BRAWNIN: A sORF-encoded Peptide Essential for Vertebrate Mitochondrial Complex III

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome is enriched for proteins smaller than 100 a.a. (defined as SEPs). Using a prediction and validation pipeline for small open-reading-frame (sORF)-encoded peptides (SEPs), we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN (BR), a 71 amino acid peptide encoded by the C12orf73 gene, is essential for respiratory chain complex III (CIII) assembly. In human cells, BR is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In vivo, BR is enriched in muscle tissues and its maternal zygotic deletion in zebrafish causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BR is essential for oxidative phosphorylation across vertebrate species. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism. The dataset included in this entry is for the Zebrafish and MEF BR knockouts.