Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae.
The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.
Lai-Zhang
J
J
Department of Biochemistry and Molecular Biology, The Chicago Medical School, Chicago, IL 60064, USA.
Mueller
D M
DM
D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005816:Genetic Complementation Test; D008928:Mitochondria / Q000201:enzymology; D009097:Multienzyme Complexes / Q000235:genetics; D009154:Mutation; D010641:Phenotype; D021382:Protein Sorting Signals / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases