SACCHAROMYCES-CEREVISIAE VIG9 ENCODES GDP-MANNOSE PYROPHOSPHORYLASE, WHICH IS ESSENTIAL FOR PROTEIN GLYCOSYLATION

A genomic DNA fragment that complements a newly identified protein gly cosylation-defective mutation, vig9, of Saccharomyces cerevisiae was c loned. Chromosomal integration of this fragment by homologous recombin ation indicated that it contains the wild type VIG9 gene. The nucleoti de sequence was determined. A predicted gene product showed significan t amino acid sequence homology with several bacterial enzymes that cat alyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from s ugar phosphates and (deoxy)ribonucleotide triphosphate. We examined th e enzyme activity to synthesize GDP-mannose in the cell extracts of th e 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 pyrophospho rylase of S. cerevisiae which catalyzes the production of GDP-mannose. We demonstrated the enzyme activity of Vig9 protein using a recombina nt fusion protein produced in Escherichia coli.