Subunit III of the chloroplast ATP-synthase can form a Ca 2+-binding site on the lumenal side of the thylakoid membrane

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Subunit III, the 8 kDa component of the chloroplast CF 0 H + channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca 2+-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrenedivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca 2+-ligand blot assay known to detect high affinity Ca 2+-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl 2 in the presence of 10-fold excess of MgCl 2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca 2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La 3+. Ca 2+ binding was maximum at pH 7.5–8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivativizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca 2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca 2+-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca 2+-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca 2+-binding site can form on the lumenal side of the membrane in the functional CF 0 structure.