica l mobility of isolated subunit III but did not inhibit the Ca2+ bindin g. The data indicate that the carbonyl group of the formylated N-termi nal Met-1 and probably the carboxyl group of the subunit III C-termina l Val-81 provide some of seven essential oxygen ligands normally requi red for defining a Ca2+-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptide s is essential for forming the Ca2+ binding site. Based on the accepte d models for the hairpin conformation of the subunit III, it does seem clear that the Ca2+-binding site can form on the lumenal side of the membrane in the functional CF0 structure.