A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro

A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein... The central reaction in the process of N‐linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc3Man9GlcNAc2 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Wiley

A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro

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Publisher
Wiley
Copyright
Copyright © 2013 Wiley Periodicals, Inc
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1093/emboj/16.6.1164
pmid
9135133
Publisher site
See Article on Publisher Site

Abstract

The central reaction in the process of N‐linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc3Man9GlcNAc2 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.

Journal

The EMBO JournalWiley

Published: Mar 15, 1998

Keywords: ; ; ; ;

References

  • Functional characterization of Ost3p. Loss of the 34‐kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates
    Karaoglu, D; Kelleher, DJ; Gilmore, R
  • Assembly of asparagine‐linked oligosaccharides
    Kornfeld, R; Kornfeld, S
  • Methods in Yeast Genetics: A Laboratory Course Manual
    Rose, M; Winston, F; Hieter, P
  • Nuclear pre‐mRNA splicing in yeast
    Woolford, J

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