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Solution structure and siRNA-mediated knockdown analysis of the mitochondrial disease-related protein C12orf65

Authors

  • Hiroyuki Kogure,

    1. Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
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  • Yusuke Hikawa,

    1. Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
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  • Mamoru Hagihara,

    1. Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
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  • Naoya Tochio,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Seizo Koshiba,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Yusuke Inoue,

    1. Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
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  • Peter Güntert,

    1. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, and Frankfurt Institute for Advanced Studies, Goethe University, Frankfurt am Main, Germany
    2. Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan
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  • Takanori Kigawa,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    2. Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8502, Japan
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  • Shigeyuki Yokoyama,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    2. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-Ku, Tokyo 113-0033, Japan
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  • Nobukazu Nameki

    Corresponding author
    1. Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
    • Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-Cho, Kiryu, Gunma 376-8515, Japan
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Abstract

Loss of function of the c12orf65 gene causes a mitochondrial translation defect, leading to encephalomyopathy. The C12orf65 protein is thought to play a role similar to that of ICT1 in rescuing stalled mitoribosomes during translation. Both proteins belong to a family of Class I peptide release factors (RFs), all characterized by the presence of a GGQ motif. Here, we determined the solution structure of the GGQ-containing domain (GGQ domain) of C12orf65 from mouse by NMR spectroscopy, and examined the effect of siRNA-mediated knockdown of C12orf65 on mitochondria in HeLa cells using flow cytometry. The GGQ domain, comprising residues 60–124 of the 184-residue full-length protein, forms a structure with a 310-β1-β2-β3-α1 topology that resembles the GGQ domain structure of RF more closely than that of ICT1. Thus, the GGQ domain structures of this protein family can be divided into two types, depending on the region linking β2 and β3; the C12orf65/RF type having a 6-residue π-HB turn and the ICT1 type having an α-helix. Knockdown of C12orf65 resulted in increased ROS production and apoptosis, leading to inhibition of cell proliferation. Substantial changes in mitochondrial membrane potential and mass in the C12orf65-knockdown cells were observed compared with the control cells. These results indicate that the function of C12orf65 is essential for cell vitality and mitochondrial function. Although similar effects were observed in ICT1-downregulated cells, there were significant differences in the range and pattern of the effects between C12orf65- and ICT1-knockdown cells, suggesting different roles of C12orf65 and ICT1 in rescuing stalled mitoribosomes. Proteins 2012. © 2012 Wiley Periodicals, Inc.

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