In Vitro Reconstitution of the Selection, Ubiquitination, and Membrane Extraction of a Polytopic ERAD Substrate
Secretory and membrane proteins that are destined for intracellular organelles in eukaryotes are first synthesized at the endoplasmic reticulum (ER) and are then delivered to their final destinations. The ER contains high concentrations of molecular chaperones and folding enzymes that assist substrates to acquire their native conformations. However, protein misfolding is an inevitable event especially when cells are exposed to stress or during development or aging. ER-associated degradation (ERAD) is a major mechanism to eliminate misfolded proteins from the secretory pathway. The importance of ERAD is underscored by the fact that mutations in secretory and membrane proteins or corruption of the ERAD machinery have been linked to human diseases. Many components involved in ERAD have been identified by a genetic analysis using the yeast Saccharomyces cerevisiae, and it now appears that most of these factors are conserved in higher eukaryotes. In this chapter, we describe a method to recapitulate the ubiquitination and extraction of misfolded polytopic membrane proteins in vitro using materials prepared from yeast. These techniques provide a powerful tool to further dissect the ERAD pathway into elementary steps.
Key wordsEndoplasmic reticulum ER-associated degradation ATP proteasome Ufd2 Cdc48/p97 microsomes yeast
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