Abstract
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.
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- Ballou C. E., Lipke P. N., Raschke W. C. Structure and immunochemistry of the cell wall mannans from Saccharomyces chevalieri, Saccharomyces italicus, Saccharomyces diastaticus, and Saccharomyces carlsbergensis. J Bacteriol. 1974 Feb;117(2):461–467. doi: 10.1128/jb.117.2.461-467.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ballou C. E. Some aspects of the structure, immunochemistry, and genetic control of yeast mannans. Adv Enzymol Relat Areas Mol Biol. 1974;40(0):239–270. doi: 10.1002/9780470122853.ch6. [DOI] [PubMed] [Google Scholar]
- Ballou D. L. Genetic control of yeast mannan structure: mapping genes mnn2 and mnn4 in Saccharomyces cerevisiae. J Bacteriol. 1975 Aug;123(2):616–619. doi: 10.1128/jb.123.2.616-619.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Behrens N. H., Cabib E. The biosynthesis of mannan in Saccharomyces carlsbergensis. J Biol Chem. 1968 Feb 10;243(3):502–509. [PubMed] [Google Scholar]
- Garewal H. S., Wasserman A. R. Triton X-100-4 M urea as an extraction medium for membrane proteins. I. Purification of chloroplast cytochrome b559. Biochemistry. 1974 Sep 24;13(20):4063–4071. doi: 10.1021/bi00717a001. [DOI] [PubMed] [Google Scholar]
- Jones G. H., Ballou C. E. Studies on the structure of yeast mannan. I. Purification and some properties of an alpha-mannosidase from an arthrobacter species. J Biol Chem. 1969 Feb 10;244(3):1043–1051. [PubMed] [Google Scholar]
- Lehle L., Tanner W. Membrane-bound mannosyl transferase in yeast glycoprotein biosynthesis. Biochim Biophys Acta. 1974 May 20;350(1):225–235. doi: 10.1016/0005-2744(74)90220-4. [DOI] [PubMed] [Google Scholar]
- Nakajima T., Ballou C. E. Structure of the linkage region between the polysaccharide and protein parts of Saccharomyces cerevisiae mannan. J Biol Chem. 1974 Dec 10;249(23):7685–7694. [PubMed] [Google Scholar]
- Raschke W. C., Kern K. A., Antalis C., Ballou C. E. Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J Biol Chem. 1973 Jul 10;248(13):4660–4666. [PubMed] [Google Scholar]
- Schutzbach J. S., Ankel H. Multiple mannosyl transferases in Cryptococcus laurentii. J Biol Chem. 1971 Apr 10;246(7):2187–2194. [PubMed] [Google Scholar]
- Sharma C. B., Babczinski P., Lehle L., Tanner W. The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. Eur J Biochem. 1974 Jul 1;46(1):35–41. doi: 10.1111/j.1432-1033.1974.tb03594.x. [DOI] [PubMed] [Google Scholar]