Abstract
A genomic DNA fragment that complements a newly identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae was cloned. Chromosomal integration of this fragment by homologous recombination indicated that it contains the wild type VIG9 gene. The nucleotide sequence was determined. A predicted gene product showed significant amino acid sequence homology with several bacterial enzymes that catalyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from sugar phosphates and (deoxy)ribonucleotide triphosphate. We examined the enzyme activity to synthesize GDP-mannose in the cell extracts of the wild type, vig9-1 mutant, and VIG9 transformant yeasts. Reduction of the activity in the mutant cell and its restoration by VIG9 suggested that the VIG9 gene is the structural gene for GDP-mannose pyrophosphorylase of S. cerevisiae which catalyzes the production of GDP-mannose. We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli.
Highlights
N-Linked saccharides are first transferred to proteins as a Glc3Man9GlcNAc2 unit from the Dol1-PP-intermediate when proteins are translocated into the endoplasmic reticulum
O-Linked saccharides that are composed exclusively of mannose in S. cerevisiae are transferred to proteins in the Golgi apparatus sequentially from GDP-Man except for the first mannose, which is transferred from Man-P-Dol in the endoplasmic reticulum
The mnn mutants were selected based on changes in cell surface characteristics such as binding of dyes, ion exchange matrix, or antisera [2]. [3H]Mannose suicide selection was used to obtain the alg mutants, which are defective in the formation of oligosaccharide-PP-Dol [6], and the och1 mutant [7]
Summary
N-Linked saccharides are first transferred to proteins as a Glc3Man9GlcNAc2 unit from the Dol1-PP-intermediate when proteins are translocated into the endoplasmic reticulum. The erd mutant was obtained by a retention defect of the endoplasmic reticulum resident proteins [11] Studies of these mutants and the cloned wild type genes have been helpful in elucidating the biosynthetic enzymes and intermediate structures of oligosaccharides. To increase the collection of glycosylation-defective mutants, we screened for vanadate-resistant colonies as described previously but not screened exhaustively by Ballou et al [8]. This selection is excellent because mutants are positively selected, the biochemical rationale of enrichment is not clear. We report here the cloning and analysis of VIG9, the structural gene for GDP-Man pyrophosphorylase of S. cerevisiae
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