Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway

Abstract

The yeast Saccharomyces cerevisiae temperature-sensitive lethal mutant alg1-1, has been previously shown to lack the activity necessary for the addition of the first mannose residue in the synthesis of lipid-linked precursor oligosaccharide. The gene ALG1 has been cloned by complementation of the temperature-sensitive mutation alg1-1 with a total genomic DNA library. The original DNA fragment isolated was 11,300 base pairs and has been subcloned to a 1,500-base pair fragment which is still capable of complementing alg1-1. The gene ALG1 has been mapped on chromosome II at a distance of 2.1 map units from LYS2. The ALG1 gene product has been shown to catalyze the transfer of a mannosyl residue from GDP-mannose to the lipid-linked acceptor GlcNAc2, yielding Man beta 1-4GlcNAc2-lipid, in lysates from Escherichia coli transformants. This result proves that ALG1 is the structural gene for the first mannosyltransferase in lipid-linked oligosaccharide assembly.