Further evidence for the existence of a membrane potential in Trypanosoma brucei brucei

The distribution of 137Cs+, in the presence of valinomycin, has been used to measure the magnitude of the membrane potential (delta psi) in bloodstream forms of Trypanosoma brucei brucei. Values of the delta psi falling in the range -100 mV to -160 mV were observed and the maintenance of this delta psi was sensitive to certain ionophores and protonophores.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

The mitochondrial ATP synthase of Trypanosoma brucei: developmental regulation through the life cycle.

The mitochondrial H(+)-ATPase of the parasitic protozoan Trypanosoma brucei is shown to be developmentally regulated through the T. brucei life cycle as has been shown for components of the mitochondrial electron transport chain. We have substantiated our results by assaying not only for oligomycin-sensitive ATPase activity but also by determining the level of ATP synthetic activity. These results show that the level of ATPase present in the procyclic form of T. brucei is increased by at least threefold from that of the early bloodstream form while the ATPase activity in the late bloodstream form is only about twofold higher than the early form. ATP synthesis activity shows these same results. We have determined the level of ATP synthase protein present in the life cycle stages by Western analysis employing the antibodies that we have raised against both the water soluble F1 and the membrane-associated F0 moieties which we have purified from T. brucei. The Western blots of the procyclic form show strong reactivity with both the F0 and F1 antibodies. The other two life cycle stages, the early and the late bloodstream forms, show considerably less reactivity, paralleling the activity results. Electron micrographs of the sonicated mitochondrial fraction show inverted vesicles which are studded with knobby H(+)-ATPase in the procyclic form. The early bloodstream vesicles show very few of these characteristic structures, while the late bloodstream form shows a range of vesicles from nearly nude to partially studded.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

The distribution of permeant ions demonstrates the presence of at least two distinct electrical gradients in bloodstream forms of Trypanosoma brucei.

The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in bloodstream forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide-p-trifluoromethoxyphenylhydrazone-sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (delta psi) in bloodstream forms of T. brucei; a potential across the plasma membrane (delta psi p) and an additional delta psi, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.     

Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates

Transport of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane requires a mitochondrial membrane potential. We have studied whether additional energy sources are also necessary for protein translocation. Reticulocyte lysate (containing radiolabelled precursor proteins) and mitochondria were depleted of ATP by pre-incubation with apyrase. A membrane potential was then established by the addition of substrates of the electron transport chain. Oligomycin was included to prevent dissipation of delta psi by the action of the F0F1-ATPase. Under these conditions, import of subunit beta of F1-ATPase (F1 beta) was inhibited. Addition of ATP or GTP restored import. When the membrane potential was destroyed, however, the import of F1 beta was completely inhibited even in the presence of ATP. We therefore conclude that the import of F1 beta depends on both nucleoside triphosphates and a membrane potential.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

The mechanism of stimulation of respiration by fatty acids in isolated hepatocytes

Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.     

Reversible depolarization of in situ mitochondria by oxidative stress parallels a decrease in NAD(P)H level in nerve terminals

We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress.     

The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function

Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasite's life cycle. Here we report that silencing expression of the alpha subunit of the mitochondrial F(1)-ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the gamma subunit of F(1) that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.     

Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei.

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.     

Maintenance of internal pH and an electrochemical gradient in Trypanosoma brucei

The internal pH value (pHi) of the long-slender bloodstream form of Trypanosoma brucei was estimated from the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione or 14C-labeled methyl amine between the intracellular space of the cells and the medium. The pHi of T. brucei remained relatively constant at 7.0-7.2 throughout an extracellular pH (pHo) range of 6.0-8.0. The maintenance of an internal pH more acidic than the environment appears to be a unique feature. Preincubation of T. brucei with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or CCCP + valinomycin had no appreciable effect on the delta pH across the T. brucei membrane when the external pH was 8.0. However, when the external pH was 6.0, CCCP abolished the observed delta pH. Nigericin significantly dissipated the delta pH across the T. brucei membrane at all pHo values. These data suggest that under physiological conditions, the maintenance of a delta pH across the bloodstream-form T. brucei membrane may be by a mechanism other than an energy-dependent gradient, whereas an energy-dependent pump may be needed for maintaining the pHi in an acidic environment. The electrical potential (delta psi) across the trypanosomal plasma membrane was also estimated using the lipophilic cation, [3H]tetraphenyl-phosphonium bromide. It appears dependent on both the external pH and the external salt conditions. Under ionic conditions similar to the host bloodstream, it ranges from -76 to -160 mV over an external pH range of 6.0 to 8.0, with an estimated value of -155.5 +/- 0.7 at the physiological pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes

The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria.     

Proton electrochemical potential of the inner mitochondrial membrane in isolated perfused rat hearts, as measured by exogenous probes

The membrane potential (delta psi) and delta pH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial delta psi, measured by means of the subcellular distribution of [3H]triphenylmethylphosphonium (TPMP+), was 125 +/- 7 mV (negative inside) in hearts beating at 5 Hz and 150 +/- 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane delta pH, measured by means of the subcellular distribution of low concentrations of [1-14C]propionate, was 0.63 +/- 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 +/- 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with delta psi.     

Mitochondrial protein import: involvement of the mature part of a cleavable precursor protein in the binding to receptor sites.

The precursor of F0-ATPase subunit 9 was bound to mitochondria in the absence of a mitochondrial membrane potential (delta psi). Binding was mediated by a protease-sensitive component on the mitochondrial surface. When delta psi was reestablished, bound precursor was directly imported without prior release from the mitochondrial membranes. A chimaeric protein consisting of the complete subunit 9 precursor fused to cytosolic dihydrofolate reductase (DHFR) was also specifically bound to mitochondria in the absence of delta psi. Two other fusion proteins, consisting either of the entire presequence of subunit 9 and DHFR or of part of the presequence and DHFR, were imported in the presence of delta psi. In the absence of delta psi, however, specific binding to mitochondria did not take place. We suggest that the hydrophobic mature part of subunit 9 is involved in the delta psi-independent binding of the subunit 9 precursor to receptor sites on the mitochondrial surface.     

Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.     

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.