CYTOSOLIC AND PLASTID FORMS OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE IN EUGLENA-GRACILIS ARE DIFFERENTIALLY EXPRESSED DURING LIGHT-INDUCED CHLOROPLAST DEVELOPMENT
C. Reinbothe et al., CYTOSOLIC AND PLASTID FORMS OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE IN EUGLENA-GRACILIS ARE DIFFERENTIALLY EXPRESSED DURING LIGHT-INDUCED CHLOROPLAST DEVELOPMENT, MGG. Molecular & general genetics, 245(5), 1994, pp. 616-622
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
.