Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an α1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide

The biosynthesis of asparagine-linked glycans Occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked olicyosaccharide precursor Glc(3)Man(9)GlcNAc-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize AlgI I from yeast as a mannosyltransferase catalysing the sequential transfer of two alpha 1,2-linked mannose residues from GDP-mannose to Man(3)GlcNAc(2)-PP-Dol and subsequently to Man(4)GlcNAc(2)-PP-Dol forming the Man(3)GlcNac(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic trans membrane-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-trans membrane 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, Gly(85) and Gly(87), 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, Lys(319), was identified as being important for activity, which could be involved in the binding of the phosphate of the glycosyl donor.