Subcellular distribution in rat liver of a novel broad-specificity (alpha 1----2, alpha 1----3 and alpha 1----6) mannosidase active on oligomannose glycans.

Recently, the purification to homogeneity was reported of a novel broad-specificity alpha-mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197, 229-238]. The enzyme catalyzed the ordered removal of alpha 1----2-, alpha 1----3- and alpha 1----6-linked mannose residues from MannGlcNAc oligosaccharide substrates where n = 4-9. We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a 'late' endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in 'early' endosomes but not in receptor-enriched and ligand-free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X-100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross-reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA.