Mitochondrial proteostasis regulated by CRL5(Ozz) and Alix modulates skeletal muscle metabolism and fiber type.

High-energy-demanding tissues, such as skeletal muscle, rely on mitochondrial proteostasis for proper function. Two key quality-control mechanisms -the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles- help maintain mitochondrial proteostasis, but whether these processes interact remains unclear. Here, we show that CRL5(Ozz) and its substrate, Alix, localize to mitochondria and together regulate the levels and distribution of the mitochondrial solute carrier Slc25A4, which is essential for ATP production. In Ozz(-/-) or Alix(-/-) mice, skeletal muscle mitochondria exhibit similar morphological abnormalities, including swelling and dysmorphism, along with partially overlapping metabolomic alterations. We demonstrate that CRL5(Ozz) ubiquitinates Slc25A4, targeting it for proteasomal degradation, while Alix facilitates Slc25A4 loading into exosomes for lysosomal degradation. Loss of Ozz or Alix in vivo disrupts the steady-state levels of Slc25A4, impairing mitochondrial metabolism and triggering a switch in muscle fiber composition from oxidative, mitochondria-rich slow to glycolytic fast fibers.