Mitochondrial membrane potential and aging

The mitochondrial membrane potential (or protonmotive force) is the central bioenergetic parameter that controls respiratory rate, ATP synthesis and the generation of reactive oxygen species, and is itself controlled by electron transport and proton leaks. As a consequence of extensive research, there has emerged a consensus as to how these parameters integrate. Despite this consensus, the literature contains contradictory reports on the extent to which these parameters are modified in animal models of aging. This article critically examines the basis for a number of these reports.     

Determination of the orientation of membrane vesicles derived from mitochondria

Membrane vesicles of physiological as well as inverted orientation can be isolated from mitochrondria. The presence of these vesicles in a membrane can be determined and quantitated by determining the differences between the two vesicle types in terms of rates of NADH oxidation, rates of oxidation of tricarboxylate cycle intermediates, rates of ATP hydrolysis and sensitivity to inhibitors, stimulation of respiration by exogenous cytochromec, inhibition of respiration by polycationic proteins, and visualization of the ATPase by electron microscopy. Procedures to isolate the two membrane types and characteristics of homogeneously oriented preparations are described. Differences in data obtained with homogeneous vesicle preparations and with vesicles of mixed orientation are illustrated. Nonhomogeneously oriented preparations can be enriched in the desired vesicular type by the use of immunoprecipitation, affinity chromatography, and differential centrifugation. The concept of a hybrid vesicle containing oppositely oriented regions is not supported by experimental data.     

Effective coupling of four independent membrane systems in steady-state oxidative phosphorylation

Alexandre et al. have proposed a four-system model of oxidative phosphorylation. The thermodynamic consequences of this model are explored, assuming as a first approximation that these four systems can be treated as a self-contained group. The method can be generalized, in higher approximations, to include further systems and other complications. Respiratory control is considered from the point of view of the model. Self-consistent numerical examples are given to represent mitochondrial activity in state 3 and in state 4.     

Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei

The energy-transducing N,N′-dicyclohexylcarbodiimide-sensitive (DCCD-sensitive) ATPase complex consists of two parts, a soluble catalytic protein (F₁), and an intrinsic membrane protein (F₀). The bacterial coupling factor complex, BCF₀-BCF₁, has recently been purified from Mycobacterium phlei, and used to reconstitute oxidative phosphorylation in detergent-extracted membranes. The BCF₀ moiety has been purified by being recovered from the purified BCF₀-BCF₁ complex by affinity chromatography. BCF₀ is a lipoprotein or lipoprotein complex with an approximate molecular weight of 60,000. The preparation contained 0.15 mg of phospholipid per milligram protein. There appear to be three polypeptides, with approximate molecular weights of 24,000, 18,000, and 8,000 as determined by sodium dodecylsulfate a crylamide gel electrophoresis. Purified BCF₀ conferred DCCD sensitivity on a purified BCF₁ preparation. Reconstitution of oxidative phosphorylation was achieved after incubation of detergent-extracted membranes with purified BCF₀ and purified BCF₁.     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Respiration and plasma membrane ATPase in strawberry stolons

The biochemical, physiological and anatomical properties of strawberry (Fragaria ananassa Duch.) cv. 'Cambridge Favourite' stolons were studied during growth. ATPase activity was measured, in microsomal and plasma membrane fractions, along with chlorophyll determination, in-situ photosynthesis measurements and scanning electron microscopy (SEM) and X-ray microanalysis of stolon cross-sections. Potassium-stimulated ATPase activity and proton-pumping, both together indicating the presence of plasma membrane ATPase, was greatest in the stolon tip, the tissue with the fastest growth and respiratory activity. The enzyme activity and respiration gradient from the tip of the stolon to the base was concomitant with xylem development which was more differentiated in the base than in the tip. These cross-sections also showed 30% greater amounts of calcium and potassium of the cryo-preserved basal part relative to the stolon tip. This gradient existed independent of the presence of daughter plants. A hypothesis is presented which suggests that for the long-distance longitudinal transport of nutrients this gradient between stolon tip and base is likely to be involved in stolon growth.     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Energy-dependent Solute Uptake Into the Symplast of Leaves: ATP/KCl, ATP/Sucrose, ATP/D-serine and H+/ATP Stoichiometries of Transmembrane Transport

Abstract: KCl, sucrose, D-serine and some other solutes were fed through the petiole to leaflets of Solanum tuberosum and uptake into the symplast was monitored. Solute transport was accompanied by changes in membrane potential, apoplastic pH and respiration. After termination of solute feeding, membrane potential, apoplastic pH and respiration returned to initial steady state values. From transpiration, solute uptake was calculated and compared to ATP production during stimulated respiration, assuming an ATP/CO₂ ratio of 5. On this basis, calculated ATP/KCl ratios of energized transport approached 0.5. Similar ATP/solute ratios were observed with sucrose, mannitol, methylglucose and D-serine. With glucose, many ratios were somewhat above 0.5, possibly because of some metabolization of imported glucose. We conclude that solute uptake is energized by the proton motive force across the plasma membrane. Low ATP/substrate ratios suggest that the H⁺/ATP ratio of proton export by the plasma membrane ATPase is not 1 as presently assumed but 2 in potato leaves, and that the contribution of the alternative cyanide-resistant oxidase to leaf respiration is small, if not negligible, in the dark.     

Autocatalytic cooperativity and self-regulation of ATPase pumps in membrane active transport

We investigate the effect of autocatalysis on the conformational changes of membrane pumps during active transport driven by ATP. The translocation process is described by means of an alternating access model. The usual kinetic scheme is extended by introducing autocatalytic steps and allowing for dynamic formation of enzyme complexes. The usual features of cooperative models are recovered, i.e., sigmoid shapes of flux versus concentration curves. We show also that two autocatalytic steps lead to a mechanism of inhibition by the substrate as experimentally observed for some ATPase pumps. In addition, when the formation of enzyme complexes is allowed, the model exhibits a multiple stationary states regime, which can be related to a self-regulation mechanism of the active transport in biological systems. Correspondence to: G. Weissmüller     

Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins

Many H⁺-pump rhodopsins conserve “H⁺ donor” residues in cytoplasmic (CP) half channels to quickly transport H⁺ from the CP medium to Schiff bases at the center of these proteins. For conventional H⁺ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H⁺ to the Schiff bases and then capture the other H⁺ from the medium, whereas the Lys donor first captures H⁺ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H⁺ pumps seem to have different mechanisms, which are probably optimized for their respective H⁺-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H⁺ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H⁺ donors. These differences were further examined by focusing on the activation volumes during the H⁺-transfer reactions. The results revealed essential differences between archaeal H⁺ pump (DR) and eubacterial H⁺ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H⁺-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.     

Large-scale isolation of the Neurospora plasma membrane H+-ATPase

A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O2 to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H+-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with alpha-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H+-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 mumol of P1 released/min/mg of protein at 30 degrees C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.     

Resolution of electron and proton transfer events in the electrochromism associated with quinone reduction in bacterial reaction centers

We have measured the electrochromic response of the bacteriopheophytin, BPh, and bacteriochlorophyll, BChl, cofactors during the Q_A ^–Q_B Q_AQ_B ^– electron transfer in chromatophores of Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The electrochromic response rises faster in chromatophores and is more clearly biexponential than it is in isolated reaction centers. The chromatophore spectra can be interpreted in terms of a clear kinetic separation between fast electron transfer and slower non-electron transfer events such as proton transfer or protein relaxation. The electrochromic response to electron transfer exhibits rise times of about 4 µs (70%) and 40 µs (30%) in Rb. capsulatus and 4 µs (60%) and 80 µs (40%) in Rb. sphaeroides. The BPh absorption band is shifted to nearly equivalent positions in the Q_A ^– and nascent Q_B ^– states, indicating that the electrochromic perturbation of BPh absorption from the newly formed Q_B ^– state is comparable to that of Q_A ^– . Subsequently, partial attenuation of the Q_B ^– electrochromism occurs with a time constant on the order of 200 µs. This can be attributed to partial charge compensation by H⁺ (or other counter ion) movement into the Q_B pocket. Electron transfer events were found to be slower in detergent isolated RCs than in chromatophores, more nearly monoexponential, and overlap H⁺ transfer, suggesting that a change in rate-limiting step has occurred upon detergent solubilization.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Bioenergetics of alkalophilic bacteria

Summary The central problem for organisms which grow optimally, and in some cases obligately, at pH values of 10 to 11, is the maintenance of a relatively acidified cytoplasm. A key component of the pH homeostatic mechanism is an electrogenic Na⁺/H⁺ antiporter which—by virtue of kinetic properties and/or its concentration in the membrane—catalyzes net proton uptake while the organisms extrude protons during respiration. The antiporter is also capable of maintaining a constant pH_in during profound elevations in pH_out as long as Na⁺ entry is facilitated by the presence of solutes which are taken up with Na⁺. Secondary to the problem of acidifying the interior is the adverse effect of the large pH gradient, acid in, on the total pmf of alkalophile cells. For the purposes of solute uptake and motility, the organisms appear to largely bypass the problem of a low pmf by utilizing a sodium motive force for energization. However, ATP synthesis appears not to resolve the energetics problem by using Na⁺ or by incorporating the proton-translocating ATPase into intracellular organelles. The current data suggest that effective proton pumping carried out by the alkalophile respiratory chain at high pH may deliver at least some portion of the protons to the proton-utilizing catalysts, i. e., theF ₁ F ₀-ATPase and the Na⁺/H⁺ antiporter, by some localized pathway.     

Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB

TonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB.While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy-entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications.     

Action of the herbicides neburon and siduron on membrane permeabilities in potato tuber mitochondria

The herbicides neburon and siduron are uncouplers of oxidative phosphorylation in potato tuber (Solanum tuberosum L. cv. Bintje) mitochondria. Their effect on the ion permeabilities of the mitochondrial membrane was investigated using the acid-base pulse technique, swelling experiments and integrity tests. Both herbicides permeabilize the membrane to H⁺ ions. They have no action on the permeabilities of K⁺ and Fe(CN)^3?₆. The swelling observed with Ca²⁺ was better interpreted as an effect on membrane structure than as a true swelling. Diuron, a parent compound that does not uncouple oxidative phosphorylation, does not act on Ca²⁺-induced apparent swelling.     

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

The membrane sector (V_o) of the proton pumping vacuolar ATPase (V-ATPase, V₁V_o-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained V_o two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of V_o from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free V_o and V_o as part of holo V₁V_o revealed that the cytoplasmic N-terminal domain of subunit a (a_NT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from V_o, V₁, and subunit C. Isothermal titration calorimetry using recombinant subunit d and a_NT revealed that the two proteins bind each other with a K_d of ∼5 μm. Treatment of the purified V_o sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a V_oΔd complex. Passive proton translocation assays revealed that both V_o and V_oΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V₁ from V_o during reversible enzyme dissociation plays a role in blocking passive proton translocation across free V_o and that the interaction between a_NT and d seen in free V_o functions to stabilize the V_o sector for efficient reassembly of V₁V_o.     

Aluminum impairs morphogenic transition and stimulates H(+) transport mediated by the plasma membrane ATPase of Yarrowia lipolytica

The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO₄)₂) inhibits yeast–hypha transition. Both vanadate-sensitive H⁺ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H⁺ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H⁺ efflux in solid medium. The present results suggest that alterations in the plasma membrane H⁺ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H⁺-ATPase as a key factor of this process.