Mutations on the N-terminal edge of the DELSEED loop in either the α or β subunit of the mitochondrial F1-ATPase enhance ATP hydrolysis in the absence of the central γ rotor

Abstract

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)β(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)β(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)β(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)β(3) subcomplex.

Fig 1

Suppression of ρ^o lethality by the atp1-7 and atp2-12 alleles after the deletion of ATP3 encoding the γ subunit of F₁-ATPase. (A) Growth phenotype of K. lactis cells on EB medium, which eliminates mtDNA. Cells were grown in YPD medium, diluted to an equal density in water, and spotted on YPD medium supplemented with EB. The cells were then incubated at 28°C for 7 days before being photographed. (B) Schematic showing the strategy for the disruption of the ATP3 gene encoding the γ subunit of F₁-ATPase. (C) Southern blot analysis showing the disruption of ATP3 in CK307 (atp2-12 atp3Δ::URA3) and CK308 (atp1-7 atp3Δ::URA3). WT, wild type.

Fig 2

Respiratory growth phenotype of K. lactis atp1-7 and atp2-12 mutants. Cells were streaked on the nonfermentable YPG (glycerol) medium and incubated at 28°C for 4 days, before being photographed. The double mutant CK314-2B (atp1-7 atp2-12) was transformed with the plasmids pUK-KlMGI2/HP and pCXJ22-KlATP2 expressing the wild-type ATP1 and ATP2 genes, respectively. A representative transformant was streaked on the plate and tested for respiratory growth.

Fig 3

The atp1-7 and atp2-12 mutations occur on the N-terminal edge of the DELSEED loops in the α and β subunits of F₁-ATPase. (A) Comparison of amino acid sequences from various organisms of the DELSEED loop regions where the Klatp1-7 and Klatp2-12 mutations occur. The DELSEED loop sequences are highlighted in pink. αPhe446 and βGly419 are converted to Ile and Asp (green), respectively, in the Klatp1-7 and Klatp2-12 mutants, respectively. Kl, K. lactis; Sc, S. cerevisiae; Bt, Bos taurus; Hs, Homo sapiens; Ba, thermophilic Bacillus. (B) Crystal structure of the α₃β₃ core of F₁-ATPase from thermophilic Bacillus PS3 (Protein Data Bank accession number 1SKY). For clarity, only half of the symmetric α₃β₃ core is shown. The DELSEED loops are highlighted in pink, and the N-terminal α helix preceding the DELSEED loops is shown in green. The amino acids αF398 and βG388 correspond to αF446 and βG419, respectively, which are mutated in the K. lactis atp1-7 and atp2-12 mutants, respectively. (C) Close-up view at the DELSEED loops in the α and β subunits, which may interact with each other in the γ-less enzyme.

Fig 4

The δ and ε subunits are not required for the suppressor phenotype of the mutant γ-less F₁-ATPase. The K. lactis strains were grown in YPD medium overnight, diluted in water, and spotted on YPD (glucose), YPG (glycerol), and EB media. Cells were grown for 5 days at 28°C before being photographed. The strains used are identified by their genotypes in the template on the right and consist of strains CK392/7 (atp1-7), CK401 (atp1-7 γΔ) CK408 (atp1-7 γΔ δΔ), CK409 (atp1-7 γΔ εΔ), CK412/1 (atp1-7 γΔ δΔ εΔ), CK391/18 (atp2-12), CK400 (atp2-12 γΔ), CK405 (atp2-12 γΔ δΔ), CK406 (atp2-12 γΔ εΔ), and CK410/6 (atp2-12 γΔ δΔ εΔ).

Fig 5

Purification and characterization of γ-less F₁-ATPases. (A) The chromosomal integration of atp1-7-His₆, but not ATP1-His₆ or ATP1, into atp1Δ γΔ cells suppresses the ρ^o lethal phenotype on YPD medium supplemented with EB. (B) Western blot analysis showing the copurification of Atp1-7-His₆ and Atp1-HiS₆ with the β subunit after nickel column chromatography. (C) BN-PAGE analysis of purified αF446I-His₆/β and α-His₆/β complexes. The protein samples were resuspended in Coomassie G-250 to a final concentration of 0.5% before electrophoresis. The proteins were visualized by immunoblotting using antibodies against the α and β subunits of F₁-ATPase.

Fig 6

Analysis of the γ-less F₁ complexes from isolated mitochondria. (A) Western blot analysis showing the steady-state levels of the α and β subunits in isolated mitochondria from the wild-type (PM6-7A), αΔ (CK196/1), γΔ (CK204), γΔ atp1-7 (CK308), and γΔ atp2-12 (CK307) strains. The mitochondrial matrix protein Ilv5 was used as a sample loading control. (B) BN-PAGE analysis of digitonin-solubilized mitochondria showing the aggregation of the γ-less F₁ complexes, which were visualized by immunoblotting using antibodies against the α and β subunits.

Fig 7

Analysis of γ-less F₁-ATPases by size exclusion chromatography. The α-His₆ and αF446I-His₆ pull-down products were analyzed on a calibrated S200 column. (A) Western blot analysis showing the elution profile of F₁ subunits. The wild-type F₁-ATPase was purified by chloroform extraction of submitochondrial particles and was used as a control. (B) Relative distribution of the F₁ α subunit in the fractions from size exclusion chromatography.