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Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

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Abstract

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (KRF-2008-359-C00026). This research was also supported in part by a grant (2009K001282) from the Brain Research Center of the twenty-first Century Frontier Research Program funded by the Ministry of Education, Science, and Technology, Republic of Korea (to C. C.). We acknowledge Dr. Richard J. Youle’s valuable comments on the manuscript and support in some aspects of the work. We also thank Soojay Banerjee for technical support.

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Authors and Affiliations

  1. Cell Signaling and Bioimaging Laboratory, Department of Bio and Brain Engineering, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon, 305-701, Korea

    Seung-Wook Ryu, Hyeon Joo Jeong, Myunghwan Choi & Chulhee Choi

  2. KI for Biocentury, KAIST, Daejeon, 305-701, Korea

    Seung-Wook Ryu & Chulhee Choi

  3. Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD, 21201, USA

    Mariusz Karbowski

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  1. Seung-Wook Ryu
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  2. Hyeon Joo Jeong
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  3. Myunghwan Choi
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  4. Mariusz Karbowski
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  5. Chulhee Choi
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Correspondence to Seung-Wook Ryu or Chulhee Choi.

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Supplementary Fig. 1

. The mitochondrial localization of OPA3. (A) The 20-kDa endogenous OPA3 was identified using anti-OPA3 antibody. HeLa cells were transfected with the indicated constructs, harvested, and analyzed by Western blotting using anti-OPA3 antibody (diluted 1:500). (B) Mitochondrial fraction with FLAG-OPA3 in control buffer or swelling buffer (OS) were digested by proteinase K (PK). Western blots of the separated proteins were probed with anti-FLAG, anti-Tom 20, and anti-OPA1 antibodies. (C) Mitochondrial fraction with OPA3-myc in control buffer or swelling buffer (OS) was digested by trypsin. Alkali (Na2CO3)-treated mitochondria were extracted, after ultracentrifugation to separate soluble and pelleted proteins. Western blots of the separated proteins were probed with anti-myc antibody (TIFF 20491 kb)

Supplementary Fig. 2. Effect of the patient OPA3 mutant (G93S) on mitochondrial morphology

(A) HeLa cells were transfected with OPA3-YFP or OPA3(G93S)-YFP. After 15 h, the cells were fixed and stained with fluorescently labeled Tom 20 antibody, and analyzed by confocal microscopy. Bar, 10 μm. (B) HeLa cells were transfected with the indicated constructs. Cells were treated with TRAIL for 6 h, stained with fluorescently labeled anti-cytochrome c antibody and analyzed by confocal microscopy. The number of cells displaying cytosolic cytochrome c was counted, and are shown as a percentage of the total cells counted in cell population (TIFF 19932 kb)

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Ryu, SW., Jeong, H.J., Choi, M. et al. Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation. Cell. Mol. Life Sci. 67, 2839–2850 (2010). https://doi.org/10.1007/s00018-010-0365-z

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  • DOI: https://doi.org/10.1007/s00018-010-0365-z

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