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Formation of an Energized Inner Membrane in Mitochondria with a -Deficient F₁-ATPase

Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071

Received 4 August 2005/ Accepted 27 September 2005

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 F₁F_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 F₁-ATPase are hypothesized to be required for this process in vivo. Deletion of ATP3, the structural gene for the subunit of the F₁-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 ß subunit of F₁-ATPase (atp2-227). This mutation allowed substantial ATP hydrolysis by the F₁-ATPase even in the absence of the subunit, enabling yeast to generate a twofold greater inner membrane potential in response to ATP compared to mitochondria isolated from yeast lacking the subunit and containing wild-type ß subunits. Analysis of the suppressing mutation by blue native polyacrylamide gel electrophoresis also revealed that the ₃ß₃ heterohexamer can form in the absence of the subunit.