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.
Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5 iso1 and SIR-T5 iso2 ) encoded by the human SIRT5 gene and whose C-termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N-termini, we found that the cleaved SIRT5 iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5 iso1 was localized in both mitochondria and cytoplasm. SIRT5DC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2 . These results suggest that the cytoplasmic localization of cleaved SIRT5 iso1 is dependent on the SIRT5 iso1 -specific C-terminus. Further analysis showed that the C-terminus of SIRT5 iso2 , which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5 iso1 -specific C-terminus is necessary for maintaining the stability of SIRT5 iso1 . Moreover, genome sequence analysis from each organism examined indicated that SIRT5 iso2 is a primate-specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate-specific functions via two isoforms with different intracellular localizations or stabilities.
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