Abstract
In this report, we extend our previous characterization of Dictyostelium discoideum glutathione S-transferase (DdGST) enzymes that are expressed in the eukaryotic model organism. Transcript profiling of gstA1-gstA5 (alpha class) genes in vegetative, log phase cells identified gstA2 and gstA3 with highest expression (6-7.5-fold, respectively) when compared to other gstA transcripts. Marked reductions in all gstA transcripts occurred under starvation conditions, with gstA2 and gstA3 exhibiting the largest decreases (-96% and -86.6%, respectively). When compared to their pre-starvation levels, there was also a 60 percent reduction in total GST activity. Glutathione (GSH) pull-down assay and mass spectroscopy detected three isozymes (DdGSTA1, DdGSTA2 and DdGSTA3) that were predominantly expressed in vegetative cells. Biochemical and kinetic comparisons between rDdGSTA2 and rDdGSTA3 shows higher activity of rDdGSTA2 to the CDNB (1-chloro-2,4-dinitrobenzene) substrate. RNAi-mediated knockdown of endogenous DdGSTA2 caused a 60 percent reduction in proliferation, delayed development, and altered morphogenesis of fruiting bodies, whereas overexpression of rDdGSTA2 enzyme had no effect. These findings corroborate previous studies that implicate a role for phase II GST enzymes in cell proliferation, homeostasis, and development in eukaryotic cells.