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

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

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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

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Proteomics
Glycosylation
Proteins
Electrophoresis
Ribosomes
Yeast
Mass spectrometry
Mass Spectrometry
Salts
Yeasts
Chemical analysis
Native Polyacrylamide Gel Electrophoresis
Ribosome Subunits
Puromycin
dolichyl-diphosphooligosaccharide - protein glycotransferase
Rough Endoplasmic Reticulum
Mannose
Keratinocytes
Sugars
Sodium Dodecyl Sulfate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits. / Shibatani, Toru; David, Larry; McCormack, Ashley L.; Frueh, Klaus; Skach, William.

In: Biochemistry, Vol. 44, No. 16, 26.04.2005, p. 5982-5992.

Research output: Contribution to journalArticle

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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.",
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N2 - 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.

AB - 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.

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