The transmembrane domain of murine alpha-mannosidase is 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 a nd hybrid N-glycans, This enzyme consists of a cytoplasmic tail, a transmem brane domain followed by a "stem" region and a large C-terminal catalytic d omain, To analyze the determinants of targeting, we constructed various del etion mutants of murine alpha1,2-mannosidase IB as web as alpha1,2-mannosid ase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy when expressed tran siently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosida se IB with that of the homologous yeast alpha1,2-mannosidase and deleting t he "stem'' region fn this chimera had no effect on Golgi targeting, but cau sed increased cell surface localization The N-terminal tagged protein lacki ng a catalytic domain was also localized to the Golgi, In the latter case, when the stem region was partially or comptetely removed, the protein was f ound in both the ER and the Golgi, A chimera consisting of the alpha1,2-man nosidase IB N-terminal region (eytoplasmic and transmembrane domains plus 1 0 amino acids of the "stem" region) and GFP was localized mainly to the Gol gi, Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no eff ect on Golgi localization, A GFP chimera lacking the entire cytoplasmic tai l was found in both the ER and the Golgi, These results indicate that the t ransmembrane domain of alpha1,2-mannosidase IB is a major determinant of Go lgi localization.