The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum.
The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2. alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX). Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum. 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi. The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase. The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment.
Massaad
M J
MJ
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Franzusoff
A
A
Herscovics
A
A
D001692:Biological Transport; D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005656:Fungal Proteins / Q000378:metabolism*; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008351:Mannans; D008565:Membrane Proteins; D009844:Oligosaccharides; C089137:RER1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C116695:mannosyl(9)-N-acetylglucosamine(2); D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase