Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus.
The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites. Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-(3)H]glucosamine and [2-(3)H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.
Takeuchi
K
K
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Yamazaki
H
H
Shiraishi
N
N
Ohnishi
Y
Y
Nishikawa
Y
Y
Horinouchi
S
S
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D001483:Base Sequence; D016368:Frameshift Mutation; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D050356:Lipid Metabolism; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D020103:Rhizomucor / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D014316:Tritium
D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D014316:Tritium; C083733:ALG2 protein, S cerevisiae; D008364:Mannosyltransferases; D016282:Aspartic Acid Endopeptidases; D005944:Glucosamine; D008358:Mannose