Overexpression of HUT1 gene stimulates in vivo galactosylation by enhancing UDP-galactose transport activity in Saccharomyces cerevisiae.
Transfer of activated sugar-nucleotides from the cytoplasm to the lumen of the Golgi is an essential requirement for glycosylation of glycoproteins, proteoglycans and glycosphingolipids. Although mannosylation is the major modification in the yeast Saccharomyces cerevisiae, several reports suggest the presence of galactose residues on yeast proteins and sphingolipids. We have detected alpha-galactosylated O-linked chitinase by lectin blotting from cells that functionally express the gma12(+) gene, encoding alpha 1,2-galactosyltransferase from Schizosaccharomyces pombe. This result implies the presence of a UDP-galactose transporter in S. cerevisiae. A conserved gene, HUT1, which encodes a putative multi-transmembrane protein, was cloned and characterized for its possible involvement in galactosylation. The HUT1 gene is not essential and is expressed at a relatively low level under the physiological conditions we examined. The disruption of this gene did not show any apparent impairments in glycosylation. However, a temperature- and concentration-dependent increase in UDP--galactose transport activity was detected from cells overexpressing HUT1 in the presence of gma12(+). The surface of these cells was confirmed to carry galactose residues by staining with FITC-conjugated alpha-galactose-specific lectin. These results suggest a role for Hut1p in the transport of UDP--galactose from the cytosol into the Golgi lumen in S. cerevisiae.
Department of Molecular Biology, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305-8566, Japan.
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Kainuma
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Chiba
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Takeuchi
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Jigami
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002688:Chitinases / Q000378:metabolism; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D033704:Nucleotide Transport Proteins; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D000099237:UDP-Galactose Translocators
D002688:Chitinases; D005700:Galactosyltransferases; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033704:Nucleotide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D000099237:UDP-Galactose Translocators; C433884:HUT1 protein, S cerevisiae; C117616:gma12 protein, S pombe