CHARACTERIZATION OF ALG2 ENCODING A MANNOSYLTRANSFERASE IN THE ZYGOMYCETE FUNGUS RHIZOMUCOR PUSILLUS

ALG2 of Saccharomyces cerevisiae encodes the glycosyltransferase that mannosylates Man(2)GlcNAc(2)-dolichol diphosphate (PP-Dol) and Man(1)G lcNAc(2)-PP-Dol to form Man(3)GlcNAc(2)-PP-Dol. The genomic DNA and cD NA encoding an ALG2 homologue were cloned from the zygomycete fungus, Rhizomucor pusillus, and their nucleotide sequences were determined. T he cloned cDNA under the control of the yeast GAL1 promoter complement ed the temperature-sensitive (ts) growth of the alg2-1 mutant of S. ce revisiae, indicating that it represented a functional ALG2 homologue o f R. pusillus. Five introns intervened the R. pusillus alg2 encoding a 455-amino-acid (aa) protein that showed end-to-end similarity in aa s equence to yeast Alg2 and contained a dorichol-binding consensus seque nce (Val/Ile-x-Phe-x-x-Ile, where x is any aa) very near its C-terminu s. The yeast alg2-1 gene had two mutation points at( 377)Gly to Arg an d (386)Gln to Lys. alg2-2 also contained two mutations at (54)Glu to L ys and (377)Gly to Arg. Site-directed mutagenesis of the fungal Alg2 a nd determination of their phenotypes in the yeast alg2-1 mutant showed that a mutation at (368)Gly (equivalent to 377Gly Of yeast Alg2) to A rg resulted in generation of a ts enzyme. The fungal Alg2 containing a mutation at the position corresponding to (54)Glu or (386)Gln of yeas t Alg2 still complemented the ts growth of yeast alg2-1. (C) 1998 Else vier Science B.V. All rights reserved.