Yeast protein degradation assays

For radioactive pulse-chase experiments, 10 OD₆₀₀-ml equivalents of yeast cells were grown in SD or minimal rich media to logarithmic phase. Cells were harvested by centrifugation in a 15-ml capacity tube and washed twice with 1 ml of SD media lacking all amino acids that had been warmed to 30°C; the culture was transferred to a 2-ml screw-cap microcentrifuge tube during the first wash. Washed cells were then resuspended in 0.2 ml SD media lacking the amino acid methionine. A volume of EXPRE³⁵S³⁵S protein labeling mix (Perkin Elmer; NEG072007MC) yielding ∼0.1 mCi of ³⁵S was then added, and cells were vortexed for 10 s and then incubated at 30°C for 6–10 min. Labeled cells were harvested by centrifugation and resuspended in 0.45 ml of chase buffer (SD media plus 10 mM excess methionine) with 0.5 mg/ml cycloheximide (except where noted in figure legends). A 0.1-ml aliquot was immediately taken as timepoint zero, added to 0.1 ml of 2× lysis buffer (90 mM HEPES, pH 7.5, 2% SDS, 30 mM dithiothreitol [DTT]), and placed on ice until the end of the chase. Subsequent timepoints were collected in the same manner. At the end of the chase, all samples were boiled for 8 min and then frozen at -80°C. Samples were thawed, 1 ml of Triton lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) was added, and the tubes were vortexed for 20 s. Samples were clarified by centrifugation at 21,130 × g (Eppendorf 5424) for 10 min, and the supernatant was transferred to fresh microcentrifuge tubes. Aliquots of each sample were spotted on filter paper to quantify trichloroacetic acid–precipitable radioactivity, and these values were used to add equivalent amounts of total radioactive protein to fresh microcentrifuge tubes. Polyclonal rabbit antibody to MATalpha2 or monoclonal mouse antibody to the HA epitope (Sigma) was added and samples were incubated at 4°C for 2 h with mixing. Samples were then mixed with Protein A resin (for rabbit antibodies; Repligen) or Protein G resin (for mouse antibodies; Santa Cruz) and incubated at 4°C for 1 h with mixing. Samples were washed four to five times with immunoprecipitation wash buffer (30 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 0.1% SDS) before proteins were eluted by adding 0.06 ml SDS–PAGE sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 143 mM β-mercaptoethanol, and bromophenol blue to color) and boiling for 5 min. Samples (approximately one-half of each eluate) were subjected to SDS–PAGE, gels were fixed and dried, and the dried gels were exposed to a phosphoimager screen for 1–3 d. Screens were imaged on a STORM860 (GE, Marlborough, MA), and data were analyzed using ImageQuant 5.2 software (GE).

Cycloheximide-chase experiments were carried out as previously described (Hickey and Hochstrasser, 2015 ). Independent yeast transformants were used for replication of all protein degradation experiments.