The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.
Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655-0103.
S
S
Silberstein
P G
PG
Collins
D J
DJ
Kelleher
P J
PJ
Rapiejko
R
R
Gilmore
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001483:Base Sequence; D005656:Fungal Proteins / Q000302:isolation & purification; D005800:Genes, Fungal / Q000235:genetics*; D005804:Genes, Lethal / Q000235:genetics; D015698:Genomic Library; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008565:Membrane Proteins; D009844:Oligosaccharides; C034241:ribophorin; D001216:Asparagine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase