Translocation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase precursor into isolated chloroplasts
- 46 Downloads
A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 126.96.36.199) from potato (Solanum tuberosum L.) presumably specifies a chloroplast transit sequence near its 5′-end. In order to show the function of this transit sequence, we constructed a plasmid that contains the entire coding region of the cDNA downstream from a T7 promoter. Using this plasmid as template, DAHP synthase mRNA was synthesized in vitro with T7 RNA polymerase. The resulting mRNA served as template for the in vitro synthesis of a 59-kDa polypeptide. This translation product was identified as the DAHP synthase precursor by immunoprecipitation with a monospecific polyclonal antibody raised against pure tuber DAHP synthase and by radiosequencing of the [3H]leucine-labeled translation product. Incubation of the 59-kDa polypeptide with isolated spinach (Spinacia oleracea L.) chloroplasts resulted in a 53-kDa polypeptide that was resistant to protease treatment. Fractionation of chloroplasts, reisolated after import, showed the mature DAHP synthase in the stroma fraction. Incubation of the 59-kDa polypeptide with a chloroplast precursor-processing enzyme cleaved the precursor between Ser49 and Ala50, generating a mature DAHP synthase of 489 residues. The uptake of the DAHP synthase precursor into isolated chloroplasts was inhibited by anti-DAHP synthase, and the precursor was not processed cotranslationally by canine microsomal membranes. We conclude that the transit sequence is able to direct DAHP synthase into chloroplasts.
Unable to display preview. Download preview PDF.