Cytosolic action of phytochelatin synthase.

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.

Dissection of glutathione conjugate turnover in yeast.

Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a gamma-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a gamma-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the gamma-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the gamma-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced gamma-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway.

Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae.

Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.

YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

In Saccharomyces cerevisiae the pyruvate dehydrogenase (PDH) complex is regulated by reversible phosphorylation of its Pda1p subunit. We here provide evidence that Pda1p is phosphorylated by the mitochondrial kinase Yil042cp. Deletion of YOR090c, encoding a putative mitochondrial phosphatase, results in a decreased PDH activity, indicating that Yor090cp acts as the corresponding PDH phosphatase. We demonstrate by means of blue native gel electrophoresis and tandem affinity purification that both enzymes are associated with the PDH complex.