USP16 regulates mitochondrial function by deubiquitinating TOM40, a core component of the translocase in the mitochondrial outer membrane.

Polycomb Repressive Complex 1 (PRC1) and its antagonistic H2A deubiquitinase USP16 have been regarded as nuclear proteins acting on chromatin. However, our recent studies revealed that a fraction of PRC1 and USP16 also localizes to mitochondria, where PRC1 plays a critical role in maintaining mitochondrial function. The role of USP16 in this context, however, has not yet been investigated. This study shows that USP16 interacts with and stabilizes TOM40, the core subunit of the TOM complex. USP16 deubiquitinates K48-linked ubiquitin chains from lysine 175, 184, and 309 of TOM40. USP16 knockout disrupts mitochondrial homeostasis, resulting in impaired membrane potential, elevated reactive oxygen species, reduced ATP synthesis, and altered oxygen consumption rates. Expression of FLAG-tagged USP16 restores TOM40 protein levels and rescues mitochondrial function. TOM40 re-expression in USP16 KO cells partially restores mitochondrial function, confirming its essential role. In summary, this study reveals that USP16 regulates mitochondrial function through TOM40.