Solution Structure of the Catalytic Domain of the Mitochondrial Protein ICT1 That Is Essential for Cell Vitality

Yoshihiro Handa, Yusuke Hikawa, Naoya Tochio, Hiroyuki Kogure, Makoto Inoue, Seizo Koshiba, Peter Güntert, Yusuke Inoue, Takanori Kigawa, Shigeyuki Yokoyama, Nobukazu Nameki

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38 Citations (Scopus)

Abstract

The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a β1-β2-α1-β3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.

Original languageEnglish
Pages (from-to)260-273
Number of pages14
JournalJournal of Molecular Biology
Volume404
Issue number2
DOIs
Publication statusPublished - 2010 Nov 26
Externally publishedYes

Keywords

  • Flow cytometry
  • GGQ motif
  • Mitoribosome
  • Peptidyl-tRNA hydrolysis
  • Protein structure

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Handa, Y., Hikawa, Y., Tochio, N., Kogure, H., Inoue, M., Koshiba, S., Güntert, P., Inoue, Y., Kigawa, T., Yokoyama, S., & Nameki, N. (2010). Solution Structure of the Catalytic Domain of the Mitochondrial Protein ICT1 That Is Essential for Cell Vitality. Journal of Molecular Biology, 404(2), 260-273. https://doi.org/10.1016/j.jmb.2010.09.033