A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro.
The central reaction in the process of N-linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc(3)Man(9)GlcNAc(2) from the lipid dolicholpyrophosphate to selected asparagine residues, is catalyzed by the oligosaccharyltransferase (OTase). This enzyme consists of multiple subunits; however, purification of the complex has revealed different results with respect to its protein composition. To determine how many different loci are required for OTase activity in vivo, we performed a novel, specific screen for mutants with altered OTase activity. Based on the synthetic lethal phenotype of OTase mutants in combination with a deficiency of dolicholphosphoglucose biosynthesis which results in non-glucosylated lipid-linked oligosaccharide, we identified seven complementation groups with decreased OTase activity. Beside the known OTase loci, STT3, OST1, WBP1, OST3, SWP1 and OST2, a novel locus, OST5, was identified. OST5 is an intron-containing gene encoding a putative membrane protein of 9.5 kDa present in highly purified OTase preparations. OST5 protein is not essential for growth but its depletion results in a reduced OTase activity. Suppression of an ost1 mutation by overexpression of OST5 indicates that this small membrane protein directly interacts with other OTase components, most likely with Ost1p. A strong genetic interaction with a stt3 mutation implies a role in complex assembly.
Reiss
G
G
Mikrobiologisches Institut, ETH Zürich, Switzerland.
te Heesen
S
S
Gilmore
R
R
Zufferey
R
R
Aebi
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D006602:Hexosyltransferases; D007438:Introns; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013489:Suppression, Genetic; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004271:DNA, Fungal; D008565:Membrane Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase