Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae.
The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.
Grondin
B
B
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Herscovics
A
A
D000818:Animals; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000648:ultrastructure; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000648:ultrastructure; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D012697:Serine Endopeptidases / Q000378:metabolism; D014158:Transcription, Genetic; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D006596:Hexosaminidases; D008361:Mannosidases; D043323:alpha-Mannosidase; D012697:Serine Endopeptidases; D019286:Endopeptidase K