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:dolic hol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was i solated by screening a Schizosaccharomyces pombe genomic DNA library i n lambda phage under low-stringency hybridization using the Saccharomy ces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S-pombe DNA re vealed a 1338-bp open reading frame (ORF) encoding a hydrophobic prote in 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. Over expression of the gpt gene in S-pombe cells increased resistance to tu nicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold in crease in poly(A)(+) RNA hybridizing to the gpt probe. Northern analys is 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 e ither the intact S-pombe gpt ORF or the CHO L-G1PT cDNA. The S-pombe g pt gene was mapped to chromosome 2 near top1 and ade1.