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

2017 ◽  
Vol 114 (34) ◽  
pp. 9086-9091 ◽  
Author(s):  
Jiuya He ◽  
Joe Carroll ◽  
Shujing Ding ◽  
Ian M. Fearnley ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Domain ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Helical Structure ◽  
Cyclophilin D ◽  
Membrane Domain ◽  
Peripheral Stalk ◽  
Subunit B

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.

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

2019 ◽  
Vol 116 (26) ◽  
pp. 12816-12821 ◽  
Author(s):  
Joe Carroll ◽  
Jiuya He ◽  
Shujing Ding ◽  
Ian M. Fearnley ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Domain ◽  
Total Concentration ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Peripheral Stalk ◽  
Associated Proteins ◽  
Pore Opening ◽  
Membrane Bound

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.

Abstract 331: Impact of Acetylated Cyclophilin D on the Mitochondrial Permeability Transition Pore

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Gisela Beutner ◽  
Jacob Perkins ◽  
Ronak A Sardari ◽  
George A Porter
Keyword(s):  
Atp Synthase ◽  
Cyclosporine A ◽  
Matrix Protein ◽  
Mitochondrial Permeability Transition ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Retention Capacity ◽  
Transport Chain

Background: The mitochondrial matrix protein cyclophilin D (CypD) is a key regulator of mitochondrial function. CypD controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is regulated by several post-translational modifications including acetylation of lysine 166 in the mouse. Objective: To investigate how acetylation at lysine 166 of CypD specifically in the heart modifies its ability to regulate the PTP and the ATP synthase. Results: We generated a conditional cardiac knock-in mouse model where lysine 166 has been mutated into glutamine (CypD K166Q ) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of native CypD. Results show that mitochondrial oxygen consumption was not affected by the expression of CypD K166Q . The calcium retention capacity (CRC) was measured with Arsenazo III and decreased significantly when CypD K166Q was expressed. The CypD inhibitor cyclosporine A significantly increased the CRC in WT mice. However, cyclosporine A was did not inhibit CypD in the hearts of mice expressing only CypD K166Q or in addition to wild-type CypD. The ability of the ATP synthase to create dimers or oligomers was assessed by western blotting and the hydrolysis of ATP in in-gel assays and shows that expression of CypD K166Q decreased the assembly of the ATP synthase into dimers or oligomers. Conclusions: Our data show that the expression of CypD K166Q increases the sensitivity of PTP opening to calcium and limits the assembly of ATP synthase into oligomers.

scholarly journals Effect of anions on Cyclophilin D binding to F-ATP synthase: Implications for the permeability transition pore

2018 ◽  
Vol 1859 ◽  
pp. e111-e112
Author(s):  
Giovanna Lippe ◽  
Gabriele Coluccino ◽  
Valentina Giorgio ◽  
Federico Fogolari ◽  
Valeria Petronilli ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D

scholarly journals Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy

2016 ◽  
Vol 113 (45) ◽  
pp. 12709-12714 ◽  
Author(s):  
Kutti R. Vinothkumar ◽  
Martin G. Montgomery ◽  
Sidong Liu ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Domain ◽  
Lateral Force ◽  
Membrane Domain ◽  
Subunit A ◽  
Mechanical Coupling ◽  
Peripheral Stalk ◽  
Mitochondrial Atp Synthase ◽  
A Subunit ◽  
Pichia Angusta

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3β3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator’s integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ∼30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine.

scholarly journals Atomistic simulations indicate the c-subunit ring of the F1Fo ATP synthase is not the mitochondrial permeability transition pore

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Wenchang Zhou ◽  
Fabrizio Marinelli ◽  
Corrine Nief ◽  
José D Faraldo-Gómez
Keyword(s):  
Atp Synthase ◽  
Mitochondrial Permeability Transition Pore ◽  
Catalytic Domain ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Inner Mitochondrial Membrane ◽  
Ion Conductance ◽  
Mitochondrial Permeability ◽  
C Subunit

Pathological metabolic conditions such as ischemia induce the rupture of the mitochondrial envelope and the release of pro-apoptotic proteins, leading to cell death. At the onset of this process, the inner mitochondrial membrane becomes depolarized and permeable to osmolytes, proposedly due to the opening of a non-selective protein channel of unknown molecular identity. A recent study purports that this channel, referred to as Mitochondrial Permeability Transition Pore (MPTP), is formed within the c-subunit ring of the ATP synthase, upon its dissociation from the catalytic domain of the enzyme. Here, we examine this claim for two c-rings of different lumen width, through calculations of their ion conductance and selectivity based on all-atom molecular dynamics simulations. We also quantify the likelihood that the lumen of these c-rings is in a hydrated, potentially conducting state rather than empty or blocked by lipid molecules. These calculations demonstrate that the structure and biophysical properties of a correctly assembled c-ring are inconsistent with those attributed to the MPTP.

scholarly journals Formation of High-Conductive C Subunit Channels upon Interaction with Cyclophilin D

2021 ◽  
Vol 22 (20) ◽  
pp. 11022
Author(s):  
Giuseppe Federico Amodeo ◽  
Natalya Krilyuk ◽  
Evgeny V. Pavlov
Keyword(s):  
Lipid Bilayers ◽  
Atp Synthase ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Inner Mitochondrial Membrane ◽  
C Subunit ◽  
High Concentrations ◽  
Main Component

The c subunit of the ATP synthase is an inner mitochondrial membrane (IMM) protein. Besides its role as the main component of the rotor of the ATP synthase, c subunit from mammalian mitochondria exhibits ion channel activity. In particular, c subunit may be involved in one of the pathways leading to the formation of the permeability transition pore (PTP) during mitochondrial permeability transition (PT), a phenomenon consisting of the permeabilization of the IMM due to high levels of calcium. Our previous study on the synthetic c subunit showed that high concentrations of calcium induce misfolding into cross-β oligomers that form low-conductance channels in model lipid bilayers of about 400 pS. Here, we studied the effect of cyclophilin D (CypD), a mitochondrial chaperone and major regulator of PTP, on the electrophysiological activity of the c subunit to evaluate its role in the functional properties of c subunit. Our study shows that in presence of CypD, c subunit exhibits a larger conductance, up to 4 nS, that could be related to its potential role in mitochondrial toxicity. Further, our results suggest that CypD is necessary for the formation of c subunit induced PTP but may not be an integral part of the pore.

scholarly journals TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

2021 ◽  
Author(s):  
Giuseppe Cannino ◽  
Andrea Urbani ◽  
Marco Gaspari ◽  
Mariaconcetta Varano ◽  
Alessandro Negro ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Inhibitory Effect ◽  
Cyclophilin D ◽  
Electrophysiological Measurements ◽  
Permeability Transition Pore Opening ◽  
Pore Opening ◽  
Client Proteins ◽  
Oligomycin Sensitivity Conferring Protein

AbstractBinding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

scholarly journals A Novel Conceptual Model for the Dual Role of FOF1-ATP Synthase in Cell Life and Cell Death

2020 ◽  
Vol 11 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Sunil Nath
Keyword(s):  
Cell Death ◽  
Atp Synthase ◽  
Mitochondrial Permeability Transition ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Scientific Debate ◽  
Cell Life ◽  
Energy Conserving

AbstractThe mitochondrial permeability transition (MPT) has been one of the longstanding enigmas in biology. Its cause is currently at the center of an extensive scientific debate, and several hypotheses on its molecular nature have been put forward. The present view holds that the transition arises from the opening of a high-conductance channel in the energy-transducing membrane, the permeability transition pore (PTP), also called the mitochondrial megachannel or the multiconductance channel (MMC). Here, the novel hypothesis is proposed that the aqueous access channels at the interface of the c-ring and the a-subunit of FO in the FOF1-ATP synthase are repurposed during induction of apoptosis and constitute the elusive PTP/ MMC. A unifying principle based on regulation by local potentials is advanced to rationalize the action of the myriad structurally and chemically diverse inducers and inhibitors of PTP/MMC. Experimental evidence in favor of the hypothesis and its differences from current models of PTP/MMC are summarized. The hypothesis explains in considerable detail how the binding of Ca2+ to a β-catalytic site (site 3) in the F1 portion of ATP synthase triggers the opening of the PTP/MMC. It is also shown to connect to longstanding proposals within Nath’s torsional mechanism of energy transduction and ATP synthesis as to how the binding of MgADP to site 3 does not induce PTP/MMC, but instead catalyzes physiological ATP synthesis in cell life. In the author’s knowledge, this is the first model that explains how Ca2+ transforms the FOF1-ATP synthase from an exquisite energy-conserving enzyme in cell life into an energy-dissipating structure that promotes cell death. This has major implications for basic as well as for clinical research, such as for the development of drugs that target the MPT, given the established role of PTP/MMC dysregulation in cancer, ischemia, cardiac hypertrophy, and various neurodegenerative diseases.

scholarly journals Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel

2019 ◽  
Vol 294 (28) ◽  
pp. 10987-10997 ◽  
Author(s):  
Lishu Guo ◽  
Michela Carraro ◽  
Andrea Carrer ◽  
Giovanni Minervini ◽  
Andrea Urbani ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Site Directed Mutagenesis ◽  
Critical Residue ◽  
Subunit E ◽  
Mitochondrial Megachannel ◽  
High Conductance

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

scholarly journals Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

2017 ◽  
Vol 114 (13) ◽  
pp. 3409-3414 ◽  
Author(s):  
Jiuya He ◽  
Holly C. Ford ◽  
Joe Carroll ◽  
Shujing Ding ◽  
Ian M. Fearnley ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Mitochondrial Permeability Transition ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Targeting ◽  
Inner Membrane ◽  
C Subunit ◽  
Mitochondrial Targeting Sequences

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

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