The genetic interaction of kar2 and wbp1 mutations. Distinct functions of binding protein BiP and N-linked glycosylation in the processing pathway of secreted proteins in Saccharomyces cerevisiae.
The endoplasmic binding protein BiP and N-linked glycosylation are proposed to be essential components in the processing pathway of secreted protein. In Saccharomyces cerevisiae, BiP is encoded by the KAR2 gene; WBP1 encodes an essential component of the N-oligosaccharyltransferase complex. wbp1 mutations result in reduced oligosaccharyltransferase activity and a temperature-sensitive phenotype. We show that a combination of kar2 and wbp1 mutations results in a synthetic phenotype with a strongly reduced growth rate at the permissive temperature. To investigate the role of N-linked glycosylation in BiP function, the processing of non-glycosylated carboxypeptidase was followed in different kar2 strains at the permissive temperature. In all kar2 strains, the processing of non-glycosylated carboxypeptidase Y was drastically reduced. A specific BiP/non-glycosylated carboxypeptidase Y complex was detected in kar2-159 and kar2-203 cells whereas the kar2-1 mutation did not result in such a complex. Our data show that BiP and N-linked glycosylation are directly involved in the processing of secreted proteins. The results support the hypothesis that BiP stabilizes the folding-competent and assembly-competent state of a polypeptide, whereas N-linked oligosaccharides are structural components required in the folding process after the polypeptide is released from BiP.
te Heesen
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Institut für Molekularbiologie I, Universität Zürich, Switzerland.
Aebi
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D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005800:Genes, Fungal; D005838:Genotype; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014166:Transferases / Q000378:metabolism*
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008565:Membrane Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase