title: Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.
abstract: The oligosaccharyltransferase (OST) complex catalyses the N-glycosylation of polypeptides entering the endoplasmic reticulum, a process essential for the productive folding and trafficking of many secretory and membrane proteins. In eukaryotes, the OST typically comprises a homologous catalytic STT3 subunit complexed with several additional components that are usually conserved, and that often function to modulate N-glycosylation efficiency. By these criteria, the status of keratinocyte-associated protein 2 (KCP2) was unclear: it was found to co-purify with the canine OST suggesting it is part of the complex but, unlike most other subunits, no potential homologues are apparent in Saccharomyces cerevisiae. In this study we have characterised human KCP2 and show that the predominant species results from an alternative initiation of translation to form an integral membrane protein with three transmembrane spans. KCP2 localises to the endoplasmic reticulum, consistent with a role in protein biosynthesis, and has a functional KKxx retrieval signal at its cytosolic C-terminus. Native gel analysis suggests that the majority of KCP2 assembles into a distinct ~500 kDa complex that also contains several bona fide OST subunits, most notably the catalytic STT3A isoform. Co-immunoprecipitation studies confirmed a robust and specific physical interaction between KCP2 and STT3A, and revealed weaker associations with both STT3B and OST48. Taken together, these data strongly support the proposal that KCP2 is a newly identified subunit of the N-glycosylation machinery present in a subset of eukaryotes.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, UK.
authors.forename: Peristera
authors.initials: P
authors.lastname: Roboti
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D000818:Animals; D055162:Biocatalysis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005057:Eukaryotic Cells / Q000201:enzymology; D006602:Hexosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits; D006602:Hexosyltransferases; C581362:KRTCAP2 protein, human; C581363:OST48 protein, human; C099573:STT3A protein, human; C551978:STT3B protein, human; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase