Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae.
The mitochondrial F(1)F(0)-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of approximately 600 kDa. F(1)-ATPase is composed of alpha(3)beta(3)gammadeltaepsilon with an overall molecular mass of 370 kDa. The genes encoding bovine F(1)-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (DeltaalphaDeltabetaDeltagammaDeltadeltaDeltaepsilon). This strain expressing bovine F(1) is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F(1)-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F(1)- or F(1)F(0)-ATP synthase.
Puri
Neeti
N
Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, 3333 Greenbay Road, North Chicago, IL 60064, USA.
Lai-Zhang
Jie
J
Meier
Scott
S
Mueller
David M
DM
D025181:ATP Synthetase Complexes / Q000737:chemistry; D000818:Animals; D002244:Carbon / Q000378:metabolism; D002417:Cattle; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D008929:Mitochondria, Heart / Q000378:metabolism; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D002244:Carbon; D025181:ATP Synthetase Complexes; D006180:Proton-Translocating ATPases