Defining and Manipulating the Fate of Glutathione and Glutathione S-Conjugates

Rutgers University New Brunswick, New Brunswick NJ

Investigators

Abstract

This project focuses on the hydrolysis of glutathione (gamma-Glu-Cys-Gly) and glutathione-S-conjugates in plants, a poorly understood pathway that likely plays a central role in at least two important physiological processes. The first is detoxification of xenobiotics (e.g., herbicides), initiated when they are conjugated with glutathione. Glutathione-S-conjugates are processed after their formation. However, the processing pathway and enzymes are not known with certainty, nor is it known what role processing plays in xenobiotic detoxification. Similarly, although glutathione has been hypothesized to be a major transport/storage form of cysteine in plants, how cysteine is released from glutathione is not well understood. Cleavage of the unique gamma-glutamyl bond of glutathione by a gamma-glutamyl transpeptidase may be the first step in degradation of glutathione and modification of glutathione-S-conjugates. Arabidopsis thaliana contains four putative gamma-glutamyl transpeptidase genes, GGT1, GGT2, GGT3 and GGT4. GGT1 and GGT2 share high coding-sequence homology and exon-intron structure. GGT3 differs markedly from GGT1 and GGT2 in that it lacks the exons encoding the amino terminal ~half of the proteins. GGT4 shares less than 60% coding sequence homology with the other three GGT genes. The functional roles of the structurally different GGT genes and the function of the GGT proteins are the foci of this research. The catalytic properties of recombinant GGT proteins will be studied to determine how the missing amino terminal domain of GGT3 and the divergent coding sequence of GGT4 affect enzymatic function. Transgenic Arabidopsis expressing GGT constructs carrying a tagged sequence will be used to determine whether the amino terminal domain of GGT3 affects its subcellular compartmentation. The hexa-His sequence will also serve as a tag for purification of individual GGT proteins from the plant as a means to explore the subunit composition of gamma-glutamyl transpeptidase. The physiological role of GGT proteins will be explored using two complementary strategies. First, spatial and temporal expression will be studied to determine in which tissues and subcellular compartments the enzymes are expressed. Then, as time and funding permit, the consequence of altering the expression of individual GGT's in transgenic Arabidopsis will be studied. This research should increase our understanding of how plants process foreign compounds such as herbicides and how they maintain the proper internal balance of nutrients.