Isolation and Analysis of Protein Aggregates

50 OD₆₀₀ units of logarithmically growing cells in YPD medium were harvested, and cell pellets were frozen in liquid N₂. For preparation of cell lysates, the pellets were resuspended in lysis buffer (20 mM Na-phosphate, pH 6.8, 10 mM DTT, 1 mM EDTA, 0.1% Tween, 1 mM PMSF, protease inhibitor cocktail [Roche], 3 mg/ml zymolyase T20, and 1.25 U/ml benzonase) and incubated at room temperature for 20 min. Chilled samples were treated by tip sonication (Branson; eight times at level 4 and duty cycle 50%) and centrifuged for 20 min at 200 g at 4°C. Supernatants were adjusted to identical protein concentrations, and aggregated proteins were pelleted at 16,000 g for 20 min at 4°C. After removing supernatants, aggregated proteins were washed twice with 2% NP-40 (in 20 mM Na-phosphate, pH 6.8, 1 mM PMSF, and protease inhibitor cocktail), sonicated (six times at level 4 and duty cycle 50%), and centrifuged at 16,000 g for 20 min at 4°C. Aggregated proteins were washed in NP-40–deficient buffer (sonication, four times at level 2 and duty cycle 65%), boiled in SDS sample buffer, separated by SDS-PAGE (14%), and analyzed by Coomassie staining. Experiments were performed at least three times with similar results. Identification of isolated aggregates by mass spectrometry was performed according to previously described protocols (Kramer et al., 2002 (link); Wegrzyn et al., 2006 (link)). For significant protein identification, a score threshold of 200 was chosen.
For radioactive labeling of newly synthesized proteins, cells were starved for 1 h in methionine-free SC medium. The cells were labeled with 20 µCi/ml [³⁵S]methionine for 1 min, chilled quickly, and treated with 300 µg/ml cycloheximide to stop protein translation. ³⁵S-labeled aggregates were isolated, separated via SDS-PAGE, and visualized by autoradiography in FLA-9000 (Fujifilm).