Abstract Mitochondrial alternative open reading frames (ORFs) substantially broaden the functional scope traditionally attributed to mitochondrial DNA, encoding peptides and proteins that participate in diverse cellular processes. These newly identified ORFs are embedded within annotated sequences, both coding and non-coding, and reveal layers of overlapping genetic information. We report the discovery of MTALTCO1, a 259 amino-acid protein, the longest mitochondrial alternative protein identifie