Characterisation of a highly diverged mitochondrial ATP synthase peripheral stalk subunit b in Trypanosoma brucei

The mitochondrial F1Fo ATP synthase of Trypanosoma brucei has been studied in detail. Whereas its F1 moiety is relatively highly conserved in structure and composition, the same is not the case for the Fo part and the peripheral stalk. A core subunit of the latter, the normally conserved subunit b, could not be identified in trypanosomes suggesting that it might be absent. Here we have identified a 17 kDa mitochondrial protein of the inner membrane that is essential for normal growth, efficient oxidative phosphorylation and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicate that the protein is associated with the F1Fo ATP synthase. Its ablation reduces the levels of Fo subunits, but not those of F1, and disturbs the cell cycle. HHpred analysis showed that the protein has structural similarities to subunit b of other species, indicating that the Fo part of the trypanosomal ATP synthase does contain a highly diverged subunit b. Thus, the Fo part of the trypanosomal ATPase synthase may be more widely conserved than initially thought.

Mitochondrial ATP Synthase Subunit d, a Component of the Peripheral Stalk, is Essential for Growth and Heat Stress Tolerance in Arabidopsis thaliana

SUMMARYAs rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNAi to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V of the electron transport chain). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and provide new information about complex V assembly and quality control, as well as support the importance of mitochondrial respiration in normal plant growth and heat stress tolerance.SIGNIFICANCE STATEMENTThe energy converter, mitochondrial ATP synthase, is critical for all organisms, but the functional importance of ATP synthase subunit d remains largely unknown in plants. We demonstrate the contributions of subunit d to plant growth, development, and heat stress tolerance, as well as to ATP synthase stability, ROS signaling and mitochondrial dysfunction regulation.

Assembly of the peripheral stalk of ATP synthase in human mitochondria

The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F1-catalytic, peripheral stalk (PS), and c8-rotor ring modules. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, imported into the mitochondrial matrix, and assembled into the complex with the mitochondrial gene products ATP6 and ATP8. Intermediate vestigial ATPase complexes formed by disruption of nuclear genes for individual subunits provide a description of how the various domains are introduced into the enzyme. From this approach, it is evident that three alternative pathways operate to introduce the PS module (including associated membrane subunits e, f, and g). In one pathway, the PS is built up by addition to the core subunit b of membrane subunits e and g together, followed by membrane subunit f. Then this b-e-g-f complex is bound to the preformed F1-c8module by subunits OSCP and F6. The final component of the PS, subunit d, is added subsequently to form a key intermediate that accepts the two mitochondrially encoded subunits. In another route to this key intermediate, first e and g together and then f are added to a preformed F1-c8-OSCP-F6-b-d complex. A third route involves the addition of the c8-ring module to the complete F1-PS complex. The key intermediate then accepts the two mitochondrially encoded subunits, stabilized by the addition of subunit j, leading to an ATP synthase complex that is coupled to the proton motive force and capable of making ATP.

Structure of the dimeric ATP synthase from bovine mitochondria

The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer–monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer–monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.

Persistence of the permeability transition pore in human mitochondria devoid of an assembled ATP synthase

The opening of the permeability transition pore, a nonspecific channel in inner mitochondrial membranes, is triggered by an elevated total concentration of calcium ions in the mitochondrial matrix, leading to disruption of the inner membrane and necrotic cell death. Cyclosporin A inhibits pore opening by binding to cyclophilin D, which interacts with the pore. It has been proposed that the pore is associated with the ATP synthase complex. Previously, we confirmed an earlier observation that the pore survives in cells lacking membrane subunits ATP6 and ATP8 of ATP synthase, and in other cells lacking the enzyme’s c8rotor ring or, separately, its peripheral stalk subunits b and oligomycin sensitive conferral protein. Here, we investigated whether the pore is associated with the remaining membrane subunits of the enzyme. Individual deletion of subunits e, f, g, and 6.8-kDa proteolipid disrupts dimerization of the complex, and deletion of DAPIT (diabetes-associated protein in insulin sensitive tissue) possibly influences oligomerization of dimers, but removal of each subunit had no effect on the pore. Also, we removed together the enzyme’s membrane bound c8ring and the δ-subunit from the catalytic domain. The resulting cells assemble only a subcomplex derived from the peripheral stalk and membrane-associated proteins. Despite diminished levels of respiratory complexes, these cells generate a membrane potential to support uptake of calcium into the mitochondria, leading to pore opening, and retention of its characteristic properties. It is most unlikely that the ATP synthase, dimer or monomer, or any component, provides the permeability transition pore.

Assembly of the membrane domain of ATP synthase in human mitochondria

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F1-c8 complex inhibited by the ATPase inhibitor protein IF1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast Fo membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme’s peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme’s catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O, had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.