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In Vitro Dissection of Protein Translocation into the Mammalian Endoplasmic Reticulum

  • Ajay Sharma
  • Malaiyalam Mariappan
  • Suhila Appathurai
  • Ramanujan S. Hegde
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.

Key words

in vitro translation microsomes membrane proteins reconstitution proteoliposomes protease protection protein topology 

Notes

Acknowledgments

Work in the Hegde lab is supported by the NICHD Intramural Research Program of the National Institutes of Health.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ajay Sharma
    • 1
  • Malaiyalam Mariappan
    • 1
  • Suhila Appathurai
    • 1
  • Ramanujan S. Hegde
    • 1
  1. 1.Cell Biology and Metabolism ProgramNational Institute of Child Health and Human Development, National Institutes of HealthBethesdaUSA

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