Translocation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase precursor into isolated chloroplasts

Abstract

A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) 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 [(3)H]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.