title: (Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization.
abstract: Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.
authors.affiliation: Institut Für Physiologische Chemie, Universität Bonn Nussallee 11, 53115 Bonn, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Hardt
authors.forename: Burga
authors.initials: B
authors.lastname: Kalz-Fuller
authors.forename: Raquel
authors.initials: R
authors.lastname: Aparicio
authors.forename: Christof
authors.initials: C
authors.lastname: Volker
authors.forename: Ernst
authors.initials: E
authors.lastname: Bause
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D001120:Arginine; C042059:glucosidase I; D000520:alpha-Glucosidases