title: Analysis of structural signals conferring localisation of pig OST48 to the endoplasmic reticulum.
abstract: Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the co-translational transfer of GlcNAc2-Man9-Glc3 from Dol-PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ER-targeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man9-mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the C-terminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48AK) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ER-residency on OST48. The double-lysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48AK-3, when co-expressed in COS-1 cells together with myc-tagged ribophorin 1, was quantitatively retained in the ER. By contrast, co-expression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMOdeltaK-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the C-terminus of the Man9-mannosidase luminal domain. Thus ER-localisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the double-lysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in re-capture of molecules which have escaped from the ER.
authors.affiliation: Institut für Physiologische Chemie, Universität Bonn, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Hardt
authors.forename: R
authors.initials: R
authors.lastname: Aparicio
authors.forename: W
authors.initials: W
authors.lastname: Breuer
authors.forename: E
authors.initials: E
authors.lastname: Bause
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002240:Carbohydrate Sequence; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000502:physiology; D006602:Hexosyltransferases; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D013552:Swine / Q000378:metabolism*; D014162:Transfection; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; C034241:ribophorin; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase