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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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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.
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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.

Mentions: The protective effects of both the Hsp70 and Hsp60 chaperone systems on aggregation of mitochondrial proteins raised the question of whether especially newly imported proteins are vulnerable to aggregation, as both chaperone systems are responsible for efficient folding reactions inside the organelle (Cheng et al., 1989; Horst et al., 1997). To differentiate between proteins that were recently imported and proteins that have already adopted their native conformation under steady-state conditions, we synthesized and labeled the aggregation-prone model protein Ilv2 by in vitro translation in rabbit reticulocyte lysate in the presence of [35S]-methionine. Radioactively labeled Ilv2 can be imported into isolated mitochondria and detected by SDS–PAGE followed by autoradiography. Directly after import, we subjected mitochondria to a 20-min heat treatment at 37°C to induce aggregation, isolated the aggregates by detergent lysis and a high-velocity spin, and finally detected newly imported [35S]-Ilv2 by autoradiography and steady-state Ilv2 by immunodecoration with a specific antiserum. Because the amounts of imported labeled preproteins are usually too low to be detected by Western blot, both species of the same protein can be distinguished. Indeed, whereas a considerable amount of total Ilv2 remained soluble in wild-type mitochondria following heat treatment, newly imported Ilv2 was found almost exclusively in the pellet (Figure 7A, lanes 8 and 9). This indicates that newly imported Ilv2 is more vulnerable to aggregation, possibly because it has not yet folded to its native conformation. Consequently, as unfolded polypeptides should be stabilized by the mtHsp70 system, we also investigated aggregation of newly imported Ilv2 in ssc1–3 mitochondria, where the temperature-sensitive mtHsp70 had been inactivated by a short heat treatment. Whereas both newly imported and total Ilv2 aggregated completely after treatment at 37°C in ssc1–3 (Figure 7A, lanes 11 and 12), a significant fraction of imported Ilv2 was found in the pellet even without any heat treatment (Figure 7A, lanes 5 and 6). We thus conclude that Ssc1 not only has a stabilizing effect on fully folded Ilv2, but is also required to prevent aggregation of still unfolded or incompletely folded protein directly after the import process. In addition, we also investigated aggregation of newly imported Ilv2 in mitochondria from the pim1Δ mutant strain, as also Pim1 was shown to protect steady state Ilv2 from aggregation (see Figure 6). However, no significant difference concerning the aggregation behavior between newly imported and steady-state Ilv2 could be detected in pim1Δ (Figure 7B), indicating that the protease is not directly involved in the protection of newly imported proteins against aggregation.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: The protective effects of both the Hsp70 and Hsp60 chaperone systems on aggregation of mitochondrial proteins raised the question of whether especially newly imported proteins are vulnerable to aggregation, as both chaperone systems are responsible for efficient folding reactions inside the organelle (Cheng et al., 1989; Horst et al., 1997). To differentiate between proteins that were recently imported and proteins that have already adopted their native conformation under steady-state conditions, we synthesized and labeled the aggregation-prone model protein Ilv2 by in vitro translation in rabbit reticulocyte lysate in the presence of [35S]-methionine. Radioactively labeled Ilv2 can be imported into isolated mitochondria and detected by SDS–PAGE followed by autoradiography. Directly after import, we subjected mitochondria to a 20-min heat treatment at 37°C to induce aggregation, isolated the aggregates by detergent lysis and a high-velocity spin, and finally detected newly imported [35S]-Ilv2 by autoradiography and steady-state Ilv2 by immunodecoration with a specific antiserum. Because the amounts of imported labeled preproteins are usually too low to be detected by Western blot, both species of the same protein can be distinguished. Indeed, whereas a considerable amount of total Ilv2 remained soluble in wild-type mitochondria following heat treatment, newly imported Ilv2 was found almost exclusively in the pellet (Figure 7A, lanes 8 and 9). This indicates that newly imported Ilv2 is more vulnerable to aggregation, possibly because it has not yet folded to its native conformation. Consequently, as unfolded polypeptides should be stabilized by the mtHsp70 system, we also investigated aggregation of newly imported Ilv2 in ssc1–3 mitochondria, where the temperature-sensitive mtHsp70 had been inactivated by a short heat treatment. Whereas both newly imported and total Ilv2 aggregated completely after treatment at 37°C in ssc1–3 (Figure 7A, lanes 11 and 12), a significant fraction of imported Ilv2 was found in the pellet even without any heat treatment (Figure 7A, lanes 5 and 6). We thus conclude that Ssc1 not only has a stabilizing effect on fully folded Ilv2, but is also required to prevent aggregation of still unfolded or incompletely folded protein directly after the import process. In addition, we also investigated aggregation of newly imported Ilv2 in mitochondria from the pim1Δ mutant strain, as also Pim1 was shown to protect steady state Ilv2 from aggregation (see Figure 6). However, no significant difference concerning the aggregation behavior between newly imported and steady-state Ilv2 could be detected in pim1Δ (Figure 7B), indicating that the protease is not directly involved in the protection of newly imported proteins against aggregation.

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