Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase

Abstract

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.

FIG. 1.

Suppression of the atp3Δ slow-growth phenotype by atp2-227. The indicated strains were streaked on YPD and YPEG media (A) and on synthetic dextrose SD medium lacking or containing 25μg/ml EtBr (B) and incubated for 5 days at 30°C. Growth of yeast in the presence of EtBr induces the quantitative loss of mtDNA (12, 31). Strains: wild type (WT), PTY44; atp3Δ, JTY3; atp2-227 atp3Δ, JTY6; atp2-227, TCY55.

FIG. 2.

Rescue of atp3Δ slow-growth phenotype by atp2-227. atp2Δ atp3Δ yeast cells were transformed with pRS316 (vector control), pATP2 (wild-type ATP2), and pATP2-227 (atp2-227). Transformed strains were grown on plates of SD medium lacking uracil (URA) and lacking (ρ⁺) or containing (ρ°) 25 μg/ml EtBr and then streaked to SD medium lacking uracil and incubated for 5 days at 30°C to compare growth.

FIG. 3.

atp2-227 encodes a change from glycine to serine at residue 227 of the β-subunit of the F₁-ATPase. (A) Predicted amino acid sequence of residues 201 to 249 of the yeast wild-type ATP2 and atp2-227 alleles aligned with the comparable region of bovine ATP2 using CLUSTALW (33). Black boxes indicate nonconserved amino acid substitutions, and gray boxes indicate conservative substitutions. The arrow marks position 227. (B) A three-dimensional rendering of the bovine F₁-ATPase active site with the equivalent glycine residue (yeast residue 227 is equivalent to bovine βGLY193) was created using the program VMD, version 1.8.2 (15).

FIG. 4.

F₁F_o-ATPase assembly in atp2-227, atp3Δ, and atp3Δ atp2-227 yeast. A total of 100 μg (ρ⁺ yeast) or 150 μg (ρ° yeast) of mitochondria isolated from the indicated strains in 1% digitonin buffer was resolved by BN-PAGE on a 4 to 10% polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with antibodies against Atp2p. The band corresponding to the monomer of the F₁F_o-ATPase migrated with a molecular weight slightly smaller than thyroglobulin (669 kDa; data not shown). Strains: wild type (WT) ρ⁺, PTY44 ρ⁺; atp2-227 atp3Δ ρ°, JTY6; atp3Δ ρ°, JTY3; atp2-227 ρ⁺, TCY55; WT ρ°, PTY44 ρ°.

FIG. 5.

atp2-227 atp3Δ ρ° and atp2-227 ρ° yeast require the α subunit (ATP1) of the F₁-ATPase for vigorous growth. The indicated strains were streaked to SD medium lacking (A) and containing (B) 25 μg/ml EtBr and incubated for 5 days at 30°C. Strains: wild type (WT), PTY44; atp3Δ, JTY3; atp2-227, TCY55; atp2-227 atp3Δ, JTY6; atp1Δ, TCY46; atp1Δ atp2-227, TCY103; atp1Δ atp3Δ, TCY64; atp1Δ atp2-227atp3Δ, TCY102.

FIG. 6.

atp2-227 atp3Δ ρ° yeast generates a greater inner mitochondrial membrane potential in response to ATP compared to atp3Δ ρ° yeast. At the indicated times, 250 μg of mitochondria isolated from the indicated strains (M), ATP to a final concentration of 1 mM (A), and 50 ng of valinomycin in 95% ethanol (V) were added to a final volume of 2.5 ml of reaction buffer containing 1 μM rhodamine 123 (Molecular Probes) (11, 14). The relative fluorescence was monitored using a FluoroMax-2 spectrofluorometer operating in the steady-state mode. The samples were excited at 502 nm, and emission was measured at 525 nm. Strains: wild type (WT) ρ⁺, PTY44 ρ⁺; WT ρ°, PTY44 ρ°; atp3Δ ρ°, JTY3; atp2-227 atp3Δ ρ°, JTY6; atp2Δ atp3Δ ρ⁺, TCY60; atp2-227 ρ⁺, TCY55.