Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits

Toru Shibatani, Larry L. David, Ashley L. McCormack, Klaus Frueh, William R. Skach

Research output: Contribution to journalArticlepeer-review

108 Scopus citations

Abstract

Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugars to nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. Nine OST subunits have been identified in yeast. However, the composition and organization of mammalian OST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate that mammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct protein complexes that range in size from ∼500 to 700 kDa. These complexes exhibit different ribosome affinities and subunit compositions. The major complex, OSTC I, had an apparent size of ∼500 kDa and was readily released from ribosome translocon complexes after puromycin treatment under physiological salt conditions. Two additional complexes were released only after treatment with high salt: OSTCII (∼600 kDa) and OSTCIII (∼700 kDa). Both remained stably associated with heterotrimeric Sec61αβγ, while OSTCIII also contained the tetrameric TRAP complex. All known mammalian OST subunits (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) were present in all complexes. In addition, two previously uncharacterized proteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which is weakly homologous to the C-terminal half of yeast Ost3p and Ostop. The second protein (14 kDa) was tentatively identified as keratinocyte-associated protein 2 (KCP2) and has no previously known function. Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.

Original languageEnglish (US)
Pages (from-to)5982-5992
Number of pages11
JournalBiochemistry
Volume44
Issue number16
DOIs
StatePublished - Apr 26 2005

ASJC Scopus subject areas

  • Biochemistry

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