title: Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an alpha1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide.
abstract: The biosynthesis of asparagine-linked glycans occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize Alg11 from yeast as a mannosyltransferase catalysing the sequential transfer of two alpha1,2-linked mannose residues from GDP-mannose to Man3GlcNAc2-PP-Dol and subsequently to Man4GlcNAc2-PP-Dol forming the Man5GlcNAc2-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic transmembrane-spanning helices. Using Alg11 topology reporter fusion constructs, we show that only the N-terminal domain fulfils this criterion. Surprisingly, this domain can be deleted without disturbing glycosyltransferase function and membrane association, indicating also that the other two hydrophobic domains contribute to ER localization, but in a non-transmembrane manner. By site-directed mutagenesis we investigated amino acids important for transferase activity. We demonstrate that the first glutamate residue in the EX7E motif, conserved in a variety of glycosyltransferases, is more critical than the second, and loss of Alg11 function occurs only when both glutamate residues are exchanged, or when the mutation of the first glutamate residue is combined with replacement of another amino acid in the motif. This indicates that perturbations in EX7E are not restricted to the second glutamate residue. Moreover, Gly85 and Gly87, within a glycine-rich domain as part of a potential flexible loop, were found to be required for Alg11 function. Similarly, a conserved lysine residue, Lys319, was identified as being important for activity, which could be involved in the binding of the phosphate of the glycosyl donor.
authors.affiliation: Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93053 Regensburg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Absmanner
authors.forename: Verena
authors.initials: V
authors.lastname: Schmeiser
authors.forename: Michael
authors.initials: M
authors.lastname: Kämpf
authors.forename: Ludwig
authors.initials: L
authors.lastname: Lehle
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008364:Mannosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D008070:Lipopolysaccharides; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C023023:lipid-linked oligosaccharides; C547472:Alg11 protein, S cerevisiae; D008364:Mannosyltransferases