Asparagine-Linked Glycosylation in Schizosaccharomyces pombe: Functional Conservation of the First Step in Oligosaccharide-Lipid Assembly

Jun Zou, Jane R. Scocca, Sharon 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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Keywords

  • Hamster
  • N-acetylglucosaminyl-PP-dolichol
  • Saccharo-myces cerevisiae
  • Schizosaccharomyces pombe

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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