Asparagine-Linked Glycosylation in Schizosaccharomyces pombe

Functional Conservation of the First Step in Oligosaccharide-Lipid Assembly

J. Zou, J. R. Scocca, S. S. Krag

Research output: Contribution to journalArticle

Abstract

The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in λ phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S. pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852. The S. pombe protein was 50% identical to the S. cerevisiae protein and 43% identical to the protein from Chinese hamster ovary (CHO) cells. Overexpression of the gpt gene in S. pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe. Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene. The gpt gene is essential for viability of S. pombe. Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S. pombe gpt ORF or the CHO L-G1PT cDNA. The S. pombe gpt gene was mapped to chromosome 2 near top1 and ade1.

Original languageEnglish (US)
Pages (from-to)487-496
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume317
Issue number2
DOIs
StatePublished - Mar 10 1995

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Glycosylation
Schizosaccharomyces
Asparagine
Oligosaccharides
Conservation
Genes
Lipids
Open Reading Frames
Cricetulus
Dolichol Phosphates
Schizosaccharomyces pombe Proteins
Ovary
Saccharomyces cerevisiae Proteins
Tunicamycin
Bacteriophages
Gene encoding
Uridine Diphosphate
Acetylglucosamine
Cell membranes
Chromosomes

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Asparagine-Linked Glycosylation in Schizosaccharomyces pombe : Functional Conservation of the First Step in Oligosaccharide-Lipid Assembly. / Zou, J.; Scocca, J. R.; Krag, S. S.

In: Archives of Biochemistry and Biophysics, Vol. 317, No. 2, 10.03.1995, p. 487-496.

Research output: Contribution to journalArticle

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abstract = "The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in λ phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S. pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852. The S. pombe protein was 50{\%} identical to the S. cerevisiae protein and 43{\%} identical to the protein from Chinese hamster ovary (CHO) cells. Overexpression of the gpt gene in S. pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe. Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene. The gpt gene is essential for viability of S. pombe. Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S. pombe gpt ORF or the CHO L-G1PT cDNA. The S. pombe gpt gene was mapped to chromosome 2 near top1 and ade1.",
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