THE YEAST CWH41 GENE ENCODES GLUCOSIDASE-I

N-Glycosylation in the yeast Saccharomyces cerevisiae entails the synt hesis of a Glc(3)Man(9)GlcNAc(2) oligosaccharide precursor which is su bsequently transferred to suitable protein accepters, a process which is conserved among all eukaryotes, Processing of the oligosaccharide o ccurs immediately following this transfer, the first step being the re moval of the terminal alpha-1,2-linked glucose by glucosidase I in the endoplasmic reticulum, Although yeast glucosidase I has been isolated , the yeast gene encoding this enzyme has not yet been identified, In the present work, it is shown that Cwh4lp, a yeast endoplasmic reticul um protein previously identified as being required for normal cell wal l beta-1,6-glucan synthesis (Jiang, Sheraton, Ram, Dijkgraaf, Klis, an d Bussey (1996) J, Bacteriol,, 178, 1162-1171), has significant amino acid similarity to the product of the human glucosidase I cDNA, Tetrad analysis for glucosidase I activity ill vitro and in vivo was done on the progeny from the spores obtained from the heterozygous diploid, c wh4l Delta::HIS3. It is shown that, unlike CWH41 cells, cell extracts obtained from cwh41 Delta null mutants are unable to release glucose r esidues from the synthetic trisaccharide substrate alpha-D-Glc 1-->2 a lpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)(8) COOCH3, in vitro, Following 1 h labeling of cells with [H-3]mannose, analysis by high pressure liquid chromatography of the labeled N-linked oligosaccharides, combined wit h treatment with jack bean or mannosidase and yeast glucosidase I, sho ws that the oligosaccharides isolated from a cwh41 Delta null mutant a re fully glucosylated, retaining the three terminal glucose residues, whereas the oligosaccharides from CWH41 cells do not have any glucose residues, These results showing a lack of glucosidase I activity in cw h41 Delta null mutants both in vitro and in vivo are consistent with t he structural evidence that CWH41 encodes the yeast glucosidase I.