Abstract
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 CRL5Ozz 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 CRL5Ozz 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.
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Data availability
All data supporting the findings of this study are available in the article and its supplementary information. Additional information can be obtained from the corresponding authors upon request. Metabolomics data have been uploaded to the Metabolomics Workbench; the accession number is: ST004190. All numerical source data underlying Figs. 3–6 and Supplementary Figs. S3, S4, S6, and S7, together with the complete metabolomic dataset, are supplied in the Supplementary Data. Uncropped blot and gel images can be found in the Supplementary Information.
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Acknowledgements
We would like to thank Dr. George Campbell of the St. Jude Cell and Tissue Imaging Center for help with image acquisitions. We gratefully acknowledge Dr. Tommaso Nastasi for his valuable contributions during the initial phase of this project and Dr. Angela McArthur (St. Jude Department of Scientific Editing) for editing the manuscript. The C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/GanlH mice were obtained from the MRC Harwell Institute, which distributes this strain on behalf of the European Mouse Mutant Archive (EMMA: www.infrafrontier.eu). The repository number is EM:11343. The C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/GanlH mice were originally produced at The University of Dundee. A. d’Azzo holds an endowed chair in Genetics and Gene Therapy from the Jewelry Charity Fund. This study was funded in part by the National Institutes of Health grants AR049867, GM104981, and CA021764, the Assisi Foundation of Memphis, and the American Lebanese Syrian Associated Charities (ALSAC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Y.C. conceived the study, designed the experiments, analyzed the data, generated the mouse models, coordinated the efforts of all authors, and drafted the manuscript. G.P. designed, performed, and analyzed the soleus metabolomics and TCA data, and helped with the interpretation of the data and explanation of the methodology. E.G. maintained the animal colonies and performed surgeries. D.v.d.V. performed the isolation, purification, and analyses of exosomes from WT and mutant muscles, and helped with statistical analyses, and the editing of the manuscript and figures. K.V., J.J., and K.K. performed the 3D segmentation, rendering, and statistical analyses of muscle mitochondria from WT and mutant mice. J.W. wrote the macros for ImageJ/Fiji software used to analyze and quantify mitophagy, to determine the MyHC content in myofibers from WT and mutant muscle,s and to quantify ATPase activity in different fiber types. R. W. and C. R. obtained and processed TEM and SEM images. R.R. and S.R. helped with the mitochondrial OCR assays and the analyses of ΔΨm. X.Q. purified anti-Ozz and anti-Alix antibodies, prepared and maintained primary myoblast cultures, and performed Alix overexpression studies in C2C12 cells. J.D. performed proteomic analyses of C2C12 overexpressing tagged Ozz. J.T.O. and T.B. provided intellectual insights. G.C.G. provided intellectual insights, advised on the CRISPR/CAS9 mutagenesis, and edited the manuscript. A. d’A. conceived the study, designed the experiments, analyzed the data, oversaw all experiments, coordinated the efforts of all authors, secured the funding for this project, and wrote and edited the manuscript.
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Campos, Y., Palacios, G., Gomero, E. et al. Mitochondrial proteostasis regulated by CRL5Ozz and Alix modulates skeletal muscle metabolism and fiber type. Commun Biol (2025). https://doi.org/10.1038/s42003-025-09363-3
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DOI: https://doi.org/10.1038/s42003-025-09363-3
