Tetsuya Nohara

Isolation and characterization of the cDNA for pea chloroplast SecA Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts

We report here the isolation of the cDNA for pea chloroplast SecA. Pea SecA encodes a polypeptide of 1,011 amino acids and shows high sequence similarity with cyanobacterial SecA. Pea SecA was synthesized as a larger precursor and was imported into isolated chloroplasts in vitro. The purified pea SecA, which was expressed in Escherichia coli cells, stimulated the in vitro import of the 33 kDa protein of the oxygen‐evolving complex into thylakoids. These results indicate that higher plant chloroplasts contain a bacterial‐type SecA protein‐dependent system for the intraorganellar protein transport into thylakoids.

Sec-dependent Pathway and ΔpH-dependent Pathway Do Not Share a Common Translocation Pore in Thylakoidal Protein Transport

Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the ΔpH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the ΔpH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the ΔpH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the ΔpH-dependent pathway.

Cloning and Characterization of Chloroplast Sec-Y Proteins

The membrane structures of chloroplasts resemble those of cyanobacteria. They are surrounded by double-membrane envelope and have internal thylakoids. However, many chloroplast proteins are encoded by the nuclear genome, are synthesized outside the chloroplasts, and are imported into chloroplasts [1]. Once inside the chloroplasts, proteins are transported to their final destinations such as thylakoids [1, 2]. Since this intra-chloroplast protein transport resembles the intracellular protein transport in cyanobacteria which is facilitated by the bacterial “Sec” system [3–6], characterization of the chloroplast version of the Sec system in intraorganellar protein transport attracted much attention [7, 8]. Previously, we isolated pea SecA cDNA and A rabidopsis thaliana SecY cDNA, and demonstrated that recombinant SecA stimulates the transport of 33 kDa protein in oxygen harvesting PSII complex into thylakoids [7, 9, 10].