The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization.
Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.
Becker
B
B
McGill Cancer Centre, McGill University Montréal, Québec, Canada. b.becker@uni-koeln.de
Haggarty
A
A
Romero
P A
PA
Poon
T
T
Herscovics
A
A
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase