In the yeast Saccharomyces cerevisiae, the enzyme γ-glutamyl transpeptidase (γ-GT; EC 220.127.116.11) 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 Σ1278b, under conditions of γ-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of γ-GT displayed a Km (app) of 18±2mM and a Vmax (app) of 48.5±5nmol of GSH/min per mg of protein. Vacuoles containing γ-GT displayed practically the same Km, but a higher Vmax (app) (150±12nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking γ-GT showed no increase in Vmax (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 Vmax (app)-increasing effect of γ-GT concerns only the YCF1 system. γ-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 γ-GT was also measured. It is proposed that γ-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 γ-GT physiology and GSH metabolism.
- peptide thiol
Abbreviations used: ALP, alkaline phosphatase; Blast, basic local alignment search tool; CGase, l-Cys-Gly dipeptidase; γ-GT, γ-glutamyl transpeptidase; ORF, open reading frame; SBM, standard basal medium; V-ATPase, vacuolar (H+) ATPase; YCF1, yeast cadmium factor 1.
- The Biochemical Society, London ©2001