5- enol Pyruvylshikimate-3-phosphate synthase (EPS P synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate ( N -phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPS P synthase from Petunia hybrida . DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPS P synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5′ open reading frame of the cDNA, the transit peptide of pre-EPS P synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPS P synthase into chloroplasts in vitro , we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for in vitro translation. The translation products, when analyzed by NaDodSO 4 /PAGE autoradiography, indicate a relative molecular mass for pre-EPS P synthase of ≈55 kDa. Uptake studies with intact chloroplasts, in vitro , indicate that pre-EPS P synthase was rapidly taken up into chloroplasts and proteolytically cleaved to the mature ≈48-kDa enzyme. The transit peptide was shown to be essential for import of the precursor enzyme into the chloroplast. To our knowledge, post-translational import into chloroplasts of a precursor enzyme involved in amino acid biosynthesis has not been reported previously. Furthermore, enzymatic analysis of translation products indicates that pre-EPS P synthase is catalytically active and has a similar sensitivity to the herbicide glyphosate as the mature enzyme. To our knowledge, pre-EPS P synthase represents the only example of a catalytically competent chloroplast-precursor enzyme.

Della-Cioppa, G., Bauer, S. C., Klein, B. K., Shah, D. M., Fraley, R. T., & Kishore, G. M. (1986). Translocation of the precursor of 5- enol pyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proceedings of the National Academy of Sciences, 83(18), 6873–6877.