Cell fractionation

Yeast cells grown at 30°C or heat shocked (37°C + MG132) in the absence or presence of CHX were harvested at 1,150 g for 5 min, washed with 1 ml of ice-cold 50 mM Tris/HCl, pH 8.5, and resuspended in 250 μl of 50-mM Tris/HCl, pH 8.5, and 500 mM NaCl supplemented with cOmplete protease inhibitor cocktail (Sigma-Aldrich). Cells were frozen in liquid nitrogen, and cell lysis was performed in 2 ml Eppendorf Safe-Lock tubes by mixer milling (MM 400; Retsch) at 30 Hz for 90 s and five cycles. The lysate was thawed and precleared at 3,000 g for 1 min at 4°C. The total cell lysate was decanted into a prechilled new reaction tube followed by separation of soluble and insoluble (P20) fractions at 16,000 g for 20 min at 4°C. The soluble fraction was removed and subjected to a second round of centrifugation at 100,000 g (P100) for 20 min at 4°C. Pellets isolated at 16,000 g (P₂₀)/100,000 g (P₁₀₀) were washed with 50 mM Tris, pH 8.5, and 150 mM NaCl supplemented with cOmplete protease inhibitor cocktail and spun down again for 20 min at the appropriate g. Pellets were resuspended in 50 mM Tris, pH 8.5, 150 mM NaCl, 8 M urea, 2% (wt/vol) SDS, and 2 mM DTT by shaking in a thermomixer (Eppendorf) at 37°C for 5 min. All fractions were mixed with 6× protein loading buffer (50 mM Tris/HCl, pH 6.8, 30% [vol/vol] glycerol, 10% [wt/vol] SDS, 5% β-mercaptoethanol, and 0.05% [wt/vol] Bromphenol blue). Distribution of mCherry-VHL and Hsp42 among soluble and insoluble fractions was monitored by SDS-PAGE and Western blotting.