Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca²⁺ gradients, induced by the toxin in T corn was prevented by N,N′-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N′-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

A Comparison of Purified Host Specific Toxin from Helminthosporium maydis, Race T, and Its Acetate Derivative on Oxidation by Mitochondria from Susceptible and Resistant Plants

NADH or succinate oxidation and malate oxidation were differentially affected in mitochondria from both susceptible and resistant corn by a purified and chemically characterized preparation of host-specific toxin from Bipolaris (Helminthosporium) maydis, race T. NADH and succinate oxidation by susceptible T corn mitochondria were stimulated 50 to 200% with apparent uncoupling from the cytochrome chain at approximately 10(-9)m toxin (5 to 20 ng/ml). Significant inhibition of malate oxidation was observed at slightly higher toxin concentrations, but oxidation was still coupled to ADP utilization. Inhibition of malate oxidation also was observed in N corn (resistant) and soybean mitochondria at approximately 1,000-fold greater concentrations, but stimulation of NADH and succinate oxidation was not found at any toxin concentration tested.A fully acetylated toxin derivative at approximately 1 microgram per milliliter also caused stimulation of NADH or succinate oxidation in T corn mitochondria, but not those of N corn or soybean mitochondria at 100 micrograms per milliliter. Malate oxidation was inhibited to the same extent by toxin acetate with mitochondria from T corn, N corn, and soybean. The blocking of hydroxyl groups in race T toxin by acetyl functions eliminated selectivity toward malate oxidation only. The data suggest that inhibition of malate oxidation is either a separate or secondary effect of selective action of toxin on T corn mitochondria, perhaps by interference with transport in or out of the matrix. Sensitivity of T, but not N, corn mitochondria to purified toxin decays within minutes after pellets are suspended in aqueous osmotica, with no obvious change in mitochondrial integrity. The action of race T toxin seems to involve a labile process, such as ion gradient(s), or an unstable structural conformation of T corn mitochondria.     

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.     

Voltage-dependent channels found in the membrane fraction of corn mitochondria

Transmembrane channels have been found in the membrane fraction of corn (Zea mays W64AN) mitochondria that exhibit a remarkable resemblance to the voltage dependent anion-selective channels (VDAC) located in the outer membrane of animal (Rattus norvegicus), protist (Paramecium aurelia), and fungal (Neurospora crassa) mitochondria. The channels in corn were demonstrated to be essentially identical to VDAC channels in three characteristic properties: (a) single channel conductance magnitude, (b) weak anion selectivity, and (c) nature of voltage dependence. These findings led us to conclude that the channels present in corn mitochondria are VDAC channels. This discovery may have repercussions concerning the regulation and function of higher plant mitochondria, and the causation of higher plant excitability.     

The in vivo response of corn mitochondria to Bipolaris (Helminthosporium) maydis (race T) toxin

The addition of 2-deoxyglucose to tissue elicits an in vivo mitochondrial conformation response (contraction) that can be viewed ultrastructurally and is indicative of the phosphorylative capability of mitochondria. Utilizing this technique toxin from Bipolaris (Helminthosporium) maydis race T was found to penetrate leaf and root tissue of Texas male-sterile cytoplasm corn (Zea mays L. W64A) only slowly, but once in cells the toxin had a rapid deleterious effect on mitochondrial function. It is concluded that B. maydis (race T) toxin has effects on in vivo mitochondria similar to those reported after in vitro experimentation and that mitochondria are a primary site of toxin action. These observations are followed by the suggestion that susceptibility or resistance to B. maydis (race T) is conferred in corn by a cytoplasmically inheritable character associated with mitochondria.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

Mode of Methomyl and Bipolaris maydis (race T) Toxin in Uncoupling Texas Male-Sterile Cytoplasm Corn Mitochondria

Bipolaris maydis race T toxin (BmT), and its functional analog, methomyl, uncoupled Texas male-sterile (T) cytoplasm mitochondria by decreasing the resistance of the inner membrane to protons. However, unlike protonophoric or ionophoric agents, BmT toxin and methomyl induced irreversible swelling. Packed volume measurements showed that mitochondrial volume was irreversibly increased by methomyl and BmT toxin indicating that mitochondria no longer functioned as differentially permeable osmometers. The decreased resistance of inner mitochondrial membranes to protons and the loss of osmotic volume regulation suggests that methomyl and BmT toxin induced the formation of hydrophilic pores in T mitochondrial inner membranes.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation

Isolated corn mitochondria (Zea mays cv. B73 × Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Δ¹-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.     

Evidence that the crystalline arrays in the outer membrane of Neurospora mitochondria are composed of the voltage-dependent channel protein

Antibodies were raised in rabbits against the outer membrane of Neurospora mitochondria. Antibodies were obtained that were specific for this membrane's major polypeptide ($\text{M, 31 000}$) and its slower-migrating derivatives on SDS-polyacrylamide gels. These antibodies inhibited the insertion into phospholipid bilayers of voltage-dependent ion channels from detergent extracts of the mitochondrial outer membranes. The same antibodies bound preferentially to membranes containing crystalline surface arrays in outer mitochondrial membrane fractions. These results indicate that the 31 kDa polypeptide is a component both of the ion channels and of the membrane arrays, suggesting identity between the functional and structural entities.     

Isolation and properties of the outer membrane of plant mitochondria.

The outer membrane of turnip (Brassica rapa L.) mitochondria was isolated by incubating the mitochondria with a dilute digitonin solution and differential centrifuging. The outer membrane fraction was not contaminated by inner membrane enzymes and lacked an NADPH-cytochrome c reductase. However it possessed very active NADH-cytochrome c, dichloroindophenol and ferricyanide reductases which were insensitive to antimycin A, Amytal and low (less than 10 μm) concentrations of Dicumarol. p-Chloromercuribenzoate (ClHgBzO^?) and high concentrations (greater than 10 μm) of Dicumarol inhibited the reductases, ClHgBzO^? almost completely. Preincubation of the outer membrane with NADH protected it from ClHgBzO^? inhibition. An acid phosphatase and an NADPH-ferricyanide reductase were also detected, but the latter was only loosely bound to the membrane. The NADH dehydrogenase of the outer membrane was insensitive to ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetate (1 mm) and was not stimulated by CaCl₂ (0.5 mm), thus differing from the external NADH oxidase of the inner membrane (Coleman, J. O. D., and Palmer, J. M. (1971) FEBS Lett., 17, 203–208). Respiratory-linked oxidation of exogenous NADH by intact mitochondria showed a similar pattern of inhibition by ClHgBzO^? as did the outer membrane, but was inhibited strongly by low concentrations of Dicumarol (5 μm inhibited by 70%).     

Effects of Methomyl and Helminthosporium maydis Toxin on Matrix Volume, Proton Motive Force, and NAD Accumulation in Maize (Zea mays L.) Mitochondria

Methomyl and Helminthosporium maydis race T toxin block oxidative phosphorylation in mitochondria isolated from maize plants with Texas male sterile cytoplasm (T) but not in mitochondria isolated from those with Normal cytoplasm (N) (Bednarski, Izawa, Scheffer 1977 Plant Physiol 59: 540-545). Moreover, they have been reported to cause specific swelling in T mitochondria (Miller, Koeppe 1971 Science 173: 67-69; Koeppe, Cox, Malone 1978 Science 201: 1227-1229). We could not detect, by direct volume measurements, any change induced by these compounds in the mitochondrial matrix space. We show here that the proton motive force, which in maize mitochondria is composed of a large transmembrane potential and of a low transmembrane pH difference, is absent in T mitochondria incubated in the presence of methomyl or of Helminthosporium maydis race T toxin, while it is unchanged in N mitochondria. Methomyl and Helminthosporium maydis race T toxin induce, independently of the collapse of the proton motive force, a release of the cofactors NAD and coenzyme A from the mitochondrial matrix space. In particular, we show that NAD is transported in maize mitochondria, and that this transport, which is not dependent on the proton motive force, is inhibited by methomyl or Helminthosporium maydis race T toxin.     

N‐terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase‐9‐dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N‐terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N‐terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagicEscherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N‐terminal region in addressing certain RTX toxins to mitochondria.     

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.