The processing α1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum

The yeast alpha 1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man(9)GlcNAc(2) to a single isomer of Man(8)GlcNAc(2). alpha 1,2-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 alpha 1,2-mannosidase, indirect immunofluorescence showed that alpha 1,2-mannosidase localization is abnormal in rer1 cells and that the alpha 1,2-mannosidase localizes in the vacuoles of rer1/Delta pep4 cells whereas in wild-type and Delta pep4 cells it is found in the endoplasmic reticulum, S-35-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha 1,2-mannosidase, then with either alpha 1,2-mannosidase antibodies or antibodies to alpha 1,6-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 alpha 1,2-mannosidase tvas immunoprecipitated by al,6-mannose antibodies in wild-type, Delta pep4 and rer1/Delta pep4 cells with endogenous levels of alpha 1,2-mannosidase, and in wild-type, Delta pep4, rer1 and rer1/Delta pep4 cells overexpressing alpha 1,2-mannosidase. The alpha 1,2-mannosidase of rer1/Delta pep4 cells had a slower mobility on the gels than alpha 1,2-mannosidase precipitated from wild-type or Delta pep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles. It is concluded that the endoplasmic reticulum localization of alpha 1,2-mannosidase in wild-type cells depends an Rer1p for retrieval from an early Golgi compartment.