The UDP-Glc:Glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress.
Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28 degreesC. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28 degreesC and did not grow at 37 degreesC. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37 degreesC and had, when grown at 28 degreesC, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by GT and GII.
Fanchiotti
S
S
Instituto de Investigaciones Bioquímicas Fundación Campomar, 1405 Buenos Aires, Argentina.
Fernández
F
F
D'Alessio
C
C
Parodi
A J
AJ
D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism; D010641:Phenotype; D017510:Protein Folding; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D013696:Temperature
D009844:Oligosaccharides; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase