Interaction of the endoplasmic reticulum alpha 1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system.
The alpha1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi alpha1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Deltapep4 cells and in the vacuoles of rer1/Deltapep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of beta-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.
Massaad
M J
MJ
McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada.
Herscovics
A
A
D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D005656:Fungal Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D008361:Mannosidases / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C089137:RER1 protein, S cerevisiae; C071239:SEC12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase