gamma-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.
Mehdi
K
K
Université Libre de Bruxelles, Laboratoire de Physiologie et d'Ecologie Microbiennes, Institut Pasteur de Bruxelles, 642 Rue Engeland, B-1180 Brussels, Belgium.
Thierie
J
J
Penninckx
M J
MJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D008954:Models, Biological; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C089179:YCF1 protein, S cerevisiae; D005723:gamma-Glutamyltransferase; D005978:Glutathione