A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP

Abstract

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30 degrees C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M(r) of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.