Saccharomyces cerevisiae VIG9 Encodes GDP-mannose Pyrophosphorylase, Which Is Essential for Protein Glycosylation

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 GDPmannose. We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli.Most secretory and membrane proteins of eukaryotic cells are modified by glycosylation. Glycosylation endows protein with a wide variety of characteristics that are important in their cellular functions. Glycosylation occurs in the secretory pathway, and three types of modification are known: (i) attachment of N-linked saccharides to asparagine residues; (ii) attachment of O-linked saccharides to serine or threonine residues; and (iii) attachment of glycosylphosphatidylinositol anchors at the COOH termini. This is also the case with the yeast Saccharomyces cerevisiae, although mannose is the main component of N-and O-linked saccharides, and varieties of sugars are found in those of higher eukaryotes. In yeasts, the fourth type of protein-saccharide linkage was suggested for the covalent linkage between the cell wall proteins and glucans, although details are yet to be studied (1). Several excellent reviews on glycosylation in yeasts have been published (2)(3)(4)(5).N-Linked saccharides are first transferred to proteins as a Glc 3 Man 9 GlcNAc 2 unit from the Dol 1 -PP-intermediate when proteins are translocated into the endoplasmic reticulum. They are then trimmed to form the core N-linked saccharide, Man 8 GlcNAc 2 , which is common in eukaryotes. After proteins are transported from the endoplasmic reticulum to the Golgi apparatus in S. cerevisiae, core N-linked saccharides receive various quantities of mannose, depending on the protein species, one by one from GDP-Man. O-Linked saccharides that are composed exclusively of mannose in S. cerevisiae are also 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. As mannose is the major component of both N-and O-linked saccharides in S. cerevisiae, formation of GDP-Man, the activated form of mannose, should be vitally important.In S. cerevisiae, genetic approaches have been utilized in many scientific studies. To study the mechanism and function...

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

“…2). The Vig9 protein has been demonstrated to have GTP:␣-D-mannose-1-phosphate guanylyltransferase (MPG; EC 2.7.7.13) activity in vitro (19). CYT1 also shows similarity to bacterial MPGs and to the sequence of an N-terminal peptide from the small subunit of porcine MPG (data not shown).…”

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

“…Mutations that block early steps in the assembly of core glycans (e.g. alg1, alg2, alg4, and vig9) are lethal (40,41) as is the knock-out mutation of the yeast PMM gene SEC53 (6). Furthermore, glycosylation mutants with defects in the oligosaccharyltransferase protein complex were reported to undergo programmed cell death (42,43).…”

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins

“…Since then, the enzyme has been purified from a number of organisms and tissues such as Pseudomonas aeruginosa [19], Mycobacterium smegmatis [20], mammary gland [21] and pig liver [22]. The gene for this enzyme was cloned from Saccaromyces cerevisiae and expressed in Escherichia coli cells [23]. Interestingly, GMPP from P. aeruginosa is a bifunctional enzyme that has both GMPP and phosphomannose isomerase activities [19].…”