CHLOROPLAST SECA FUNCTIONS AS A MEMBRANE-ASSOCIATED COMPONENT OF THE SEC-LIKE PROTEIN TRANSLOCASE OF PEA-CHLOROPLASTS

Protein cross-linking studies with a thylakoid membrane translocation intermediate were used to de monstrate that chloroplast SecA functions as a membrane-associated component of the Sec-like ATP-dependent prot ein translocase of pea chloroplasts, In assays with isolated thylakoid s, it was observed that translocation of the 33-kDa protein of the oxy gen-evolving complex of photosystem II (OE33) decreased when the ATP c oncentration was low, and that the protein accumulated as a bound prec ursor. The bound precursor was able to be translocated into the lumen when the ATP concentration was raised, indicating that the precursor w as bound to the translocation apparatus. Inclusion of apyrase in the i mport reaction prevented translocation but did not affect precursor bi nding to the membrane. When this translocation intermediate was treate d with the cross-linking agent disuccinimidyl suberate. a single predo minant crosslinked product of 120 kDa was produced, This conjugate cou ld be immunoprecipitated with antibodies to pea chloroplast SecA, iden tifying the cross-linking partner as SecA. This provides direct eviden ce for a functional interaction between a thylakoid precursor protein and a component of the thylakoid protein-translocation apparatus.