Characterization and expression of Saccharomyces cerevisiae alpha-mannosidase involved in processing of N-linked oligosaccharides (original) (raw)

Abstract

The specific $ alpha$-mannosidase of the yeast Saccharomyces cerevisiae involved in the early processing of N-linked oligosaccharides converts Man$ sb9$GlcNAc$ sb2$ to Man$ sb8$GlcNAc$ sb2$ prior to elongation of the sugar chains with mannose residues. This membrane enzyme is believed to be associated with elements of the endoplasmic reticulum. The membrane topology of the yeast $ alpha$-mannosidase was determined by cell-free transcription and translation in a reticulocyte lysate in the presence of dog pancreas microsomes. It was demonstrated that the $ alpha$-mannosidase has a type II membrane topology in vesicles similar to that of all the Golgi glycosyltransferases and glycosidases cloned to date. The enzyme was expressed as a fusion protein in E. coli to produce antisera that will be used to immunolocalize the enzyme in yeast cells. The yeast $ alpha$-mannosidase gene lacking the NH2-terminal hydrophobic signal/anchor sequence was fused to the COOH-terminus of the Schistosoma j...

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