A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

The longest mitochondrial protein in metazoans is encoded by the male-transmitted mitogenome of the bivalve Scrobicularia plana.

Cytochrome c oxidase subunit II (COX2) is one of the three mitochondrially encoded proteins of the complex IV of the respiratory chain that catalyses the reduction of oxygen to water. The cox2 gene spans about 690 base pairs in most animal species and produces a protein composed of approximately 230 amino acids. We discovered an extreme departure from this pattern in the male-transmitted mitogenome of the bivalve Scrobicularia plana with doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA), which possesses an important in-frame insertion of approximately 4.8 kb in its cox2 gene. This feature-an enlarged male cox2 gene-is found in many species with DUI; the COX2 protein can be up to 420 amino acids long. Through RT-PCRs, immunoassays and comparative genetics, the evolution and functionality of this insertion in S. plana were characterized. The in-frame insertion is conserved among individuals from different populations and bears the signature of purifying selection seemingly indicating maintenance of functionality. Its transcription and translation were confirmed: this gene produces a polypeptide of 1892 amino acids, making it the largest metazoan COX2 protein known to date. We hypothesize that these extreme modifications in the COX2 protein affect the metabolism of mitochondria containing the male-transmitted mtDNA in Scrobicularia plana.