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BIOCHEMICAL AND STRUCTURAL STUDIES OF THE OLIGOSACCHARYL TRANSFERASE COMPLEX
Author(s) -
Chavan Manasi Manoj,
Chen Zhiqiang,
Li Hua,
Li Huilin,
lennarz William
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1021
Subject(s) - protein subunit , digitonin , endoplasmic reticulum , transferase , dimer , biochemistry , chemistry , enzyme , glycosylation , translocon , biology , stereochemistry , membrane protein , membrane , gene , organic chemistry
Oligosaccharyl transferase (OT), an enzyme complex made up of 9 transmembrane proteins catalyzes N‐glycosylation in the lumen of the endoplasmic reticulum. Several lines of evidence, including our previous split‐ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one‐step immunoaffinity technique. The digitonin‐solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25 Å resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation. (This work was supported by National Institutes of Health Grant GM33185 (to W.J.L.), Brookhaven National Laboratory Laboratory Directed Research and Development Fund 06‐06 (to H.L.), and Department of Energy Grant KP1102010 (to H.L.).