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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.
Newly synthesized secretory and membrane proteins that fail to achieve their native conformations are retained in the endoplasmic reticulum (ER) and may be degraded. This process is referred to as ER-associated degradation (ERAD). From studies over the past 13 years, it is now clear that ERAD substrates are first recognized in the ER and are then retrotranslocated back to the cytoplasm where they are ubiquitinated and degraded by the proteasome (1–5). Earlier genetic studies using the yeast Saccharomyces cerevisiae have identified core components required for ERAD, including membrane-associated E2/E3 ubiquitination enzymes, cytoplasmic and luminal chaperones, and the proteasome. Although the detailed mechanism for substrate recognition and retrotranslocation is not yet clear, current evidence suggests that, depending on the location of the misfolded lesion, molecular chaperones and chaperone-like lectins either in the ER or in the cytoplasm help select ERAD substrate (6–9). To further dissect the ERAD reaction into elementary steps and to characterize the functions of known and novel components, it is vital to biochemically reconstitute ERAD.
Ste6p is a yeast a-factor mating pheromone transporter that is synthesized in the ER and is delivered to and functions at the plasma membrane. A mutant form of Ste6p, which is called Ste6p*, is retained in the ER and is degraded by the proteasome via ERAD (10). Ste6p* has 12 transmembrane domains and is structurally similar to the cystic fibrosis transmembrane conductance regulator (CFTR), which is also an ERAD substrate and which when mutated results in cystic fibrosis. Genetic analysis has shown that Ste6p* degradation is slowed when specific E2 ubiquitin-conjugating enzymes (Ubc6p and Ubc7p), E3 ubiquitin ligases (Doa10p and Hrd1p), cytoplasmic Hsp70 and Hsp40 chaperones (Ssa1p and Ydj1p/Hlj1p), and a AAA-ATPase Cdc48p are disabled (6, 11). Although the ERAD pathway for Ste6p* is relatively well-defined, until recently it was not clear how this substrate is selected for ubiquitination and whether it is degraded in the cytoplasm or at the ER membrane.
We recently reconstituted the ubiquitination and extraction of Ste6p* using materials prepared from yeast (12). This assay has proven that Ssa1p is essential for ubiquitination. Moreover, ubiquitinated Ste6p* is extracted from the ER membrane to the cytosol in an ATP- and Cdc48p-dependent manner. We also discovered that Ufd2p, an E4 polyubiquitin chain-extending enzyme, elongates ubiquitin chains. Theoretically, this assay can be applied to any misfolded membrane protein that can be expressed in yeast. This assay also has the potential to further dissect the pathway of these ERAD substrates using yeast genetic mutants.
In vitro ubiquitination of Ste6p* and CFTR depends on relevant ubiquitination enzymes (e.g., Ubc6p/7p, Hrd1p/Doa10p, and Ufd2p) and Hsp70 and Hsp40 molecular chaperones (e.g., Ssa1p and Ydj1p/Hlj1p). To assay the effects of these agents, yeast microsomes are prepared in one of three different ways from mutant cells and isogenic wild-type cells expressing Ste6p* or CFTR. When microsomes are prepared from deletion mutant cells (e.g., ubc6Δubc7Δ, hrd1Δ, doa10Δ, hrd1Δdoa10Δ, and ufd2Δ) and isogenic wild-type strains, the cell walls are first digested with lyticase at room temperature or at 30°C for <1 h before the preparation of cell homogenate (see Section 3.1.1). However, during this incubation at the permissive temperature, the temperature-sensitive defect may be lost. Therefore, when microsomes are instead prepared from temperature-sensitive mutants (e.g., ssa1–45, a mutant form of SSA1, and the ydj1–151/hlj1Δ strains) and isogenic wild-type strains, cells are grown at a permissive temperature of 26°C and then are shifted to a restrictive temperature of 37°C. Cells are then collected on ice and are physically disrupted with glass beads by keeping them on ice to strictly control the temperature (see Section 3.1.2 or 3.1.3)(see Note 4).
The following procedure, used routinely in our laboratory, is based on a protocol previously described (21).
The in vitro ubiquitinated Ste6p* and the presence of unmodified Ste6p* can be detected by autoradiography and by western blotting, respectively. The following procedure results in sample volumes of approximately 28 μL, but 12-μL samples are sufficient for autoradiography or western blotting. The same protocol can be used to detect in vitro ubiquitinated CFTR.
The membrane extraction assay is similar to the ubiquitination assay except that each sample is separated into membrane and cytosolic fractions by centrifugation after the ubiquitination reaction.
1We typically store 125I-labeled ubiquitin at −80°C in aliquots of 20 μL. Although repeated freeze and thaw cycles (~3 times) do not seem to be detrimental to activity, best results are seen when the reagent is used within 2 months (half-life of 125I is ~60 days) after preparation. Non-labeled ubiquitin is also stored at −80°C in aliquots of 20 μL.
2Protein concentration of the cytosol is measured by the Bradford method with the protein assay kit (Bio-Rad). Bovine serum albumin (BSA) is used as the standard. No detectable loss of the activity was seen when cytosol was stored at −80°C for up to approximately 12 months.
3The in vitro ubiquitination of Ste6p* requires physiological temperature and does not occur on ice. The optimal temperature is 23°C and the extent of ubiquitination becomes inefficient at higher temperatures (e.g., 37°C), possibly because the misfolded substrate protein aggregates. However, the phenotype of some temperature-sensitive mutant alleles (e.g., ssa1–45), which is most evident at 37°C in vivo, is exhibited at 23°C in the in vitro reaction.
4Microsomes prepared from homogenates after spheroplast formation (see Section 3.1.1) or after glass beads disruption in a medium scale (see Section 3.1.3) are more E3 ligase enzyme-dependent than microsomes prepared from homogenates after a small-scale glass bead disruption (see Section 3.1.2).
5The addition of an inhibitor for deubiquitination (ubiquitin aldehyde) or a proteasome inhibitor (MG132 “n-cbz-leu-leu-leu-al”) does not result in increased ubiquitin chain extension. In addition, higher concentrations of cytosol (>~8 mg/mL) decrease the signal intensity.
6Cytosol at a final concentration of 1–2 mg/mL results in a low-molecular weight ubiquitinated species, but addition of more cytosol (at a final concentration of 4–6 mg/mL) “shifts” the ubiquitinated species to a higher molecular weight. The use of a 6% gel is critical to differentiate these two species, and this molecular weight shift is due to Ufd2p in the cytosol (12).
7All reaction samples containing 125I-labeled ubiquitin should be shielded by an approximately 1-mm lead plate to prevent excess exposure. The aliquots of 125I-labeled ubiquitin should be stored in a substantially more shielded lead container. Radioactivity at each step of this protocol should be surveyed with a γ-detecting monitor, and all items that contact 125I should be properly disposed.