CYTOSOLIC AND PLASTID FORMS OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE IN EUGLENA-GRACILIS ARE DIFFERENTIALLY EXPRESSED DURING LIGHT-INDUCED CHLOROPLAST DEVELOPMENT

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5. 1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glyc ine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein, The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the prot ein and mRNA levels during light-induced chloroplast development, as d emonstrated by the determination of their enzyme activities after non- denaturing polyacrylamide gel electrophoresis and Northern hybridizati on analysis with a Saccharomyces cerevisiae ARO1 gene probe. The al om protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPS P synthase and its mRNA are induced by light, the increase in mRNA abu ndance preceding accumulation of the protein. The two enzymes are loca lized in different subcellular compartments, as demonstrated by compar ing total protein patterns with those of isolated organelles. Glyphosa te-adapted wild-type cells and glyphosate-tolerant cells of a plastid- free mutant of E. gracilis, W(10)BSmL, were used for organelle isolati on and protein extraction, as these cell lines overproduce EPSP syntha se and the al om protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartment alization of the monofunctional EPSP synthase. These conclusions are f urther supported by the observation that EPSP synthase precursor, prod uced by in vitro translation of the hybrid-selected mRNA, was efficien tly taken up and processed to mature size by isolated chloroplasts fro m photoautotrophic wild-type E. gracilis cells, while the in vitro-syn thesized arom protein was not sequestered by isolated Euglena plastids .