Asparagine-linked glycosylation in Schizosaccharomyces pombe: functional conservation of the first step in oligosaccharide-lipid assembly.
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 lambda 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.
Zou
J
J
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Scocca
J R
JR
Krag
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009844:Oligosaccharides / Q000378:metabolism*; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D004271:DNA, Fungal; D009844:Oligosaccharides; D012333:RNA, Messenger; D014415:Tunicamycin; D001216:Asparagine; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase