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Studying Endoplasmic Reticulum Function In Vitro Using siRNA

  • Cornelia M. Wilson
  • Stephen High
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

In eukaryotic cells, N-glycosylation is typically the most common protein modification that occurs in the endoplasmic reticulum (ER) lumen. N-glycosylation is facilitated by a large heterologous protein complex called the oligosaccharyltransferase (OST) that allows the attachment of a high mannose oligosaccharide from a dolichol pyrophosphate donor en bloc onto suitable asparagine residues of newly synthesized nascent chains during translocation into the ER lumen (1). While the complexity of the OST is highly conserved in eukaryotes, the role of its different subunits is poorly defined. We have investigated the function of three OST subunits, the ER translocon-associated component ribophorin I, and two isoforms of the presumptive catalytic subunit, STT3. We use a combination of siRNA-mediated knockdown of individual proteins combined with a semi-permeabilized mammalian cell system to provide a robust read out for OST subunit function during N-glycosylation of model substrates in vitro. This approach is equally applicable to the study of other cellular components.

Key words

Endoplasmic reticulum N-glycosylation oligosaccharyltransferase semi-permeabilized cells siRNA 

Notes

Acknowledgements

We are eternally grateful to all our colleagues who have contributed their time and materials towards this chapter. The work described in this chapter was supported by grant funding from the Biotechnology and Biological Sciences Research Council.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Cornelia M. Wilson
    • 1
  • Stephen High
    • 2
  1. 1.Faculty of MedicineUniversity of LimogesLimogesFrance
  2. 2.Faculty of Life SciencesUniversity of ManchesterManchesterUK

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