Characterization of alg2 encoding a mannosyltransferase in the zygomycete fungus Rhizomucor pusillus.

Abstract

ALG2 of Saccharomyces cerevisiae encodes the glycosyltransferase that mannosylates Man2GlcNAc2-dolichol diphosphate (PP-Dol) and Man1GlcNAc2-PP-Dol to form Man3GlcNAc2-PP-Dol. The genomic DNA and cDNA encoding an ALG2 homologue were cloned from the zygomycete fungus, Rhizomucor pusillus, and their nucleotide sequences were determined. The cloned cDNA under the control of the yeast GAL1 promoter complemented the temperature-sensitive (ts) growth of the alg2-1 mutant of S. cerevisiae, indicating that it represented a functional ALG2 homologue of R. pusillus. Five introns intervened the R. pusillus alg2 encoding a 455-amino-acid (aa) protein that showed end-to-end similarity in aa sequence to yeast Alg2 and contained a dolichol-binding consensus sequence (Val/Ile-x-Phe-x-x-Ile, where x is any aa) very near its C-terminus. The yeast alg2-1 gene had two mutation points at 377Gly to Arg and 386Gln to Lys. alg2-2 also contained two mutations at 54Glu to Lys and 377Gly to Arg. Site-directed mutagenesis of the fungal Alg2 and determination of their phenotypes in the yeast alg2-1 mutant showed that a mutation at 368Gly (equivalent to 377Gly of yeast Alg2) to Arg resulted in generation of a ts enzyme. The fungal Alg2 containing a mutation at the position corresponding to 54Glu or 386Gln of yeast Alg2 still complemented the ts growth of yeast alg2-1.