Subunit III of the chloroplast ATP-synthase can form a Ca2+-binding site on the lumenal side of the thylakoid membrane

Abstract

Subunit III, the 8 kDa component of the chloroplast CF₀ H⁺ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca²⁺-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 ⁴⁵Ca²⁺-ligand blot assay known to detect high affinity Ca²⁺-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with ⁴⁵CaCl₂ in the presence of 10-fold excess of MgCl₂ and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca²⁺ 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 La³⁺. Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca²⁺-binding site. Based on the accepted 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.