Recent studies have revealed that rabbit reticulocyte lysate (RRL) efficiently reconstitutes endoplasmic reticulum-associated degradation (ERAD) of mutant and misfolded membrane proteins. When supplemented with canine pancreas microsomal membranes, the RRL system faithfully carries out ER targeting, translocation, glycosylation, and membrane integration events and therefore provides a ready source of 35S-labeled protein with defined transmembrane topology. These substrates can be rapidly isolated in native ER membranes which, when incubated in RRL lacking exogenous hemin, are degraded in an ATP-dependent manner by the ubiquitin-proteasome pathway. Because the newly translated protein is the only source of radiolabel, degradation can be followed to its end state by conversion into trichloroacetic acid (TCA)-soluble peptide fragments. A particularly useful aspect of this system is that both membrane-associated and cytosolic components are amenable to biochemical and pharmacological manipulation. Here we describe techniques for preparing translation- and degradation-competent RRL, affinity depletion, identification of cytosolic factors involved in degrading the cystic fibrosis transmembrane conductance regulator (CFTR), and reconstitution of ERAD by add-back of purified recombinant proteins. These techniques provide a powerful tool for dissecting components involved in ubiquitination, degradation, and in particular, extraction of transmembrane ERAD substrates.
ASJC Scopus subject areas
- Molecular Biology