Abstract
Glutathionylation of compounds is an important reaction in the detoxification of electrophilic xenobiotics and in the biosynthesis of endogenous molecules. The glutathione conjugates (GS conjugates) are further processed by peptidic cleavage reactions. In animals and plants, gamma-glutamyl transpeptidases initiate the turnover by removal of the glutamate residue from the conjugate. Plants have a second route leading to the formation of gamma-glutamylcysteinyl (gamma-GluCys) conjugates. Phytochelatin synthase (PCS) is well known to mediate the synthesis of heavy metal-binding phytochelatins. In addition, the enzyme is also able to catabolize GS conjugates to the gamma-GluCys derivative. In this study, we addressed the cellular compartmentalization of PCS and its role in the plant-specific gamma-GluCys conjugate pathway in Arabidopsis (Arabidopsis thaliana). Localization studies of both Arabidopsis PCS revealed a ubiquitous presence of AtPCS1 in Arabidopsis seedlings, while AtPCS2 was only detected in the root tip. A functional AtPCS1:eGFP (enhanced green fluorescent protein) fusion protein was localized to the cytosolic compartment. Inhibition of the vacuolar import of GS-bimane conjugate via azide treatment resulted in both a strong accumulation of gamma-GluCys-bimane and a massive increase of the cellular cysteine to GS-bimane ratio, which was not observed in PCS-deficient lines. These findings support a cytosolic action of PCS. Analysis of a triple mutant deficient in both Arabidopsis PCS and vacuolar gamma-glutamyl transpeptidase GGT4 is consistent with earlier observations of an efficient sequestration of GS conjugates into the vacuole and the requirement of GGT4 for their turnover. Hence, PCS contributes specifically to the cytosolic turnover of GS conjugates, and AtPCS1 plays the prominent role. We discuss a potential function of PCS in the cytosolic turnover of GS conjugates.
Highlights
Glutathionylation of compounds is an important reaction in the detoxification of electrophilic xenobiotics and in the biosynthesis of endogenous molecules
While AtGGT4 plays a role in GS conjugate catabolism in the vacuole, AtGGT1 and AtGGT2 have an active site in the apoplast (Ohkama-Ohtsu et al, 2007a) and could be involved in the secretion of conjugates coupled with the cleavage of the glutamyl moiety from g-GluCys and GS conjugates
Studies with the model xenobiotic monochlorobimane (MCB) suggest that AtPCS1 rather than AtPCS2 is the dominant player, as it has been shown that functional disruption of AtPCS1 but not of AtPCS2 leads to a decrease in the turnover of the fluorescent GS-bimane conjugate to g-GluCys-bimane in Arabidopsis (Blum et al, 2007)
Summary
Glutathionylation of compounds is an important reaction in the detoxification of electrophilic xenobiotics and in the biosynthesis of endogenous molecules. Inhibition of the vacuolar import of GS-bimane conjugate via azide treatment resulted in both a strong accumulation of g-GluCys-bimane and a massive increase of the cellular cysteine to GS-bimane ratio, which was not observed in PCS-deficient lines. These findings support a cytosolic action of PCS. A second mode of GS conjugate catabolism is initiated by the removal of the C-terminal Gly to the corresponding g-GluCys product (Fig. 1) This catabolic step is common for GS conjugate degradation in plants (Lamoureux and Rusness, 1986, 1993). Assessment of the PCS contribution to GS conjugate catabolism has been hampered by the existence of a second GGT-initiated pathway
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