title: Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form.
abstract: Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-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-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato Gto. CP 36000, Mexico.
authors.forename: Héctor M
authors.initials: HM
authors.lastname: Mora-Montes
authors.affiliation: Robert Koch-Institut, FG16, Nordufer 20, D-13353 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Bader
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato Gto. CP 36000, Mexico.
authors.forename: Everardo
authors.initials: E
authors.lastname: López-Romero
authors.affiliation: Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14-740, México DF 07000, Mexico.
authors.forename: Samuel
authors.initials: S
authors.lastname: Zinker
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato Gto. CP 36000, Mexico.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ponce-Noyola
authors.affiliation: Robert Koch-Institut, FG16, Nordufer 20, D-13353 Berlin, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hube
authors.affiliation: School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
authors.forename: Neil A R
authors.initials: NAR
authors.lastname: Gow
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato Gto. CP 36000, Mexico.
authors.forename: Arturo
authors.initials: A
authors.lastname: Flores-Carreón
mesh_terms: D002176:Candida albicans / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D012995:Solubility; D043323:alpha-Mannosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D008985:Monensin; D043323:alpha-Mannosidase; D010447:Peptide Hydrolases