title: Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis.
abstract: In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.
authors.affiliation: Departments of Pathology, Molecular and Cellular Biology, Molecular Physiology, and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Departments of Pathology, Molecular and Cellular Biology, Molecular Physiology, and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Termine
authors.affiliation: Departments of Pathology, Molecular and Cellular Biology, Molecular Physiology, and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Swulius
authors.affiliation: Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: Departments of Pathology, Molecular and Cellular Biology, Molecular Physiology, and Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the. Electronic address: rsifers@bcm.tmc.edu.
authors.forename: Richard N
authors.initials: RN
authors.lastname: Sifers
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D011817:Rabbits; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D008361:Mannosidases; D046988:Proteasome Endopeptidase Complex; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; C061553:ATP dependent 26S protease