Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: the alg2 mutation.

In the yeast , the mutation causes temperature-sensitive growth and abnormal accumulation of the lipid-linked oligosaccharide ManGlcNAc-PP-Dol (Jackson ., 272, 203–209, 1989; Huffaker and Robbins, , 80, 7466–7470, 1983). A gene having the function and genomic location of ALG2 was cloned from libraries based on the multicopy plasmid YEp24 and on the centromere plasmid YCp50. mutants transformed with plasmids containing ALG2 regained the capacity to grow and to synthesize lipid-linked oligosaccharides normally at the previously non-permissive temperature. ALG2 was essential for viability in haploid and diploid yeast. The ALG2 gene was transcribed into a single mRNA of 1.7 kb in size. The stability of ALG2 mRNA, assessed after thermal inactivation of RNA polymerase II in an mutant (Herrick ., 10, 2269–2284, 1990) was very low, with a t of <5 min. The ALG2 transcript accumulation was growth dependent, and it was at least an order of magnitude lower in stationary phase cells compared to exponentially growing cells. The putative translation product of ALG2 contained a potential dolichol recognition domain similar to that found in all three glycosyl-transferases of the lipid-linked pathway that have been sequenced. The severity of the phenotype associated with mutants suggested that the conversion of ManGlcNAc-PP-Dol to ManGlcNAc-PP-Dol, which is ALG2-dependent, results most likely in formation of the 1→3 branch of the core that is elongated during outer chain formation (Ballou , 87, 3368–3372, 1990; Ballou , 83, 3081–3085, 1986). [ABSTRACT FROM PUBLISHER]