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Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria.

Kitamura N, Nakamura Y, Miyamoto Y, Miyamoto T, Kabu K, Yoshida M, Futamura M, Ichinose S, Arakawa H - PLoS ONE (2011)

Bottom Line: Deficiency of NIX also completely impaired MALM.The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria.These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.

View Article: PubMed Central - PubMed

Affiliation: Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, Tokyo, Japan.

ABSTRACT
Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.

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NIX interacts with the coiled-coil regions of Mieap via its BH3 domain.(A) Deletion mutants of Mieap. The expression vectors for two deletion mutants (Δ273 and Δ103-260) of Mieap were prepared, as well as Ad-Mieap-α ˜full. IF experiment was carried out with anti-Mieap antibody (Mieap: green) and DsRed-mito (Mito: red). HCT116 cells were infected with Ad-Mieap vectors at an MOI of 5. Scale bar  = 10 µm. (B) Deletion mutants of NIX. The expression vectors for NIX-full and three deletion mutants (NIXΔ188-219, NIXΔ1-119, and NIXΔBH3) of NIX were prepared (upper). The subcellular localization of NIX-full and NIX mutants was shown (lower). Red box indicates transmembrane region. Green box indicates BH3 region. IF experiment was carried out with anti-FLAG antibody (NIX: green) and DsRed-mito (Mito: red). HCT116 cells were transfected by the plasmids designated to express NIX and NIX mutants. Scale bar  = 10 µm. (C–G) NIX interacts with Mieap via the coiled-coil domains of Mieap and the BH3 domain of NIX. Immunoprecipitation experiment with various forms of Mieap and NIX was carried out to characterize the interaction of Mieap and NIX (C, E, F, and G). IP: immunoprecipitaion, IB: immunoblotting, pre: cell lysate from HCT116 cells before immunoprecipitaion, post: cell lysate from HCT116 cells after immunoprecipitation, mIgG: mouse IgG, rIgG: rabbit IgG.
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pone-0016060-g003: NIX interacts with the coiled-coil regions of Mieap via its BH3 domain.(A) Deletion mutants of Mieap. The expression vectors for two deletion mutants (Δ273 and Δ103-260) of Mieap were prepared, as well as Ad-Mieap-α ˜full. IF experiment was carried out with anti-Mieap antibody (Mieap: green) and DsRed-mito (Mito: red). HCT116 cells were infected with Ad-Mieap vectors at an MOI of 5. Scale bar  = 10 µm. (B) Deletion mutants of NIX. The expression vectors for NIX-full and three deletion mutants (NIXΔ188-219, NIXΔ1-119, and NIXΔBH3) of NIX were prepared (upper). The subcellular localization of NIX-full and NIX mutants was shown (lower). Red box indicates transmembrane region. Green box indicates BH3 region. IF experiment was carried out with anti-FLAG antibody (NIX: green) and DsRed-mito (Mito: red). HCT116 cells were transfected by the plasmids designated to express NIX and NIX mutants. Scale bar  = 10 µm. (C–G) NIX interacts with Mieap via the coiled-coil domains of Mieap and the BH3 domain of NIX. Immunoprecipitation experiment with various forms of Mieap and NIX was carried out to characterize the interaction of Mieap and NIX (C, E, F, and G). IP: immunoprecipitaion, IB: immunoblotting, pre: cell lysate from HCT116 cells before immunoprecipitaion, post: cell lysate from HCT116 cells after immunoprecipitation, mIgG: mouse IgG, rIgG: rabbit IgG.

Mentions: To characterize the interaction between Mieap and NIX, two Mieap mutants and two NIX mutants were generated (Figure 3A and 3B). While both Mieap mutants were unable to induce MALM (Figure S1), the MieapΔ273 mutant clearly localized to, and entered, the mitochondria without lysosomal accumulation, whereas the MieapΔ103–260 did not (Figure 3A). These findings suggest that the coiled-coil motif of Mieap plays a pivotal role in the targeting of and entry to the damaged mitochondria. As reported previously, NIXΔ1–119 and NIX localized to the mitochondrial outer membrane, but NIXΔ188–219 did not (Figure 3B) [9].


Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria.

Kitamura N, Nakamura Y, Miyamoto Y, Miyamoto T, Kabu K, Yoshida M, Futamura M, Ichinose S, Arakawa H - PLoS ONE (2011)

NIX interacts with the coiled-coil regions of Mieap via its BH3 domain.(A) Deletion mutants of Mieap. The expression vectors for two deletion mutants (Δ273 and Δ103-260) of Mieap were prepared, as well as Ad-Mieap-α ˜full. IF experiment was carried out with anti-Mieap antibody (Mieap: green) and DsRed-mito (Mito: red). HCT116 cells were infected with Ad-Mieap vectors at an MOI of 5. Scale bar  = 10 µm. (B) Deletion mutants of NIX. The expression vectors for NIX-full and three deletion mutants (NIXΔ188-219, NIXΔ1-119, and NIXΔBH3) of NIX were prepared (upper). The subcellular localization of NIX-full and NIX mutants was shown (lower). Red box indicates transmembrane region. Green box indicates BH3 region. IF experiment was carried out with anti-FLAG antibody (NIX: green) and DsRed-mito (Mito: red). HCT116 cells were transfected by the plasmids designated to express NIX and NIX mutants. Scale bar  = 10 µm. (C–G) NIX interacts with Mieap via the coiled-coil domains of Mieap and the BH3 domain of NIX. Immunoprecipitation experiment with various forms of Mieap and NIX was carried out to characterize the interaction of Mieap and NIX (C, E, F, and G). IP: immunoprecipitaion, IB: immunoblotting, pre: cell lysate from HCT116 cells before immunoprecipitaion, post: cell lysate from HCT116 cells after immunoprecipitation, mIgG: mouse IgG, rIgG: rabbit IgG.
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getmorefigures.php?uid=PMC3022033&req=5

pone-0016060-g003: NIX interacts with the coiled-coil regions of Mieap via its BH3 domain.(A) Deletion mutants of Mieap. The expression vectors for two deletion mutants (Δ273 and Δ103-260) of Mieap were prepared, as well as Ad-Mieap-α ˜full. IF experiment was carried out with anti-Mieap antibody (Mieap: green) and DsRed-mito (Mito: red). HCT116 cells were infected with Ad-Mieap vectors at an MOI of 5. Scale bar  = 10 µm. (B) Deletion mutants of NIX. The expression vectors for NIX-full and three deletion mutants (NIXΔ188-219, NIXΔ1-119, and NIXΔBH3) of NIX were prepared (upper). The subcellular localization of NIX-full and NIX mutants was shown (lower). Red box indicates transmembrane region. Green box indicates BH3 region. IF experiment was carried out with anti-FLAG antibody (NIX: green) and DsRed-mito (Mito: red). HCT116 cells were transfected by the plasmids designated to express NIX and NIX mutants. Scale bar  = 10 µm. (C–G) NIX interacts with Mieap via the coiled-coil domains of Mieap and the BH3 domain of NIX. Immunoprecipitation experiment with various forms of Mieap and NIX was carried out to characterize the interaction of Mieap and NIX (C, E, F, and G). IP: immunoprecipitaion, IB: immunoblotting, pre: cell lysate from HCT116 cells before immunoprecipitaion, post: cell lysate from HCT116 cells after immunoprecipitation, mIgG: mouse IgG, rIgG: rabbit IgG.
Mentions: To characterize the interaction between Mieap and NIX, two Mieap mutants and two NIX mutants were generated (Figure 3A and 3B). While both Mieap mutants were unable to induce MALM (Figure S1), the MieapΔ273 mutant clearly localized to, and entered, the mitochondria without lysosomal accumulation, whereas the MieapΔ103–260 did not (Figure 3A). These findings suggest that the coiled-coil motif of Mieap plays a pivotal role in the targeting of and entry to the damaged mitochondria. As reported previously, NIXΔ1–119 and NIX localized to the mitochondrial outer membrane, but NIXΔ188–219 did not (Figure 3B) [9].

Bottom Line: Deficiency of NIX also completely impaired MALM.The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria.These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.

View Article: PubMed Central - PubMed

Affiliation: Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, Tokyo, Japan.

ABSTRACT
Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.

Show MeSH
Related in: MedlinePlus