Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase.
Eukaryotic cells require mitochondrial compartments for viability. However, the budding yeast Saccharomyces cerevisiae is able to survive when mitochondrial DNA suffers substantial deletions or is completely absent, so long as a sufficient mitochondrial inner membrane potential is generated. In the absence of functional mitochondrial DNA, and consequently a functional electron transport chain and F(1)F(o)-ATPase, the essential electrical potential is maintained by the electrogenic exchange of ATP(4-) for ADP(3-) through the adenine nucleotide translocator. An essential aspect of this electrogenic process is the conversion of ATP(4-) to ADP(3-) in the mitochondrial matrix, and the nuclear-encoded subunits of F(1)-ATPase are hypothesized to be required for this process in vivo. Deletion of ATP3, the structural gene for the gamma subunit of the F(1)-ATPase, causes yeast to quantitatively lose mitochondrial DNA and grow extremely slowly, presumably by interfering with the generation of an energized inner membrane. A spontaneous suppressor of this slow-growth phenotype was found to convert a conserved glycine to serine in the beta subunit of F(1)-ATPase (atp2-227). This mutation allowed substantial ATP hydrolysis by the F(1)-ATPase even in the absence of the gamma subunit, enabling yeast to generate a twofold greater inner membrane potential in response to ATP compared to mitochondria isolated from yeast lacking the gamma subunit and containing wild-type beta subunits. Analysis of the suppressing mutation by blue native polyacrylamide gel electrophoresis also revealed that the alpha(3)beta(3) heterohexamer can form in the absence of the gamma subunit.
Smith
Christopher P
CP
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
Thorsness
Peter E
PE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D004272:DNA, Mitochondrial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005800:Genes, Fungal; D016153:Genes, Suppressor; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000502:physiology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000172:deficiency / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D012694:Serine / Q000378:metabolism
D004272:DNA, Mitochondrial; D021122:Protein Subunits; D012694:Serine; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D006180:Proton-Translocating ATPases