What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15 bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes.