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Vol. 20, Issue 21, 4524-4530, November 1, 2009

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Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

Ryo Yonashiro^*, Ayumu Sugiura^*, Misako Miyachi^*, Toshifumi Fukuda^*, Nobuko Matsushita^*, Ryoko Inatome^*, Yoshinobu Ogata^*, Takehiro Suzuki^,, Naoshi Dohmae^,, and Shigeru Yanagi^*

*Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan; Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan; and CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Submitted February 9, 2009; Revised August 21, 2009; Accepted August 27, 2009
Monitoring Editor: Donald D. Newmeyer

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin–proteasome pathway.

Address correspondence to: Shigeru Yanagi (syanagi{at}toyaku.ac.jp).

Abbreviations used: ALS, amyotrophic lateral sclerosis; CHX, cycloheximide; ERAD, endoplasmic reticulum–associated degradation.