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 cotranslational transfer of GlcNAc[2]Man[9]Glc[3] 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 ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation 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 doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.