The Experts below are selected from a list of 138 Experts worldwide ranked by ideXlab platform
Kathleen Postle - One of the best experts on this subject based on the ideXlab platform.
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cytoplasmic membrane Protonmotive Force energizes periplasmic interactions between exbd and tonb
Molecular Microbiology, 2009Co-Authors: Anne A Ollis, Marta Manning, Kiara Held, Kathleen PostleAbstract:Summary The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the Protonmotive Force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB–ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7–ExbD–ExbB and T7–ExbD–TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7–ExbD trimers.
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Protonmotive Force exbb and ligand bound fepa drive conformational changes in tonb
Molecular Microbiology, 1999Co-Authors: Ray A Larsen, Michael G Thomas, Kathleen PostleAbstract:: TonB couples the cytoplasmic membrane Protonmotive Force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-delta V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-delta V17, for which both the conserved motif and the register of the predicted transmembrane domain alpha-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-delta V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-delta V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.
Martin D Brand - One of the best experts on this subject based on the ideXlab platform.
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Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top-Down Elasticity Analysis
FEBS Journal, 1994Co-Authors: Adolf Kesseler, Martin D BrandAbstract:The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely Protonmotive Force. The three subsystems were substrate oxidation, which produces Protonmotive Force, and the proton leak reactions and the phosphorylation reactions, which consume Protonmotive Force. By measuring the effect of cadmium on the kinetic response of each subsystem to Protonmotive Force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of Protonmotive Force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.
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effect of Protonmotive Force on the relative proton stoichiometries of the mitochondrial proton pumps
Biochemical Journal, 1991Co-Authors: Roderick P Hafner, Martin D BrandAbstract:Abstract The rate of phosphorylation of ADP by isolated mitochondria respiring on succinate was set by addition of ATP, ADP or ADP plus malonate. We measured the rates of phosphorylation and respiration and the Protonmotive Force under each of these conditions. We measured the oxygen consumption required to drive the proton leak at the Protonmotive Force reached under each condition and subtracted it from the respiration rate during phosphorylation to determine the oxygen consumption driving phosphorylation. By dividing the rate of phosphorylation by the rate of respiration driving phosphorylation we calculated the mechanistic P/O ratio (number of molecules of ADP phosphorylated per oxygen atom reduced). This ratio was the same at high, intermediate and low values of Protonmotive Force, indicating that the relative stoichiometries of the mitochondrial Protonmotive-Force-producing and Protonmotive-Force-consuming pumps (i.e. H+/O:H+/ATP) are independent of the Protonmotive Force. This greatly weakens the case for a decrease in stoichiometry, or 'slip', in the mitochondrial proton pumps at high Protonmotive Force.
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analysis of the control of respiration rate phosphorylation rate proton leak rate and Protonmotive Force in isolated mitochondria using the top down approach of metabolic control theory
FEBS Journal, 1990Co-Authors: Roderick P Hafner, Guy C Brown, Martin D BrandAbstract:The rate of respiration of isolated mitochondria was set at different values by addition of either oligomycin or an ADP-regenerating system (glucose and different amounts of hexokinase). We measured the relationship between respiration rate and membrane potential as respiration was titrated by the addition of malonate under each condition. We used the flux control summation and connectivity theorems and the branching theorem of metabolic control theory to calculate the control over respiration rate exerted by the respiratory chain (and associated reactions), phosphorylating system (and associated reactions) and proton leak at each respiration rate. The analysis also yielded the flux control coefficients of these three reactions over phosphorylation rate and proton leak rate and their concentration control coefficients over Protonmotive Force. We found that respiration rate was controlled largely by the proton leak under non-phosphorylating conditions, by the phosphorylating system at intermediate rates and by both the phosphorylating system and the respiratory chain in state 3. The rate of phosphorylation was controlled largely by the phosphorylating system itself in state 4 and at intermediate rates, while state 3 control was shared between the phosphorylating system and the respiratory chain; the proton leak had insignificant control. In all states the phosphorylating system had large negative control over the proton leak; the chain and the proton leak both had large positive control coefficients. The Protonmotive Force was controlled by the chain and by the phosphorylating system; the proton leak had little control.
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Thyroid-hormone control of state-3 respiration in isolated rat liver mitochondria.
Biochemical Journal, 1990Co-Authors: Roderick P Hafner, Guy C Brown, Martin D BrandAbstract:Oxidative phosphorylation can be treated as two groups of reactions; those that generate Protonmotive Force (dicarboxylate carrier, succinate dehydrogenase and the respiratory chain) and those that consume Protonmotive Force (adenine nucleotide and phosphate carriers. ATP synthase and proton leak). Mitochondria from hypothyroid rats have lower rates of respiration in the presence of ADP (state 3) than euthyroid controls. We show that the kinetics of the Protonmotive-Force generators are unchanged in mitochondria from hypothyroid animals, but the kinetics of the Protonmotive-Force consumers are altered, supporting proposals that the important effects of thyroid hormone on state 3 are on the ATP synthase or the adenine nucleotide translocator.
Shoji Mizushima - One of the best experts on this subject based on the ideXlab platform.
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in vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a Protonmotive Force dependent manner
Journal of Biological Chemistry, 1992Co-Authors: Midori Kato, Hajime Tokuda, Shoji MizushimaAbstract:Abstract The effect of charges existing on the mature domain of secretory proteins on the efficiency and Protonmotive Force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the Protonmotive Force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the Protonmotive Force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.
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In vitro translocation of bacterial secretory proteins and energy requirements.
Journal of Bioenergetics and Biomembranes, 1990Co-Authors: Shoji Mizushima, Hajime TokudaAbstract:The recent establishment ofin vitro assay systems has made biochemical studies on the process of membrane translocation of secretory proteins possible. This review summarizes what we have learned, using thesein vitro systems, concerning the biochemical process of protein translocation, with special reference to energy requirements. Both ATP and the Protonmotive Force participate in the translocation reaction. The requirement of ATP is obligatory, whereas that of the Protonmotive Force differs, in terms of its level, with the secretory protein species. The possible roles of ATP and the Protonmotive Force in protein translocation are discussed with special reference to the function of SecA, an essential component of the secretory machinery. The effect of positive charges, which precede or follow the hydrophobic domain of signal peptides, on translocation is also discussed.
Andrew P Wojtovich - One of the best experts on this subject based on the ideXlab platform.
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optogenetic control of mitochondrial Protonmotive Force to impact cellular stress resistance
EMBO Reports, 2020Co-Authors: Brandon J Berry, Adam J Trewin, Alexander S Milliken, Aksana Baldzizhar, Andrea M Amitrano, Andrew P WojtovichAbstract:: Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called Protonmotive Force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria-targeted light-activated proton pump that we name mitochondria-ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose-dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy-sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well-characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.
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controlling the mitochondrial Protonmotive Force with light to impact cellular stress resistance
bioRxiv, 2019Co-Authors: Brandon J Berry, Adam J Trewin, Alexander S Milliken, Aksana Baldzizhar, Andrea M Amitrano, Andrew P WojtovichAbstract:ABSTRACT Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called the Protonmotive Force (PMF) to drive diverse functions and make ATP. Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, reversible method to increase the PMF. To address this issue, we used an optogenetic approach and engineered a mitochondria-targeted light-activated proton pumping protein we called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling outcomes.
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use the Protonmotive Force mitochondrial uncoupling and reactive oxygen species
Journal of Molecular Biology, 2018Co-Authors: Brandon J Berry, Adam J Trewin, Andrea M Amitrano, Andrew P WojtovichAbstract:Abstract Mitochondrial respiration results in an electrochemical proton gradient, or Protonmotive Force (pmf), across the mitochondrial inner membrane. The pmf is a form of potential energy consisting of charge (∆ψm) and chemical (∆pH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Although capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review, we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models—mitochondria and cells—only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo.
Arnold J M Driessen - One of the best experts on this subject based on the ideXlab platform.
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in vitro pore forming activity of the lantibiotic nisin role of Protonmotive Force and lipid composition
FEBS Journal, 1993Co-Authors: Maria Garcia J Garcera, Arnold J M Driessen, Marieke G L Elferink, Wil N KoningsAbstract:Nisin is a lantibiotic produced by some strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of Gram-positive bacteria. Nisin dissipates the membrane potential (DELTApsi) and induces efflux of low-molecular-mass compounds. Evidence has been presented that a DELTApsi is needed for nisin action. The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein. Nisin-induced efflux of carboxyfluorescein was observed in the absence of a DELTApsi from liposomes composed of Escherichia coli lipids or dioleoylglycerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios. The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios. Both DELTApsi (inside negative) and DELTApH (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values. Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory. It is concluded that a DELTApsi is not essential, but that the total Protonmotive Force stimulates the action of nisin.
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bacterial protein translocation kinetic and thermodynamic role of atp and the Protonmotive Force
Trends in Biochemical Sciences, 1992Co-Authors: Arnold J M DriessenAbstract:Abstract The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have now clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the Protonmotive Force (Δp).
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precursor protein translocation by the escherichia coli translocase is directed by the Protonmotive Force
The EMBO Journal, 1992Co-Authors: Arnold J M DriessenAbstract:The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a Protonmotive Force (delta p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive. delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. delta psi and delta pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p. These results support a model [Schiebel, E., Driessen, A.J.M., Hartl, F.-U. and Wickner, W. (1991) Cell, 64, 927-939] in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving Force in precursor protein translocation.
