The coupling of mitoproteolysis and oxidative phosphorylation enables tracking of an active mitochondrial state through MitoTimer fluorescence.

The regulation of mitochondria function and health is a central node in tissue maintenance, ageing as well as the pathogenesis of various diseases. However, the maintenance of an active mitochondrial functional state and its quality control mechanisms remain incompletely understood. By studying mice with a mitochondria-targeted reporter that shifts its fluorescence from "green" to "red" with time (MitoTimer), we found MitoTimer fluorescence spectrum was heavily dependent on the oxidative metabolic state in the skeletal muscle fibers. The mitoproteolytic activity was enhanced in an energy dependent manner, and accelerated the turnover of MitoTimer protein and respiratory chain substrate, responsible for a green predominant MitoTimer fluorescence spectrum under the oxidative conditions. PGC1α, as well as anti-ageing regents promoted enhanced mitoproteolysis. In addition, cells with the green predominant mitochondria exhibited lower levels of MitoSox and protein carbonylation, indicating a favorable redox state. Thus, we identified MitoTimer as a probe for mitoproteolytic activity in vivo and found a heightened control of mitoproteolysis in the oxidative metabolic state, providing a framework for understanding the maintenance of active oxidative metabolism while limiting oxidative damages.