Showing papers on "Electrochemical gradient published in 2018"
TL;DR: The mechanism by which protons find a path through the key enzyme involved in plant energy generation is elucidated and the cryo-EM structure of the intact cF1Fo ATP synthase in lipid nanodiscs is presented.
Abstract: INTRODUCTION Green plant chloroplasts convert light into chemical energy, and adenosine triphosphate (ATP) generated by photosynthesis is the prime source of biologically useful energy on the planet. Plants produce ATP by the chloroplast F 1 F o ATP synthase (cF 1 F o ), a macromolecular machine par excellence, driven by the electrochemical proton gradient across the photosynthetic membrane. It consists of 26 protein subunits, 17 of them wholly or partly membrane-embedded. ATP synthesis in the hydrophilic α 3 β 3 head (cF 1 ) is powered by the cF o rotary motor in the membrane. cF o contains a rotor ring of 14 c subunits, each with a conserved protonatable glutamate. Subunit a conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and e transmits the torque from the F o motor to the catalytic cF 1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b′, and δ act as a stator to prevent unproductive rotation of cF 1 with cF o . All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF 1 F o has a redox switch that inhibits adenosine triphosphatase (ATPase) activity in the dark. RATIONALE Understanding the molecular mechanisms of this elaborate nanomachine requires detailed structures of the whole complex, ideally at atomic resolution. Because of the dynamic nature of this membrane protein complex, crystallization has been difficult and no high-resolution structure of an entire, functional ATP synthase is available. We reconstituted cF 1 F o from spinach chloroplasts into lipid nanodiscs and determined its structure by cryo–electron microscopy (cryo-EM). Cryo-EM is the ideal technique for this study because it can deliver high-resolution structures of large, dynamic macromolecular assemblies that adopt a mixture of conformational states. RESULTS We present the cryo-EM structure of the intact cF 1 F o ATP synthase in lipid nanodiscs at a resolution of 2.9 A (cF 1 ) to 3.4 A (cF o ). In the cF 1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase head binds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α helices of the peripheral stalk subunits b and b′ clamp the integral membrane subunit a in its position next to the c-ring rotor, thus connecting cF 1 to cF o . Subunit γ has an L-shaped double hairpin with a redox sensor that can form a disulfide bond and a chock that blocks rotation to avoid wasteful ATP hydrolysis at night. Protons are translocated through access routes in subunit a in all rotary ATPases. We observe a hydrophilic channel on the lumenal surface that connects to the glutamate residues on the c-ring rotor that carry protons for an almost full rotation before releasing them into the stroma through another hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane. CONCLUSION We observe three cF 1 F o conformations, each with the central rotor stalled in a different position. Ring rotation is unexpectedly divided into three unequal steps. The peripheral stalk may thus act like an elastic spring, evening out the different energy contributions of each step. The features of ATP synthase nanomachines are remarkably similar in chloroplasts and mitochondria, considering their evolutionary distance of a billion years or more.
265 citations
TL;DR: This review focuses on the type A cytochrome c oxidases (CcO), which are found in all mitochondria and also in several aerobic bacteria, and describes the states of the catalytic cycle and points out the few remaining uncertainties.
Abstract: This review focuses on the type A cytochrome c oxidases (CcO), which are found in all mitochondria and also in several aerobic bacteria. CcO catalyzes the respiratory reduction of dioxygen (O2) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound CcO couples the O2 reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of CcO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.
244 citations
TL;DR: Characterizing a series of hydrogenase mutants to provide direct evidence of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.
Abstract: Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest that H2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.
185 citations
TL;DR: The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions and the chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient into the major energy currency of the cell, ATP.
Abstract: INTRODUCTION The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions. The chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient, generated by the electron transport chain, into the major energy currency of the cell, ATP. The enzyme is a large (about 600,000 Da, in the monomer state) multisubunit complex, with a water soluble complex (F 1 ) that contains three active sites and a membrane complex (F o ) that contains the proton translocation pathway, principally comprised of the a subunit and a ring of 10 c subunits, the c 10 -ring (10 in yeast, 8 in mammals). F 1 has a central rotor that, at one end, is within the core of F 1 and, at the other end, is connected to the c 10 -ring of F o . During ATP synthesis, the c 10 -ring rotates, driven by the movement of protons from the cytosol to the mitochondrion, and in turn, the rotor rotates within F 1 in steps of 120 o . The rotation of the rotor causes conformational changes in the catalytic sites, which provides the energy for the phosphorylation of adenosine diphosphate (ADP), as first proposed in the binding-change hypothesis by Paul Boyer. The peripheral stalk acts as a stator connecting F 1 with F o and prevents the futile rotation of F 1 as the rotor spins within it. RATIONALE Structural studies of the ATP synthase have made steady progress since the structure of the F 1 complex was described in pioneering work by John Walker. However, obtaining a high-resolution structure of the intact ATP synthase is challenging because it is inherently dynamic. To overcome this conformational heterogeneity, we locked the yeast mitochondrial rotor in a single conformation by fusing a subunit of the stator with a subunit of the rotor, also called the central stalk. The engineered ATP synthase was expressed in yeast and reconstituted into nanodiscs. This facilitated structure determination by cryo–electron microscopy (cryo-EM) under near native conditions. RESULTS Single-particle cryo-EM enabled us to determine the structures of the membrane-embedded monomeric yeast ATP synthase in the presence and absence of the inhibitor oligomycin at 3.8- and 3.6-A resolution, respectively. The fusion between the rotor and stator caused a twisting of the rotor and a 9° rotation of the c 10 -ring, in the direction of ATP synthesis, relative to the putative resting state. This twisted conformation likely represents an intermediate state in the ATP synthesis reaction cycle. The structure also shows two proton half-channels formed largely by the a subunit that abut the c 10 -ring and suggests a mechanism that couples transmembrane proton movement to c 10 -ring rotation. The cryo-EM density map indicates that oligomycin is bound to at least four sites on the surface of the F o c 10 -ring that is exposed to the lipid bilayer; this is supported by binding free-energy molecular dynamics calculations. The sites of oligomycin-resistant mutations in the a subunit suggest that changes in the side-chain configuration of the c subunits at the a-c subunit interface are transmitted through the entire c 10 -ring. CONCLUSION Our results provide a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase. The structure provides an understanding of the mechanism of inhibition by oligomycin and suggests how extragenic mutations can cause resistance to this inhibitor. The approach presented in this study paves the way for structural characterization of other functional states of the ATP synthase, which is essential for understanding its functions in physiology and disease.
153 citations
TL;DR: Cryo-electron microscopy structures show the architecture of the respirasome with near-atomic detail, which is responsible for the folding of the membrane into cristae and thus for the huge increase in available surface that makes mitochondria the efficient energy plants of the eukaryotic cell.
Abstract: Mitochondria are the power stations of the eukaryotic cell, using the energy released by the oxidation of glucose and other sugars to produce ATP. Electrons are transferred from NADH, produced in the citric acid cycle in the mitochondrial matrix, to oxygen by a series of large protein complexes in the inner mitochondrial membrane, which create a transmembrane electrochemical gradient by pumping protons across the membrane. The flow of protons back into the matrix via a proton channel in the ATP synthase leads to conformational changes in the nucleotide binding pockets and the formation of ATP. The three proton pumping complexes of the electron transfer chain are NADH-ubiquinone oxidoreductase or complex I, ubiquinone-cytochrome c oxidoreductase or complex III, and cytochrome c oxidase or complex IV. Succinate dehydrogenase or complex II does not pump protons, but contributes reduced ubiquinone. The structures of complex II, III and IV were determined by x-ray crystallography several decades ago, but complex I and ATP synthase have only recently started to reveal their secrets by advances in x-ray crystallography and cryo-electron microscopy. The complexes I, III and IV occur to a certain extent as supercomplexes in the membrane, the so-called respirasomes. Several hypotheses exist about their function. Recent cryo-electron microscopy structures show the architecture of the respirasome with near-atomic detail. ATP synthase occurs as dimers in the inner mitochondrial membrane, which by their curvature are responsible for the folding of the membrane into cristae and thus for the huge increase in available surface that makes mitochondria the efficient energy plants of the eukaryotic cell.
113 citations
TL;DR: The structure of the obligate respiratory III2IV2 supercomplex from M. smegmatis is reported, revealing two functionally relevant conformations of the cytochrome cc subunit and a SOD subunit that may detoxify reactive oxygen species.
Abstract: In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.
91 citations
TL;DR: In this article, crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8
Abstract: The gastric proton pump—the H+, K+-ATPase—is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 A resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pK
a value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.
79 citations
TL;DR: A role for DMT1 is supported in mitochondrial Fe2+ and Mn2+ acquisition and transport was defective in rKC mitochondria with the Belgrade (G185R) mutation.
Abstract: Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.
72 citations
TL;DR: It is demonstrated that despite direct contact with the soil, the apoplastic pH close to the plasma membrane was maintained at values ranging from 6.0 to 6.4 in mature root cells, whereas the overall pH in the Apoplastic space is far more acidic.
Abstract: Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated Arabidopsis plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium–epidermis interface up to the inner cell layers that lie beyond the Casparian strip. The membrane-associated apoplastic pH was much more alkaline than the overall apoplastic space pH. Proton concentration associated with the plasma membrane was very stable, even when the growth medium pH was altered. This is in apparent contradiction with the direct connection between root intercellular space and the external medium. The plasma membrane-associated pH in the stele was the most preserved and displayed the lowest apoplastic pH (6.0 to 6.1) and the highest transmembrane delta pH (1.5 to 2.2). Both pH values also correlated well with optimal activities of channels and transporters involved in ion uptake and redistribution from the root to the aerial part. In growth medium where ionic content is minimized, the root plasma membrane-associated pH was more affected by environmental proton changes, especially for the most external cell layers. Calcium concentration appears to play a major role in apoplastic pH under these restrictive conditions, supporting a role for the cell wall in pH homeostasis of the unstirred surface layer of plasma membrane in mature roots.
60 citations
TL;DR: Overall, many critical physiological functions are maintained by the activity of the two Na+ -K+ -2Cl- cotransporters in nonpolarized cells and in epithelia.
Abstract: Two genes encode the Na+ -K+ -2Cl- cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na+ , 1K+ , and 2Cl- across the plasma membrane of cells. Na+ -K+ -2Cl- cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na+ /K+ pump. In many cells, NKCC1 accumulates Cl- above its electrochemical potential equilibrium, thereby facilitating Cl- channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca2+ channels, leading to Ca2+ influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl- conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl- . In combination with the Na+ /K+ pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl- -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K+ -driven fluid secretion in inner ear, and possibly in Na+ -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na+ /K+ pump participates in reabsorbing 30% of the glomerular-filtered Na+ . Overall, many critical physiological functions are maintained by the activity of the two Na+ -K+ -2Cl- cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.
53 citations
TL;DR: A ‘counter-charge’ model for pH sensing is proposed in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups.
Abstract: Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high pKa) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their pKa needs to be within the operational pH range. We propose a 'counter-charge' model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups.
TL;DR: A potential proton exit is identified from a buried cluster of polar residues (the proton loading site) to the P-side of CcO via paths made up of waters and conserved residues, adding to the essential electrochemical gradient.
TL;DR: It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.
TL;DR: A new overexpression system is described that has facilitated the determination of high-resolution crystal structures of Hyb OC and, hence, a prediction of the quaternary structure of the HybOCAB complex.
Abstract: Under anaerobic conditions, Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe–S protein (HybA) and an integral membrane protein (HybB). To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of Hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In the present paper, we describe a new overexpression system that has facilitated the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.
TL;DR: The Kv1.3 channel, which shows specific electrophysiological properties such as C-type and cumulative inactivation, is widely distributed throughout the whole body, but it is highly expressed in both nervous and immune system.
Abstract: Ion channels are transmembrane proteins that form aqueous pores driving ions through the plasma membrane in favor of an electrochemical gradient. Thus, voltage-dependent potassium channels (Kv) con...
TL;DR: Harmonized functioning of PM H+-ATPase and NOX appears to be justified and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.
Abstract: Plasma membrane (PM) H+-ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O2˙-, respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H+-ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H+-ATPase inhibitor). Conversely, H+-ATPase activity retarded in response to different ROS scavengers [CuCl2, N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl2 and diphenyleneiodonium (DPI)], while H2O2 promoted PM H+-ATPase activity at lower concentrations. Repressing effects of Ca+2 antagonists (La+3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H+-ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H+-ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.
TL;DR: Peroxidase reactions relieved the RISE mechanism, indicating that P700 oxidation can be induced only by the reduction of PQ to suppress the production of ROS in PSI.
Abstract: Photosynthetic organisms oxidize P700 to suppress the production of reactive oxygen species (ROS) in photosystem I (PSI) in response to the lower efficiency of photosynthesis under high light and low CO2 conditions. Previously, we found a positive relationship between reduction of plastoquinone (PQ) pool and oxidation of P700, which we named reduction-induced suppression of electron flow (RISE). In the RISE model, we proposed that the highly reduced state of the PQ pool suppresses Q-cycle turnover to oxidize P700 in PSI. Here, we tested whether RISE was relieved by the oxidation of the PQ pool, but not by the dissipation of the proton gradient (ΔpH) across the thylakoid membrane. Formation of ΔpH can also suppress electron flow to P700, because acidification on the luminal side of the thylakoid membrane lowers oxidation of reduced PQ in the cytochrome b6/f complex. We drove photosynthetic electron transport using H2O2-scavenging peroxidase reactions. Peroxidase reduces H2O2 with electron donors regenerated along the photosynthetic electron transport system, thereby promoting the formation of ΔpH. Addition of H2O2 to the cyanobacterium Synechococcus elongatus PCC 7942 under low CO2 conditions induced photochemical quenching of chlorophyll fluorescence, enhanced NADPH fluorescence and reduced P700. Thus, peroxidase reactions relieved the RISE mechanism, indicating that P700 oxidation can be induced only by the reduction of PQ to suppress the production of ROS in PSI. Overall, our data suggest that RISE regulates the redox state of P700 in PSI in cooperation with ΔpH regulation.
TL;DR: This work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium–calcium exchangers.
Abstract: Intracellular Ca2+ signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca2+ concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium-calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca2+ across the cell membrane by coupling it to the flux of Na+ in the opposite direction, down an electrochemical gradient. Na+ and Ca2+ are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na+:1Ca2+ Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium-calcium exchangers.
TL;DR: An artificially designed honeycomb multilayer for photophosphorylation to better understand the mechanism of PSII and ATP synthase integrated in one system, mimicking the photosynthetic grana structure.
Abstract: Plant thylakoid has a typical stacking structure, which is the site of photosynthesis, including light-harvesting, water-splitting and adenosine triphosphate (ATP) production. This stacking structure plays a key role in exchange of substances with extremely high efficiency and minimum energy consumption through photosynthesis. Herein we report an artificially designed honeycomb multilayer for photophosphorylation. To mimic natural thylakoid stacking structure, the multilayered Photosystem II (PSII)-ATP synthase-liposome system is fabricated via Layer-by-Layer (LbL) assembly, allowing the three-dimensional distributions of PSII and ATP synthase. Under light illumination, PSII splits water into protons and generates a proton gradient for ATP synthase to produce ATP. Moreover, it is found that the ATP production is extremely associated with the numbers of PSII layers. With such a multilayer structure assembled by LbL can be better understanding on the mechanism of PSII and ATP synthase integrated in one syst...
TL;DR: The anion transport mechanism of a synthetic molecule based on the structure of prodigiosine, a red pigment produced by bacteria, is characterized, indicating its low toxicity and capacity to transport chloride and bicarbonate, when applied at low concentration constitute a promising starting point for the development of drug candidates for CF therapy.
Abstract: Cystic fibrosis (CF) is a genetic lethal disease, originated from the defective function of the CFTR protein, a chloride and bicarbonate permeable transmembrane channel. CF mutations affect CFTR protein through a variety of molecular mechanisms which result in different functional defects. Current therapeutic approaches are targeted to specific groups of patients that share a common functional defect. We seek to develop an innovative therapeutic approach for the treatment of CF using anionophores, small molecules that facilitate the transmembrane transport of anions. We have characterized the anion transport mechanism of a synthetic molecule based on the structure of prodigiosine, a red pigment produced by bacteria. Anionophore-driven chloride efflux from large unilamellar vesicles is consistent with activity of an uniporter carrier that facilitates the transport of anions through lipid membranes down the electrochemical gradient. There are no evidences of transport coupling with protons. The selectivity sequence of the prodigiosin inspired EH160 ionophore is formate > acetate > nitrate > chloride > bicarbonate. Sulfate, phosphate, aspartate, isothionate, and gluconate are not significantly transported by these anionophores. Protonation at acidic pH is important for the transport capacity of the anionophore. This prodigiosin derived ionophore induces anion transport in living cells. Its low toxicity and capacity to transport chloride and bicarbonate, when applied at low concentration, constitute a promising starting point for the development of drug candidates for CF therapy.
TL;DR: It is concluded that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons.
TL;DR: The results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer.
Abstract: In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.
TL;DR: Cytochrome c oxidase drives aerobic respiratory chains in all organisms by transducing the free energy from oxygen reduction into an electrochemical proton gradient across a biological membrane.
Abstract: Cytochrome c oxidase (CcO) drives aerobic respiratory chains in all organisms by transducing the free energy from oxygen reduction into an electrochemical proton gradient across a biological membrane. CcO employs the so-called D- and K-channels for proton uptake, but the molecular mechanism for activation of the K-channel has remained elusive for decades. We show here by combining large-scale atomistic molecular simulations with graph-theoretical water network analysis, and hybrid quantum/classical (QM/MM) free energy calculations, that the K-channel is activated by formation of a reactive oxidized intermediate in the binuclear heme a3/CuB active site. This state induces electrostatic, hydration, and conformational changes that lower the barrier for proton transfer along the K-channel by dewetting pathways that connect the D-channel with the active site. Our combined results reconcile previous experimental findings and indicate that water dynamics plays a decisive role in the proton pumping machinery in CcO.
TL;DR: The authors show that proton uptake to the catalytic site of CytcO and presumably proton translocation was impaired by the potential, but electron transfer was not affected and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.
Abstract: The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.
TL;DR: A detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana is provided.
Abstract: Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.
TL;DR: It is shown that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14- 3-3 isoforms were analyzed for their association with AVP 1.
Abstract: Plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a crucial enzyme that exists on the tonoplast to maintain pH homeostasis across the vacuolar membrane. This enzyme generates proton gradient between cytosol and vacuolar lumen by hydrolysis of a metabolic byproduct, pyrophosphate (PP
i
). The regulation of V-PPase at protein level has drawn attentions of many workers for decades, but its mechanism is still unclear. In this work, we show that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14-3-3 isoforms were analyzed for their association with AVP1. In the presence of 14-3-3ν, -µ, -ο, and -ι, both enzymatic activities and its associated proton pumping of AVP1 were increased. Among these 14-3-3 proteins, 14-3-3 µ shows the highest stimulation on coupling efficiency. Furthermore, 14-3-3ν, -µ, -ο, and -ι exerted protection of AVP1 against the inhibition of suicidal substrate PP
i
at high concentration. Moreover, the thermal profile revealed the presence of 14-3-3ο improves the structural stability of AVP1 against high temperature deterioration. Additionally, the 14-3-3 proteins mitigate the inhibition of Na+ to AVP1. Besides, the binding sites/motifs of AVP1 were identified for each 14-3-3 protein. Taken together, a working model was proposed to elucidate the association of 14-3-3 proteins with AVP1 for stimulation of its enzymatic activity.
TL;DR: The techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors are described.
Abstract: Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.
TL;DR: It is shown that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism of transport used by PnuTSw, which contrasts sharply with the active transport mechanisms used by other bacterialThiamine transporters.
Abstract: Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.
TL;DR: Data of potato transporter overlap the mammalian ones, except for the kinetic parameters non-experimentally measurable, thus supporting the MAT in plants fulfills the same transport role.
Abstract: The Mitochondrial Ascorbic Acid Transporter (MAT) from both rat liver and potato mitochondria has been reconstituted in proteoliposomes. The protein has a molecular mass in the range of 28-35 kDa and catalyzes saturable, temperature and pH dependent, unidirectional ascorbic acid transport. The transport activity is sodium independent and it is optimal at acidic pH values. It is stimulated by proton gradient, thus supporting that ascorbate is symported with H+. It is efficiently inhibited by the lysine reagent pyridoxal phosphate and it is not affected by inhibitors of other recognized plasma and mitochondrial membranes ascorbate transporters GLUT1(glucose transporter-1) or SVCT2 (sodium-dependent vitamin C transporter-2). Rat protein catalyzes a cooperative ascorbate transport, being involved two binding sites; the measured K0.5 is 1.5 mM. Taking into account the experimental results we propose that the reconstituted ascorbate transporter is not a GLUT or SVCT, since it shows different biochemical features. Data of potato transporter overlap the mammalian ones, except for the kinetic parameters non-experimentally measurable, thus supporting the MAT in plants fulfills the same transport role.
TL;DR: In the present study the Euglena proton-pumping complexes I, III, and IV were purified from isolated mitochondria by a two-steps liquid chromatography approach and their atypical subunit composition was further resolved and confirmed using a three-steps PAGE analysis coupled to mass spectrometry identification of peptides.
Abstract: In mitochondrial oxidative phosphorylation, electron transfer from NADH or succinate to oxygen by a series of large protein complexes in the inner mitochondrial membrane (complexes I-IV) is coupled to the generation of an electrochemical proton gradient, the energy of which is utilized by complex V to generate ATP. In Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, these respiratory complexes totalize more than 40 Euglenozoa-specific subunits along with about 50 classical subunits described in other eukaryotes. In the present study the Euglena proton-pumping complexes I, III, and IV were purified from isolated mitochondria by a two-steps liquid chromatography approach. Their atypical subunit composition was further resolved and confirmed using a three-steps PAGE analysis coupled to mass spectrometry identification of peptides. The purified complexes were also observed by electron microscopy followed by single-particle analysis. Even if the overall structures of the three oxidases are similar to the structure of canonical enzymes (e.g. from mammals), additional atypical domains were observed in complexes I and IV: an extra domain located at the tip of the peripheral arm of complex I and a "helmet-like" domain on the top of the cytochrome c binding region in complex IV.