Objective: Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. Methods: We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. Results: Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. Conclusions: Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

Citrin belongs to the SLC25 transport protein family found mostly in inner mitochondrial membranes. The family prototype, the ADP-ATP carrier, delivers ATP made inside mitochondria to the cellular cytoplasm and returns ADP to the mitochondrion for resynthesis of ATP. In pre-genomic 1981, I noticed that the protein sequence of the bovine ADP-ATP carrier consists of three related sequences, each containing two transmembrane α-helices traveling in opposite senses. Colleagues and I demonstrated that two other mitochondrial carriers had similar features. From emergent genomic sequences, it became apparent that they represented a large family of transport proteins with the same characteristic threefold repeats. The human genome encodes 53 members, but the functions of many were unknown. So, colleagues and I determined how to make these proteins in Escherichia coli and introduce them into liposomes to allow exploration of their transport functions. The 27 human family members to have been thus identified include citrin and the closely related protein aralar. Both exchange aspartate from the mitochondrial matrix for cytosolic glutamate plus a proton. Citrin is expressed predominantly in liver and non-excitable tissues, whereas aralar is the dominant form in the brain. Each has a membrane extrinsic N-terminal Ca2+-binding domain, a transport domain, and a C-terminal amphipathic α-helix. Human mutations in citrin impair the urea cycle, malate-aspartate shuttle, gluconeogenesis, amino acid breakdown, and energy metabolism leading to citrin deficiency. Currently, the complex etiology of this condition is poorly understood and new knowledge would help to improve diagnosis, therapies, and finding a cure. My aims are to seek a basic understanding of the etiology of citrin deficiency and to use that knowledge in improving diagnostic procedures and in developing new treatments and a cure.

The hydrolytic activity of the ATP synthase in bovine mitochondria is inhibited by a protein called IF1, but bovine IF1 has no effect on the synthetic activity of the bovine enzyme in mitochondrial vesicles in the presence of a proton motive force. In contrast, it has been suggested based on indirect observations that human IFI inhibits both the hydrolytic and synthetic activities of the human ATP synthase and that the activity of human IF1 is regulated by the phosphorylation of Ser-14 of mature IF1. Here, we have made both human and bovine IF1 which are 81 and 84 amino acids long, respectively, and identical in 71.4% of their amino acids and have investigated their inhibitory effects on the hydrolytic and synthetic activities of ATP synthase in bovine submitochondrial particles. Over a wide range of conditions, including physiological conditions, both human and bovine IF1 are potent inhibitors of ATP hydrolysis, with no effect on ATP synthesis. Also, substitution of Ser-14 with phosphomimetic aspartic and glutamic acids had no effect on inhibitory properties, and Ser-14 is not conserved throughout mammals. Therefore, it is unlikely that the inhibitory activity of mammalian IF1 is regulated by phosphorylation of this residue.

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis . This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a “fail-safe” mechanism involving the b′-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated “fail-safe” mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c(8)-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.

The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F-1-catalytic, peripheral stalk (PS), and c(8)-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 F-1-c(8) module by subunits OSCP and F-6. 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 F-1-c(8)-OSCP-F-6-b-d complex. A third route involves the addition of the c(8)-ring module to the complete F-1-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.

The mitochondrial permeability transition pore (PTP) is a physiological phenomenon lacking a molecular basis. The phenomenon, described over 40 y ago, is that in response to elevated levels of Ca2+ ions in the mitochondrial matrix a nonspecific channel opens, water enters the mitochondria, their cristae swell, their membranes rupture, terminating ATP synthesis, and cell death by necrosis ensues (1). Pore opening can be inhibited by the binding of cyclosporin A to cyclophilin D, a prolyl cis–trans isomerase in the mitochondrial matrix that interacts with the pore without necessarily being a component. The open pore allows hydrophilic molecules up to a molecular weight of 1,500 Da to … [↵][1]1To whom correspondence may be addressed. Email: walker{at}mrc-mbu.cam.ac.uk. [1]: #xref-corresp-1-1

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.

Significance Mitochondria generate the cellular fuel, adenosine triphosphate, or ATP, to sustain complex life. Production of ATP depends on the oxidation of energy-rich compounds to produce a chemical potential difference for hydrogen ions (or proton motive force, pmf), across the inner mitochondrial membrane (IMM). Disruption of the IMM, dissipation of the pmf, and cell death occur if the total concentration of calcium inside mitochondria is elevated sufficiently to open a pore in the IMM. It has been proposed that the pore is in the membrane sector of the ATP synthase. Here, we show that five membrane proteins associated with the enzyme’s stator are not involved in the pore, and that the pore persists in the absence of the enzyme complex.

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from the pathogenic anaerobic bacterium Fusobacterium nucleatum. The enzyme can hydrolyse ATP but is partially inhibited. The structure is similar to those of the F1-ATPases from Caldalkalibacillus thermarum, which is more strongly inhibited in ATP hydrolysis, and in Mycobacterium smegmatis, which has a very low ATP hydrolytic activity. The βE-subunits in all three enzymes are in the conventional 'open' state, and in the case of C. thermarum and M. smegmatis, they are occupied by an ADP and phosphate (or sulfate), but in F. nucleatum, the occupancy by ADP appears to be partial. It is likely that the hydrolytic activity of the F. nucleatum enzyme is regulated by the concentration of ADP, as in mitochondria.

The crystal structure of the F 1 -catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α 3 β 3 -component of the catalytic domain is similar to those in active F 1 -ATPases in Escherichia coli and Geobacillus stearothermophilus . However, its ε-subunit differs from those in these two active bacterial F 1 -ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus , the α-helices adopt an “up” state where the α-helices enter the α 3 β 3 -domain and prevent the rotor from turning. The mycobacterial F 1 -ATPase is most similar to the F 1 -ATPase from Caldalkalibacillus thermarum , which also hydrolyzes ATP poorly. The β E -subunits in both enzymes are in the usual “open” conformation but appear to be occupied uniquely by the combination of an adenosine 5′-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F 1 domain in Mycobacterium tuberculosis , prevent ATP synthesis, and inhibit the growth of the pathogen.

Oxygen Production and Reduction in Artificial and Natural Systems, pp. 119-134 (2019) No AccessChapter 6: Modular Assembly of ATP SynthaseJohn E. Walker, Jiuya He, and Joe CarrollJohn E. WalkerMedical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK, Jiuya HeMedical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK, and Joe CarrollMedical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UKhttps://doi.org/10.1142/9789813276925_0006Cited by:2 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The ATP synthases in human mitochondria are assembled as separate modules representing the F1-catalytic domain, the stator and a ring of eight c-subunits, an essential part of the enzyme's rotor. Then an intermediate template structure containing the F1-domain the c8-ring and the stator is built up from these modules. Into this structure the membrane subunits ATP6 and ATP8 are inserted, ATP6 forming the transmembrane proton pathway with the adjacent c8-ring that is required for generating its rotation from the proton motive force. The association of ATP6 and ATP8 with this intermediate is stabilized by the addition of the 6.8 kDa proteolipid. The resulting complex can make ATP and form dimers at the apices of the cristae by interactions in the membrane domain between the two 6.8 kDa proteolipids, and the two ATP6 subunits. In a subsequent step subunit DAPIT (diabetes associated protein in insulin sensitive tissue) is added linking the dimers in long rows along the cristal edges. The ATP synthase in Saccharomyces cerevisiae is assembled by a similar, but non-identical, pathway. This modular assembly may reflect a modular evolution of ATP synthases, where the proto-typical complex evolved by the association of pre-existing modules incapable individually of catalyzing ATP synthesis. FiguresReferencesRelatedDetailsCited By 2Assembly of the peripheral stalk of ATP synthase in human mitochondriaJiuya He, Joe Carroll, Shujing Ding, Ian M. Fearnley and Martin G. Montgomery et al.9 November 2020 | Proceedings of the National Academy of Sciences, Vol. 117, No. 47Structure of the dimeric ATP synthase from bovine mitochondriaTobias E. Spikes, Martin G. Montgomery and John E. Walker8 September 2020 | Proceedings of the National Academy of Sciences, Vol. 117, No. 38 Oxygen Production and Reduction in Artificial and Natural SystemsMetrics History PDF download