Molecular Mechanism of Pre-Plastocyanin Import into Chloroplasts

Chloroplasts are photosynthesizing organelles found in all green plant tissue. Enclosed within the chloroplasts are thylakoids that form a closed internal membrane system. Although the chloroplast DNA encodes some of the proteins required for chloroplast function, many of its proteins are encoded by nuclear DNA, synthesized in the cytosol and post-translationally imported into the organelle. Thylakoid proteins must cross the two chloroplast envelope membranes as well as the thylakoid membrane to reach their functional location. According to the current two step model for thylakoid protein import, proteins first cross the chloroplast envelope into the stroma where a portion of their amino-terminal transit peptide is removed to yield an intermediate form. Subsequently the intermediate translocates across the thylakoid membrane, before it is further processed to its functional mature form. Plastocyanin is a copper binding protein found in the thylakoid lumen. Plastocyanin is involved in photosynthetic electron transport between cytochrome f and the photosystem I reaction center. The apoprotein is nuclear encoded and synthesized as a high molecular weight precursor in the cytoplasm. The newly synthesized plastocyanin precursor is then post-translationally imported into the chloroplast. A chimeric plastocyanin precursor containing six histidine residues at the carboxyl terminus (prPCHT) was constructed and expressed in E. coli in order to study the import pathway of plastocyanin. Following purification by affinity chromatography, the protein was biotinylated at a unique cysteine residue near the carboxyl terminal end of the fusion protein. Labeling the carboxyl terminus of the polypeptide with biotin permitted us to follow the successive changes the protein undergoes during chloroplast protein import. Short-lived intermediates in the maturation of this protein were identified during the course of chloroplast import. These results demonstrated that amino terminal processing of the protein occurred during translocation before the entire protein had entered the chloroplast. At early time points in the import pathway the amino terminus of the biotinylated prPCHT was present in the lumen of the thylakoid allowing processing by the thylakoid protease while its carboxyl terminus with the biotin label remained outside the chloroplast. A novel direct entry model for chloroplast protein import is proposed in which the thylakoid protein prPCHTB traverses the two outer envelopes and the thylakoid membrane simultaneously