A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression.
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 --> 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. 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 ATP2(3)('-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.
Supekova
Lubica
L
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Supek
Frantisek
F
Greer
John E
JE
Schultz
Peter G
PG
D020413:3' Untranslated Regions / Q000235:genetics; D000332:Aerobiosis / Q000187:drug effects; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation / Q000187:drug effects; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D020413:3' Untranslated Regions; D021382:Protein Sorting Signals; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV; D005990:Glycerol