David M. Mueller

The ATP synthase is involved in generating mitochondrial cristae morphology

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non‐essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion‐like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane.

Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase

The crystal structure of yeast mitochondrial F1 ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the α3β3 sub‐assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F1 ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F1 ATPase has an ATP analog bound and therefore this structure does not represent the ADP‐inhibited form. In addition, one of the complexes binds phosphate in the nucleotide‐free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F1 ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.

Targeted exome sequencing of suspected mitochondrial disorders

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting.

Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation

The proton pore of the F1Fo ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state. Structures are here presented of the c10 ring from Saccharomyces cerevisiae determined at pH 8.3, 6.1 and 5.5, at resolutions of 2.0 Å, 2.5 Å and 2.0 Å, respectively. The overall structure of this mitochondrial c-ring is similar to known homologs, except that the essential carboxylate, Glu59, adopts an open extended conformation. Molecular dynamics simulations reveal that opening of the essential carboxylate is a consequence of the amphiphilic nature of the crystallization buffer. We propose that this new structure represents the functionally open form of the c subunit, which facilitates proton loading and release.

Oligomycin frames a common drug-binding site in the ATP synthase.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common “drug-binding site.” We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1‐ATPase is a multimeric enzyme (α3β3γδϵ) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding α‐, β‐, γ‐ or δ‐subunits were unable to grow, while the strain with a null mutation in ϵ was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in γ or δ became 100% ρ0/ρ− and the strain with the null mutation in γ grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: ΔαΔγ, ΔβΔγ, Δatp11Δγ, ΔαΔδ, ΔβΔδ or Δatp11Δδ. These results indicate that ϵ is not an essential component of the ATP synthase and that mutations in the genes encoding the α‐ and β‐subunits and in ATP11 are epistatic to null mutations in the genes encoding the γ‐ and δ‐subunits. These data suggest that the propensity to form ρ0/ρ− mutations in the γ and δ null deletion mutant stains and the slow growing phenotypes of the null γ mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.

Asymmetric Structure of the Yeast F1 ATPase in the Absence of Bound Nucleotides

The crystal structure of nucleotide-free yeast F1 ATPase has been determined at a resolution of 3.6 Å. The overall structure is very similar to that of the ground state enzyme. In particular, the βDP and βTP subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the γ and β subunits are as important as nucleotide occupancy in determining the conformational state of the β subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the β subunits adopting the open conformation. The adenine-binding pocket of the βTP subunit is disrupted in the apoenzyme, suggesting that the βDP subunit is responsible for unisite catalytic activity.

Pigment epithelium-derived factor binds to cell-surface F1-ATP synthase

Pigment epithelium‐derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a ∼ 60 kDa PEDF‐binding protein in bovine retina with Bos taurus F1‐ATP synthase β‐subunit, and that F1Fo‐ATP synthase components have been identified recently as cell‐surface receptors, we examined the direct binding of PEDF to F1. Size‐exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real‐time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF‐binding and angiostatin‐binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF‐binding proteins of ∼ 60 kDa. Antibodies to F1β‐subunit specifically captured PEDF‐binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell‐surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell‐surface F1, these results show that PEDF is a ligand for endothelial cell‐surface F1Fo‐ATP synthase. They suggest that PEDF‐mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.

Partial Assembly of the Yeast Mitochondrial ATP Synthase 1

The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F0 into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F0. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.

Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media

Two independent cold‐sensitive pet mutants in the gene (ATP5) coding for the oligomycin sensitivity conferring protein (OSCP) have been isolated in the yeast Saccharomyces cerevisiae. The mutations in both strains alter the initiating methionine codon in the ATP5 gene: ATG to ATA (Ile) and AAG (Lys). Western blot analysis of total yeast protein after the cells were grown at 18°C, 30°C, and 37°C, indicates that the level of OSCP decreased 80% relative to the wild type strain. In addition, the level of the oligomycin‐sensitive ATPase decreased 85% relative to the wild type strain, after growth at 30°C. These findings indicate that for S. cerevisiae, the level of oxidative phosphorylation can decrease 85% without showing a large growth defect on media containing glycerol at 30°C, but not at 18°C.