Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

Abstract

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

FIGURE 1:

Identification of aggregated mitochondrial proteins. (A) Coomassie stain of an SDS gel with lysates of isolated mitochondria from S. cerevisae after treatment at the indicated temperatures and sedimentation of aggregates by ultracentrifugartion at 125,000 × g. Protein bands whose intensity increased with rising incubation temperature are marked with an asterisk (*); those whose intensity did not change with temperature are marked with #. T, total; Sup, supernatant; Pel, pellet. (B) Aggregates after treatments at 25°C or 42°C were spun down at 125,000 × g, and the indicated bands identified by mass spectrometry. (C) Quantitative analysis of mitochondrial protein aggregation on 2D-PAGE. Residual spot volume intensities were determined in the soluble fraction after heat treatment and a high-velocity spin and compared with intensities in total mitochondrial lysates. The relative difference of spot intensities in supernatant vs. total (set to 100%) for individual protein species are shown as means of three independent experiments.

FIGURE 2:

Aggregation of model proteins during heat stress. (A) Isolated mitochondria were treated at the indicated temperatures, and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (B) Spheroblasts were created from whole yeast cells to study aggregation in vivo. Cells were then stressed for 30 min at indicated temperatures, and mitochondria were isolated and analyzed as described above. (C–F) Enzyme activity assays of TCA cycle enzmes. Activity of aconitase (C), malate dehydrogenase (D), and alpha-ketoglutrarate (E) and pyruvate (F) dehydrogenase complexes were measured after treatment of isolated mitochondria at temperatures ranging from 25°C to 42°C.

FIGURE 3:

Kinetics of aggregation and decline of enzymatic activities. (A) Aggregation time course. Isolated mitochondria were incubated at 42°C for the indicated time spans, and aggregates were separated by ultracentrifugation. T, total mitochondrial lysate; Sup, supernatant; Pel, aggregate-containing pellet. (B) Decline of aconitase activity was measured in mitochondrial lysates after treatment at 25°C or 42°C for the indicated time spans. Values shown are means ± SEM of three independent experiments.

FIGURE 4:

Aggregation of model proteins during oxidative stress. (A, B) Isolated mitochondria from WT, sod1Δ and sod2Δ strains were treated at 25°C with the indicated amounts of oxidative stressor ranging from 0 to 20 mM H₂O₂ (A) or 0 to 2 mM menadione (B), and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (C) Mitochondria from wild-type (WT) or sod2Δ yeast strains were treated at the indicated temperatures, and aggregates separated by ultracentrifugation. Pellets were analyzed by SDS–PAGE and Western blotting. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.

FIGURE 5:

Protection from aggregation by the mitochondrial Hsp70 and Hsp60 chaperone systems. (A) Dependence of aggregation on mitochondrial ATP levels. Isolated mitochondria were lysed and then either supplied with 5 mM ATP (+ATP), or treated with apyrase to deplete endogenous ATP (–ATP). After heat treatment at indicated temperatures, aggregates were spun down by ultracentrifugation, and the relative amount of aggregated protein was determined. (B) Isolated mitochondria were treated at indicated temperatures and then analyzed after ultracentrifugation at 125,000 × g. Total (T), supernatant (Sup), and pellet (Pel) were subjected to SDS–PAGE, Western blotting, and immunodecoration with specific antisera against the indicated mitochondrial proteins. (C, D) Aggregation in Hsp70 mutants. Isolated mitochondria from wild-type (WT) and either conditional mutant ssc1–3 (C) or deletion mutant mdj1Δ (D) were analyzed in the aggregation assay. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate. (E) Aggregation in mitochondria from temperature-sensitive strain MIF4 after inactivation of the Hsp60 chaperone.

FIGURE 6:

Protection from aggregation by other quality control components. (A, B) Isolated mitochondria from wild-type (WT) and deletion mutants hsp78Δ (A) or pim1Δ (B, upper panels) or from yeast overexpressing Pim1 from a plasmid (B, lower panels) were analyzed in the aggregation assay. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.

FIGURE 7:

Comparison of the aggregation of newly imported and steady-state Ilv2. (A) Radioactively labeled [³⁵S]-Ilv2 was imported into isolated wild-type (WT) or ssc1–3 mitochondria, and its aggregation behavior was investigated directly after import control) or following a 20-min heat treatment at 37°C (heat shock). The solubility of endogenous steady-state Ilv2 was examined by Western blotting and immunodecoration with a specific antiserum (WB). As further controls, the soluble matrix enzyme Cit1 and the ribosomal protein Mrpl40 were also detected by immunodecoration. T, total; S, supernatant; P, pellet. (B) Quantification of the percentage of total newly imported or steady-state Ilv2 found in the aggregate pellet in wild-type (WT) or pim1Δ mitochondria.

FIGURE 8:

Overview of the protein quality control system of the mitochondrial matrix. During stress conditions, proteins become unfolded and subsequently aggregate because of irregular intermolecular interactions of exposed hydrophobic patches. mtHsp70 promotes refolding of misfolded proteins, prevents their aggregation, and cooperates with the ClpB homologue Hsp78 in disaggregation. The ATP-dependent protease Pim1 degrades misfolded proteins to protect them from aggregation, a reaction, which is assisted also by the action of Hsp78.