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

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

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.

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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.
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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.

Mentions: Mitochondria contain an elaborate network of protein quality control, consisting of chaperones and proteases. Protein quality control is an energy-consuming process, as the relevant chaperones and proteases all have intrinsic ATPase activities. To assess the impact of the protein quality control system on the aggregation of mitochondrial proteins, we therefore first tested the dependence of aggregation on mitochondrial ATP levels. We compared protein aggregation levels in yeast mitochondrial lysates supplied with ATP with corresponding samples where endogenous ATP was depleted by treatment with apyrase. Aggregation of both Ilv2 and Aco1 was indeed increased in ATP-depleted samples at 42°C (Figure 5A), indicating that ATP-dependent processes are needed to secure mitochondrial protein homeostasis. Additionally, without ATP, significantly stronger aggregation levels of Aco1 could be observed already at 37°C, although the majority of Aco1 still remained soluble at this temperature.


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

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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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.
Mentions: Mitochondria contain an elaborate network of protein quality control, consisting of chaperones and proteases. Protein quality control is an energy-consuming process, as the relevant chaperones and proteases all have intrinsic ATPase activities. To assess the impact of the protein quality control system on the aggregation of mitochondrial proteins, we therefore first tested the dependence of aggregation on mitochondrial ATP levels. We compared protein aggregation levels in yeast mitochondrial lysates supplied with ATP with corresponding samples where endogenous ATP was depleted by treatment with apyrase. Aggregation of both Ilv2 and Aco1 was indeed increased in ATP-depleted samples at 42°C (Figure 5A), indicating that ATP-dependent processes are needed to secure mitochondrial protein homeostasis. Additionally, without ATP, significantly stronger aggregation levels of Aco1 could be observed already at 37°C, although the majority of Aco1 still remained soluble at this temperature.

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

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.

Show MeSH
Related in: MedlinePlus