Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases

Using appropriate yeast strains and exogenous acceptors, we have devised specific assays for four mannosyltransferase activities involved in biosynthesis of the carbohydrate outer chain of yeast mannoproteins. The assays utilize GDP-[14C]mannose as the donor and unlabeled oligosaccharides as the acceptors, the products being neutral radioactive oligosaccharides one mannose unit larger than the acceptors. The multiglycosyltransferase system from Saccharomyces cerevisiae was solubilized in Triton X-100 and urea and purified 100-fold. Free mannose is an acceptor for the alpha1 leads to 2-mannosyltransferase, the major product being alpha[14C]Man leads to 2Man. The alpha1 leads to 6-mannooligosaccharides serve as acceptors for both the alpha1 leads to 2- and alpha1 leads to 6-transferases, but the tetrasaccharide alphaMan leads to 3alphsMan leads to 2alphaMan is a specific acceptor for the latter enzyme and yields (see article). When reduced, this same tetrasaccharide serves as the acceptor for an alpha1 leads to 3-mannosyltransferase from Saccharomyces chevalieri, yielding a pentasaccharide with two terminal 1 leads to 3 linkages. Assay of the alpha1 leads to 3-transferase in S. cerevisiae utilizes reduced alpha1 leads to 2-mannotriose as the acceptor, the product being alpha[14C]Man leads to 3alphaMan leads to 2alphaMan leads to 2Mannitol. The multienzyme system works in concert to make "mannan" in a cell-free in vitro system.