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Glycobiology, 1999, Vol. 9, No. 12 1287-1293
© 1999 Oxford University Press

Characterizationof an alg2 mutant of the zygomycetefungus Rhizomucor pusillus

Kyoko Takeuchi, Haruka Yamazaki, Norihiko Shiraishi, Yasuo Ohnishi, Yoshihisa Nishikawa2 and Sueharu Horinouchia

Department of Biotechnology, Graduate School of Agricultureand Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–8657,and 2Department of Industrial Chemistry,School of Engineering, Tokai University, Kitakaname, Hiratsuka-shi,Kanagawa 259–1292, Japan

The zygomycete fungus Rhizomucor pusillus secretesan aspartic proteinase (MPP) that contains asparagine (N)-linked oligosaccharidesat two sites. Mutant strain 1116 defective in N-glycosylationsecretes MPP with truncated oligo­saccharide chains. Lipid-linkedoligosaccharides in mutant 1116 were labeled with [6-3H]glucosamineand [2-3H]mannose, prepared by cyclesof solvent extraction, and analyzed by gel filtration chromatographyon a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116accumulated an intermediate, Man1GlcNAc2-dolicholpyrophosphate (PP-Dol), whereas wild-type strain F27 synthesizedthe fully assembled oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol.Con­sistent with this, alg2 encoding amannosyltransferase in the lipid-linked oligosaccharide biosyntheticpathway in mutant 1116 had a 5 bp insertion that generated a stop codonin the middle of the coding sequence. Transformation of mutant 1116with the intact alg2 gene on a pUC19-derived plasmidgenerated transformants that contained multicopies of alg2 atthe alg2 locus. Glycosylation of the total proteins inthe transformants was recovered to the same level as in strain F27,as determined with peroxidase-concanavalin A. These transformantsproduced MPP mainly with the same N-linked oligosaccharidesas that produced by strain F27, but still with truncated oligosaccharidesin small amounts. All of these data show that Alg2 is an {alpha}-1,3or {alpha}-1,6 mannosyltransferase that elongatesMan1GlcNAc2-PP-Dol to Man2GlcNAc2-PP-Dol.The slower growth of mutant 1116 was significantly recovered onintroduction of alg2. The viability of the alg2 mutantsof the zygomycete R.pusillus makes a contrast withthe lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.

a To whom correspondenceshould be addressed


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 J. Biol. Chem.Home page
M. Kampf, B. Absmanner, M. Schwarz, and L. Lehle
Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional {alpha}1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis
J. Biol. Chem., May 1, 2009; 284(18): 11900 - 11912.
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