single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-03, Vol.107 (11), p.5047-5052
• Main Authors: Supekova, Lubica, Supek, Frantisek, Greer, John E, Schultz, Peter G
• Format: Article
• Language: English
• Subjects: 3' Untranslated regions; 3' Untranslated Regions - genetics; Aerobiosis - drug effects; Alleles; Amino Acid Sequence; Amino Acid Substitution - genetics; Biological Sciences; Carbon sources; Cell growth; cell membranes; Cells; Coding; Cyclooxygenase-2; Cytochrome c; Cytochrome-c oxidase; Cytoplasm - drug effects; Cytoplasm - enzymology; Electron transport; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - genetics; Electron Transport Complex IV - metabolism; Evolution; Evolutionary genetics; Fermentation - drug effects; Gene expression; Gene Expression Regulation, Fungal - drug effects; Genetic mutation; Genomes; Glycerol - pharmacology; Hybrids; Hydrophobicity; Membranes; Mitochondria; Mitochondrial membranes; Mitochondrial Membranes - drug effects; Mitochondrial Membranes - enzymology; Molecular Sequence Data; Mutagenesis; mutants; Mutation; Mutation - genetics; Neurospora crassa; Phenotype; Plasmids; Protein Sorting Signals; Protein Structure, Tertiary; protein subunits; protein transport; Protein Transport - drug effects; Proteins; random mutagenesis; Saccharomyces cerevisiae; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Translation; transmembrane domain; Transmembrane domains; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1000735107

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded 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 [rightward arrow] 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. COX2W⁵⁶R 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. 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. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.