γ-glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione

In the yeast Saccharomyces cerevisiae, the enzyme gamma -glutamyl transpeptidase (gamma -GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma -GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of gamma -GT displayed a K-m (app) of 18 +/- 2 mM and a V-max (app) of 48.5 +/- 5 nmol of GSH/min per mg of protein. Vacuoles containing gamma -GT displayed practically the same K-m,but a higher V-max (app) (150 +/- 12 nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking gamma -GT showed no increase in V-max (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H+) ATPase-coupled system. The V-max (app)-increasing effect of gamma -GT concerns only the YCF1 system. gamma -GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma -GT was also measured. It is proposed that gamma -GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-Cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying gamma -GT physiology and GSH metabolism.