Protein Aggregation Assay Protocol

The assay used in this study was adapted from Koplin et al. (2010) (link). In brief, cell pellets were resuspended in zymolyase buffer (1.2 M sorbitol, 50 mM KP_i, pH 7.2, 200 mM KCl₂, 10 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride [PMSF]) containing 3 mg/ml zymolyase 20T (Amsbio, UK) and incubated for 30 min at 30°C. Washed cell pellets were resuspended in lysis buffer (0.5% Triton X-100, 50 mM KP_i, pH 7.2, 10 mM DTT, 1 mM EDTA, 1× Protease inhibitor [Roth, Germany], 1 mM PMSF). After shaking for 20 min at 4°C, cleared supernatants were centrifuged at high speed (20,000 × g) for 20 min at 4°C. The pellet was washed once in wash buffer (0.5% Triton X-100, 20 mM KP_i, pH 7.2, 1 mM PMSF) and subsequently washed in wash buffer without detergent. Aggregated proteins were analyzed by SDS–PAGE and Western blot. Aggregate pellet fractions were applied in threefold excess compared with total or supernatant fractions. Aggregation analysis in isolated mitochondria was performed as described above. For incubation of mitochondria at elevated temperatures, mitochondria were resuspended in buffer A (1.2 M sorbitol, 50 mM KP_i, pH 7.2, 100 mM KCl, 10 mM MgCl₂, 2 mM ATP, 2 mM NADH, 1 mM PMSF) before lysis.