DEVELOPMENT OF A PRECURSOR IN A SOLUBLE FORM FOR PROTEIN IMPORT INTO CHLOROPLASTS

Most of chloroplastic proteins are encoded in the nuclear genome. After majority of them are synthesized as precursors in the cytosol, precursors translocate through the outer and inner envelope membranes via translocation machinery (translocon) embedded in these membranes. Our current knowledge of translocon is mainly gained from the early stages of translocation by analysing the protein translocation intermediates (PTIs) formed in vitro under limited energy conditions. Once precursor proteins are released from the PTIs in the presence of a high level of ATP, it is impossible to suspend the movement of precursors until translocation is completed. Thus little is known regarding molecular mechanisms at the latter stages of translocation. However, if precursors are fused with the tightly folded domain to introduce steric hindrance, these precursors may plug the translocon channel under a translocation condition, as a result, PTIs at the latter stage of translocation will be formed. In order to isolate PTIs enough for biochemical analysis, a large quantity of precursors in a soluble form are required. Therefore, we made an attempt to obtain the soluble precursor protein with tightly folded domain by over expressing in Escherichia coli. Because dihydrofolate reductase (DHFR) is tightly folded in the presence of methotrexate (MTX), we designed and prepared the plasmid carrying the gene for the artificial precursor protein consisted with chloroplastic targeting sequence and various tags along with DHFR from E. coli. After over expression in E. coli cells, this precursor was recovered in a soluble fraction. When this precursor was applied for in vitro chloroplastic protein import, the precursor was imported into chloroplasts.