| Abstract
| - Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugarsto nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. NineOST subunits have been identified in yeast. However, the composition and organization of mammalianOST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodiumdodecyl sulfate−polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate thatmammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct proteincomplexes that range in size from ∼500 to 700 kDa. These complexes exhibit different ribosome affinitiesand subunit compositions. The major complex, OSTCI, had an apparent size of ∼500 kDa and was readilyreleased 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) andOSTCIII (∼700 kDa). Both remained stably associated with heterotrimeric Sec61αβγ, while OSTCIII alsocontained 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 uncharacterizedproteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which isweakly homologous to the C-terminal half of yeast Ost3p and Ost6p. The second protein (14 kDa) wastentatively 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 remarkableheterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.
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