1887

Abstract

Cytosolic -mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free -oligosaccharides. Two soluble -mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in . We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched -mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-1,2-mannosidase antibody. The -mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that 1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound 1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of -mannosidase activity in a Δ null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound 1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free -oligosaccharides revealed that cytosolic 1,2-mannosidase E-I trims free ManGlcNAc isomer B into ManGlcNAc isomer B. This is believed to be the first report demonstrating the presence of soluble 1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

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2008-12-01
2020-08-11
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