A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.     

ATP synthase from Saccharomyces cerevisiae: location of the OSCP subunit in the peripheral stalk region

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase-avidin complex, the bound avidin has been localised close to the F(1) domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F(1) domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F(1) domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of alpha-subunits, which are on top of the F(1) domain distal from the F(o) membrane domain, the OSCP extends almost 10nm along the surface of F(1) down towards F(o) where it interacts with the C terminus of the b subunit, which extends up from F(o). The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S.cerevisiae. The map reveals a marked asymmetry in the F(o) part of the complex that can be attributed to subunits in the F(o) domain.     

Two mutations in mitochondrial ATP6 gene of ATP synthase,related to human cancer,affect ROS,calcium homeostasis and mitochondrial permeability transition in yeast

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.     

Expression and purification of a novel rice (Oryza sativa L.) mitochondrial ATP synthase small subunit in Escherichia coli

To clarify the function of the rice mitochondrial ATP synthase 6 kDa subunit (RMtATP6), a method of producing large quantities of this protein is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of RMtATP6. The RMtATP6 gene (GenBank Accession No. ) was cloned into the pGEX-6p-3 vector to allow expression of RMtATP6 as a glutathione S-transferase (GST) fusion protein. The RMtATP6-GST fusion protein was purified by affinity chromatography using a glutathione-Sepharose 4B column. A Western blot analysis using anti-GST antibody showed that the fusion protein was not degraded. After enzymatic cleavage of the GST tail, the RMtATP6 protein showed a molecular weight of around 6 kDa. The predicted pI of this protein is 10.01. After improving the conditions of expression and the purification procedures, the final yield of the entire expression and purification process was about 4.6 mg of pure RMtATP6 protein per liter of bacterial culture.     

Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded F_O domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS₁₇₅N) prevents F_O-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN₁₇₅D, aN₁₇₅K, aN₁₇₅I, and aN₁₇₅T), which, in light of a recently published atomic structure of yeast F_O indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN₁₇₅ and a nearby glutamate residue (aE₁₇₂) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS₁₇₅N mutation can be suppressed by second-site suppressors (aP₁₂S, aI₁₇₁F, aI₁₇₁N, aI₂₃₉F, and aI₂₀₀M), of which some are very distantly located (by 20–30?Å) from the original mutation. The possibility to compensate through long-range effects the aS₁₇₅N mutation is an interesting observation that holds promise for the development of therapeutic molecules.     

Genetic analysis of the X-chromosomal region 1E-2A of Drosophila melanogaster

Reversion mutagenesis of three single P elements located in the cytogenetic interval 1E-2A at the tip of the X chromosome of Drosophila melanogaster was used to recover new deletions in this chromosomal region. The deletions obtained include small aberrations within region 2A and larger lesions extending from 2A into 1E and 1B. All three screens also yielded terminal deficiencies. The new deficiencies, together with previously characterized rearrangements, were analyzed for their complementation behaviour with the maternal effect locus fs(1)Nasrat and lethal loci in the region. These analyses provide an overall genetic map of the interval 1E-2A. In addition, the smaller deletions were physically mapped within cloned genomic DNA of the 2A region.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Nucleotide sequences of the genes for the alpha,beta and epsilon subunits of wheat chloroplast ATP synthase

Summary The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582)

A novel Chlamydia muridarum antigen (TC0582) was used to vaccinate BALB/c mice. Mice were also immunized with other components of the ATP synthase complex (TC0580, TC0581, and TC0584), or with the major outer membrane protein (MOMP). TC0582 was also formulated in combination with TC0580, TC0581 or MOMP. TC0582 alone, or in combination with the other antigens, elicited strong Chlamydia-specific humoral and cellular immune responses. Vaccinated animals were challenged intranasally and the course of the infection was followed for 10 days. Based on percentage change in body weight, lung weight, and number of Chlamydia inclusion forming units recovered from the lungs, mice immunized with TC0582, TC0581 or MOMP, as single antigens, showed significant protection. Mice immunized with combinations of two antigens were also protected but the level of protection was not additive. TC0582 has sequence homology with the eukaryotic ATP synthase subunit A (AtpA). Therefore, to determine if immunization with TC0582, or with Chlamydia, elicited antibodies that cross-reacted with the mouse AtpA, the two proteins were printed on a microarray. Sera from mice immunized with TC0582 and/or live Chlamydia, strongly reacted with TC0582 but did not recognize the mouse AtpA. In conclusion, TC0582 may be considered as a Chlamydia vaccine candidate.     

Solution structure of subunit F(6) from the peripheral stalk region of ATP synthase from bovine heart mitochondria

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

A comparison of the proteins found in developing wild type larvae and developing lethal mutant larvae of Drosophila melanogaster

Summary Extracts of late larval lethal mutants were compared with extracts of wild type larvae of the same developmental age on double diffusion plates using 16 different antisera. Nearly all of the mutant extracts showed relative antigen concentration differences compared with the wild type and four of the mutants lacked a protein at death found in the wild type of the same developmental age. In each case it was a different protein. The results are discussed by considering the different ways in which mutations can lead to the loss of a protein in developing systems.