To date, three of the thirteen OXPHOS genes still encoded in the mitochondria have been allotopically expressed (AE) in human cells with mutated versions of the same gene, and thereby rescued a respiratory defect: ATPase61,2, ND43, and ND14. In work funded by SENS Foundation, Corral-Debrinski’s group have used their improved AE technique of relocalizing translation of AE genes to the mitochondrial surface using the nuclear-endoded COX10 3’UTR to reverse blindess in rats caused by exposure to mutations in this gene (both genes introduced by electroporation)5. Now we have the first report of a new gene, COX2, being allotopically expressed in yeast, by mutating the gene to overcome the hydrophobicity of the mitochondrial membrane:
… Nuclear-recoded [meaning nuclear recoded, ie, allotopically-expressed -MR] Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 –> R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2(W56R) can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4.6
This is an exciting advance, albeit only in yeast. A similar amino acid change, which also reduces the protein’s hydrophobicity, has been shown to underlie the ability of the unusual case of the soybean COX2, which is a case of a native nuclear-encoded COX2 gene.7 Even in that case, the authors’ proof was only performed with isolated mitochondria in vitro, and not in intact cells. This is the first allotopic expression of this respiratory chain component in a species in which evolution has never accomplished the feat for itself. The next step, of course, is to do it in mammalian cells — preferably, of our own species. And the same broad strategy likely applies to many of the other remaining 13; indeed, during his work sponsored by SENS Foundation, Mark Hamalainen developed software that models hydrophobicity of proteins, and it predicts that a relatively small number of relatively minor amino acid changes would lower the hydrophobicity of several of the 13 nuclear-encoded ETS structural components sufficiently to make them importable when the native gene likely is not.
Additionally, and initially concerningly, the authors state that “In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3′-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP23 ′-UTR.” This might be thought to limit the potential of the exciting progress that has been made using this method by Corral-Debrinski’s group. However, Supekova et al. did not actually perform a positive control to demostrate that they could replicate her protocol to achieve import of any gene, let alone COX2: the only validation of their ability to use the general techniqe that they report performing was functional assays with the ATP23 ′-UTR itself, with no mention of any efforts to verify directly that the mRNA was actually being targeted to the mitochondrial surface, let alone that it would not be importable once there. However, it is conceivable that there are genes for which mitochondrial mRNA localization it won’t be helpful, which is fine so long as there’s an alternative solution available, as they demonstrate, in this case, that there is. And if both methods actually work, we will have our choice: we can compare the 2 methods’ resulting relative import rates, and also their level of rescue of respiration (which is likely to be higher by relocalized translation of the WT protein than by a protein with an altered AA sequence).
1: Zullo SJ, Parks WT, Chloupkova M, Wei B, Weiner H, Fenton WA, Eisenstadt JM, Merril CR. Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res. 2005 Spring;8(1):18-28. PubMed PMID: 15798371.
2. Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J, Schon EA. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet. 2002 Apr;30(4):394-9. Epub 2002 Feb 25. PubMed PMID: 11925565.
3. Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol. 2002 Nov;52(5):534-42. PubMed PMID: 12402249.
4.Bonnet C, Augustin S, Ellouze S, Bénit P, Bouaita A, Rustin P, Sahel JA, Corral-Debrinski M. The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes. Biochim Biophys Acta. 2008 Oct;1783(10):1707-17. Epub 2008 May 6. PubMed PMID: 18513491.
5. Ellouze S, Augustin S, Bouaita A, Bonnet C, Simonutti M, Forster V, Picaud S, Sahel JA, Corral-Debrinski M. Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction. Am J Hum Genet. 2008 Sep;83(3):373-87. Epub 2008 Sep 4. PMID: 18771762 [PubMed – indexed for MEDLINE]
6. Supekova L, Supek F, Greer JE, Schultz PG. A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5047-52. Epub 2010 Mar 1. PubMed PMID: 20194738.
7. Daley DO, Clifton R, Whelan J. Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10510-5. Epub 2002 Jul 25. PubMed PMID: 12142462; PubMed Central PMCID: PMC124958.