Sharon H. Ackerman

Function, structure, and biogenesis of mitochondrial ATP synthase.

Publisher Summary This chapter briefly reviews some recent advances in the areas of the subunit structure and the different functions of the ATP synthase, but focuses mainly on the roles of mitochondrial and nuclear gene products in biogenesis of the mitochondrial enzyme. Most of the discussion related to biogenesis will be centered on the ATP synthase of Saccharomyces cerevisiae as it is studies of this particular enzyme that have provided much of the currently available information on this topic. As a facultative anaerobe, S. cerevisiae is well suited for such studies because it can survive on fermentable carbon sources in the absence of mitochondrial respiration or oxidative phosphorylation. While all ATP synthases have similar gross structures and catalytic mechanism, the mitochondrial enzymes have acquired a set of subunit polypeptides during evolution that are absent in bacteria and chloroplasts. The subunit compositions of the mammalian and S. cerevisiae F1F0 are almost identical, underscoring the usefulness of yeast as a model for gaining information relevant to mitochondrial ATP synthases in higher eukaryotes. The appreciation of the importance of the ATP synthase, not only in energy metabolism, but also in other aspects of mitochondrial and cellular function, has likewise increased, mainly as a result of information gained from biochemical and genetic studies of S. cerevisiae. The dependence on the F1F0 complex of mtDNA integrity and cristae structure and the essentiality of F1 for maintaining Dc under both Δψ respiring and nonrespiring conditions are examples of the key role the ATPase plays in mitochondrial structure and function.

Atp11p and Atp12p are chaperones for F1-ATPase biogenesis in mitochondria

The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.

The α-subunit of the mitochondrial F1 ATPase interacts directly with the assembly factor Atp12p

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F1 component of the mitochondrial F1F0 ATP synthase. In this report, we show that the F1 α‐subunit co‐precipitates and co‐purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F1 α‐subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the α‐subunit. Yeast two‐hybrid screens confirm the direct association of Atp12p with the α‐subunit and indicate that the binding site for the assembly factor lies in the nucleotide‐binding domain of the α‐subunit, between Asp133 and Leu322. These studies provide the basis for a model of F1 assembly in which Atp12p is released from the α‐subunit in exchange for a β‐subunit to form the interface that contains the non‐catalytic adenine nucleotide‐binding site.

ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase

The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9‐168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the F0 component of the mitochondrial ATPase. As a consequence of the structural defect in F0, the ATPase activity of G95 mutants is not inhibited by rutamycin. The absence of subunit 9 in N9‐168 has been correlated with a lower steady‐state level of its mRNA and an increase in higher molecular weight precursor transcripts. These results suggest that the mutation is most likely to affect either translation of the ??? mRNA or processing or the primary transcript. We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin‐sensitive ATPase in G95 mutants. Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants. The nucleotide sequence of ATP13 indicates a primary translation product with an M??? of 42 897. The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria. No significant primary structure homology is detected with any protein in the most recent libraries.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

Chaperones of F1-ATPase

Mitochondrial F1-ATPase contains a hexamer of alternating α and β subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to β and α. In the absence of Atp11p and Atp12p, the hexamer is not formed, and α and β precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F1 assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486–1493) hypothesized that the chaperones themselves look very much like the α and β subunits, and proposed an exchange of Atp11p for α and of Atp12p for β; the driving force for the exchange was expected to be a higher affinity of α and β for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to β and Atp12p is bound to α, the two F1 subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F1 γ subunit that initiates the release of the chaperones from α and β and their further assembly into the mature complex.

The Molecular Chaperone, Atp12p, from Homo sapiens IN VITRO STUDIES WITH PURIFIED WILD TYPE AND MUTANT (E240K) PROTEINS

Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled α subunits of F1 and in so doing prevents the α subunit from associating with itself in non-productive complexes during assembly of the F1 moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12pE240K, which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 °C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.

The Drosophila gene 2A5 complements the defect in mitochondrial F1-ATPase assembly in yeast lacking the molecular chaperone Atp11p

Assembly of mitochondrial F1‐ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p. Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S. cerevisiae Atp11p. A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele. Furthermore, the product of this Drosophila gene was shown to interact with the S. cerevisiae F1 β subunit in the yeast two‐hybrid assay. These results indicate that Atp11p function is conserved in higher eukaryotes.

The Contribution of Coevolving Residues to the Stability of KDO8P Synthase

Background The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism. Methodology/Principal Findings The multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability) and suppression (restoring it), while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions. Conclusions/Significance Both sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein.

Accurate Simulation and Detection of Coevolution Signals in Multiple Sequence Alignments

Background While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones. Methodology/Principal Findings We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the in silico evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs. Conclusions/Significance Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most effective in each case.