Plant Physiol. (1987) 84, 670-6760032-0889/87/84/0670/07/$0 1.00/0

Dissipation of the Membrane Potential in Susceptible CornMitochondria by the Toxin of Helminthosporium maydis,Race T, and Toxin Analogs1

Received for publication December 29, 1986 and in revised form March 18, 1987

MARCIA J. HOLDEN2 AND HEVEN SZE*Department ofBotany, University ofMaryland, College Park, Maryland 20742

ABSTRACT

We have tested directly the effect of Helminthosporium maydis T(Hmt) toxin and various analogs on the membrane potential formed inmitochondria isolated from a Texas (T) cytoplasmic male-sterile and anormal (N) corn. ATP, malate or succinate generated a membranepotential (negative inside) as monitored by the absorbance change of acationic dye, safranine. The relative membrane potential (A+/) could alsobe detected indirectly as 'Ca2" uptake. Hmt toxin added to T mitochon-dria dissipated the steady state A4k similar to addition of a protonophore,carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (CpdXIII: C4,H86012 and Cpd IV: C2sH406), reduced native toxin (RT2C:C44H1013) and Pm toxin (band A: C331H600, produced by the fungus,Phyllosticta maydis) were effective in dissipating &4, and decreasing Ca2uptake 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 toxinsand analogs had no effect on A4A formed in N mitochondria. The strikingsimilarities of the HmT toxin (band 1: C41H680,3) and Cpd XIII on Tmitochondrial activities provide strong evidence supporting the correct-ness of the polyketol structure assigned to the native toxin. Since the A4,in energized mitochondria is caused mainly by the electrogenic extrusionof H', the results support the idea that HmT toxin increases membranepermeability of T mitochondria to H'. The host specificity of the toxinsuggests that an interaction with unique target site(s) on the innermitochondrial membrane ofT corn causes H' leakage.

We are interested in understanding the mode of toxin action.Most of the evidence indicates the primary target for HmT toxinaction is the mitochondrion of T corn. The toxin exerts uncou-pling effects and inhibits electron transport (6). For example,malate-dependent oxygen consumption in T mitochondria isinhibited, while the toxin stimulates respiration (02 consump-tion) and abolishes state 3/state 4 transitions when NADH orsuccinate (in nonsalt osmoticum) are substrates (10, 11, 18, 23,27, 28). Loss ofrespiratory control can be induced by compoundsthat uncouple electron transport from oxidative phosphorylationby increasing membrane permeability to protons. We haveshown previously that HmT toxin decreases Ca2+ uptake into Tmitochondria (17) and increases membrane permeability to Ca2+(14). Since Ca2+ uptake into the mitochondria is thought todepend on the membrane potential (inside negative) (8, 14, 17,26), we tested directly whether the toxin could dissipate theelectrochemical gradient of H+. Using safranine as a membranepotential probe, we show that HmT toxin dissipated the mem-brane potential in mitochondria of susceptible, but not resistant,corn. Synthetic toxin analogs, reduced native toxin and Pm toxin(produced by the fungus Phyllosticta maydis) were also effectivein dissipating the membrane potential in T mitochondria, thoughthey differed in their relative potency. Preliminary results of thisstudy have been presented (15).

MATERIALS AND METHODS

A toxin produced by the fungus, Helminthosporium maydisrace T, causes symptoms of leaf blight in corn with Texas male-sterile cytoplasm (cms-T3), while corn with N cytoplasm is insen-sitive to the toxin (6). Both toxin sensitivity and male sterilityare maternally inherited, and mitochondrial genes are thoughtto be responsible for these traits (22).

'Supported in part by the Department of Energy Contract No. DE-AS05-82ER13015 and FG05-86ER13461 to H. S. This is ScientificArticle No. A-4643, Contribution No. 7639 ofthe Maryland AgriculturalExperiment Station.

2Present address: Department of Zoology, University of Maryland,College Park, MD 20742.

3Abbreviations: cms-T, cytoplasmic male-sterile Texas; BTP, bis-tris-propane or 1,3-bis-(tris[hydroxymethyl]methylamino)propane; CCCP,carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N'-dicyclohex-ylcarbodiimide; HniT, Helminthosporium maydis, race T; Pm, Phyllos-ticta maydis; RT2C, reduced native HmT toxin; Cpd IV, synthetic toxinanalog (C25FH06); Cpd XIII, synthetic toxin analog (C41H680,2); N,normal-fertile; A#4, membrane potential.

Mitochondria Isolation. Two nearly isogenic lines of Zea maysL., W64AT (T) and W64AN (N), were used in these experiments.Etiolated seedlings (4-5 d old) were obtained by germinatingseeds between four layers of cheesecloth soaked with 0.5 mmCaSO4. Roots were cut into 1-2 cm sections and ground bymortar and pestle in a medium (10 ml/g fresh weight) of 250mM sorbitol, 25 mM Hepes-BTP (pH 7.4), 3 mm EGTA, 1 mMDTT, and 0.1 to 0.2% BSA. The homogenate was filteredthrough four layers of cheesecloth and cellular debris was re-moved by centrifugation at 1600g (3500 rpm, SS-34 rotor).Mitochondria were pelleted at 6000g (7000 rpm, SS-34 rotor)and resuspended in 250 mM sorbitol, 2.5 mm Hepes-BTP (pH7.4), 1 mM DTT, and 0.1% BSA (14, 17). Protein concentrationwas measured by the method of Bradford (5).

Determination of Membrane Potential with Safranine. A4t'formation was measured by recording the spectral shift of thelipophilic cationic dye, safranine, as in mung bean mitochondria(24). The dye accumulates in mitochondria in response to thegeneration of a potential (negative inside). The extent of thespectral shift (reflected as changes in absorbance) of safraninewas monitored with an Aminco DW-2 dual wavelength spectro-photometer using the wavelength pair 511/533 nm. An increasein absorbance (511-533 nm) was correlated with the generation

670

DISSIPATION OF MITOCHONDRIAL MEMBRANE POTENTIAL BY HmT TOXIN

of a membrane potential (inside negative) (see "Results"). Alldeterminations were performed in glass cuvettes (1 cm pathlength) at 22C. Usually, each reaction mixture (1 ml) consistedof about 500 Mg mitochondrial protein in resuspension buffer(250 mm sorbitol, 2.5 mm Hepes-BTP (pH 7.4), 1 mM DTT,0.1% BSA) and about 25 AM safranine. The dye:protein ratiowas maintained near 50 nmol/mg protein. Other ingredients,such as substrate, salt, inhibitors or toxins were added in smallvolumes (5-15 Ml) to give the final concentration noted in thefigure legends. Reactions were started by the addition ofsubstrateand usually terminated by the addition of CCCP. Toxins weredissolved in DMSO, and ionophores or water-insoluble inhibitorswere made up in ethanol or DMSO. Final DMSO or ethanolconcentrations in reaction mixtures were usually 1 or 0.5%,respectively. Up to 4% DMSO had no effect on Alp' formation(13).The total relative membrane potential was determined from

the difference of the substrate-generated absorbance change atsteady state and the level reached after the addition of theionophore. This value ranged from 0.1 to 0.21 O.D. units inindividual experiments with mitochondria from both susceptibleand resistant corn. We did not attempt to relate the absorbancechange to membrane potential in mV.Calcium Uptake. 45Ca transport into mitochondria was meas-

ured by a filtration method as previously described (14, 17).Toxin and Analogs. HmT toxin, Pm toxin, and the HmT toxin

analogs (Cpd XIII-C41H68012 and IV-C25H4406) were generousgifts of Drs. J. M. Daly, S. J. Danko, Y. Suzuki, and K. Frantzen(7, 19-21, 30, 31). Since HmT and Pm toxin preparations consistof a mixture of components, we have used band 1 (C41H68013)and band A (C33H1008) as the representative component ofHmTand Pm toxin, respectively, for comparative purposes only.

RESULTSMembrane Potential Formation in Mitochondria Can Be Mon-

itored with Safranine. To confirm that absorbance changes ofsafranine were due to the formation of a Alt in isolated cornmitochondria, we first investigated the effect of inhibitors andionophores on the safranine response induced with redox sub-strates or with ATP. Malate induced an absorbance increasewhich decayed quickly after addition of rotenone (Fig. la).Malate-induced safranine response usually would decay within2 to 4 min after substrate addition. This could be prevented byinclusion of glutamate (1 mM) in the reaction mixture (data notshown). The decay was probably the result ofinhibition ofmalatedehydrogenase by increasing oxaloacetate levels (25). Figure lademonstrates that rotenone decreased the absorbance to levelslower than the initial absorbance. This higher initial absorbancewas attributed to a potential existing prior to substrate additionand due to endogenous substrate. This idea was confirmed inFigure lb which shows that the initial absorbance could bedecreased by inhibitors of electron transport, such as rotenone(Fig. lb) or KCN (not shown). Succinate-dependent absorbanceincrease was sensitive to KCN but not rotenone (Fig. lb).ATP induced a safranine response ofsimilar magnitude to that

generated by redox substrates. The absorbance changes inducedby ATP were very stable and insensitive to inhibitors, such asKCN and rotenone (Fig. 2a). Oligomycin decreased the ATP-induced absorbance increases (Fig. 2b).

In general, the results are consistent with the idea that spectralshifts of safranine reflect qualitatively the generation or collapseof Al in mitochondria (24). Malate-, but not succinate-, gener-ated membrane potential was inhibited by rotenone (Fig. 1),while ATP-dependent Alt was sensitive to the inhibitor of theproton-pumping ATPase, oligomycin (Fig. 2). Potentials gener-ated by any substrate were dissipated quickly by CCCP or gram-icidin, while inhibitors (e.g. rotenone, oligomycin) generally

Rotenonea

Malate T CN.031O.D. CCCP

12 min

Rotenone

b/ \ ~~CCiCP

SuccinateFIG. 1. Effect of rotenone and KCN on redox substrate-induced

safranine absorbance increase in corn mitochondria (T). The reactionmixtures contained 432 Ag (a) or 600 Mg (b) mitochondrial protein inresuspension buffer (2.5 mM Hepes-BTP[pH 7.4], 250 mM sorbitol) plus5 mM MgC92, 1 mm K2HPO4, and 25 Mm safranine. To start the reaction,8 mM malate-BTP (a) or 4 mm succinate-BTP (b) was added. Finalconcentrations of additions were: 12.5 Mm rotenone, 5 Mm CCCP, 1 mMKCN.

a

ATP

.03TO.D.

,1 mm1

FIG. 2. Effect of inhibitors and CCCP on ATP-induced safranineabsorbance changes in corn mitochondria. Reaction mixture (a) con-tained 480 Mg T mitochondrial protein, 3 mMATP-BTP, 25MMsafranine,5 mM MgCl2, and 250 mM sorbitol in 2.5 mm Hepes-BTP at pH 7.4.Additions were 5 mM KCN, 12.5 uM rotenone, or 5 AM CCCP. Mixture(b) contained 450 Mg T mitochondrial protein, 20 uM safranine, I mMMgSO4, 1 mM ATP-BTP, and 250 mM sorbitol in 2.5 mmHepes-BTP atpH 7.4. Additions were 7.5 Mg/ml oligomycin and 5 gM CCCP.

caused a slower rate of decay attributable to the back leak ofprotons.Ca2 Uptake into Mitochondria Is Driven by the Membrane

Potential. Ca2e decreased Alp generated by either redox substrateor ATP (Fig. 3). The rate of A4' decay was dependent on theCa2+ concentration (Fig. 3a). If Ca2' is accumulated in cornmitochondria in response to a membrane potential (negativeinside) (8, 14, 17), and if ruthenium red inhibits Ca2" transportas in rat liver mitochondria (26), then ruthenium red should

671

6HSPlant Physiol. Vol. 84, 1987

a

b

ATP

FiG. 3. Calcium-induced collapse of the mitochondrial potential wasconcentration-dependent and prevented by ruthenium red. The reactionmixture contained 2.5 mm Hepes-BTP (pH 7.4), 250 mm sorbitol, 450Mgg protein, 1 mM MgSO4, 1 mm K2HP04, 1 mm KCN, 30 Mm safranme,and 1 mM ATP-BTP. In (a) various concentrations of CaCl2 were addedat the arrow after a steady state AA' was reached (numbers indicateconcentration in Mm). In (b) various concentrations of ruthenium redwere included in the reaction mixture (number indicates concentrationin MM), and 800 MM CaCl2 was added at the arrow. Final CCCP concen-tration was 5 MLM.

100l a b 500

j~80 .400

0

d 60 300 00

X40 20 33

20 \100

0 2 4 6 8 0 1 2 3

Ruthenium Red (pM) MgSO4 (mM)

Fwo. 4. Ruthenium red and Mg2+ decreased malate-dependent cal--ium uptake into corn mitochondria. Mitochondrial protein (100 ug)was added to a 0.5 ml reaction mixture consisting of 175 mM sorbitol,25 nm Hepes-BTP (pH 7.4), 5 mm K2HP04, 4 mM malate-BTP, 200 jM45CaC2 and various concentraions ofruthenium red (a) and MgSO4 (b).After incubation for 10 min at 22 to 24-C, aliquots were filtered. In (a),malate-dependent Ca2` uptake in the absence of ruthenium red was 484nmol/mg protein (100%).

retard the decrease of the membrane potential caused by Ca24.We show here that ruthenium red prevented the Ca2-dependentA41 decay (Fig. 3b) and inhibited Ca2+ uptake (Fig. 4a). Relativelyhigh concentrations of ruthenium red were required to retardCa-dependent A# collapse, as the Ca2' concentration used todecrease AA was 4-fold higher than that used to measure Ca?+uptake. Thus, in contrast to mung bean mitochondria (1), Ca2+uptake in corn mitochondria may more closely resemble themechanism described for mammalian mitochondria. Further-

more, our results support the idea that Ca24 uptake is potential-driven and indirectly reflects the magnitude of the A#.

Higher Ca24 concentrations were required to dissipate A4tgenerated by ATP than those formed by malate or succinate.For example, succinate-dependent AAi was completely collapsedby 100 jM CaCI2 (data not shown), whereas 800 jAM Ca2+ wasrequired to decrease the ATPdependent A# (Fig. 3a). Theseresults suggested that Mg2" (present in the ATP reaction mixture)interfered with Ca2+ uptake. This was confirmed by the directdemonstration that Mg2` decreased Ca2' uptake (Fig. 4b). Mge'also interferes with Ca24 transport in mammalian mitochondria(26). In corn mitochondria, Mg2` might compete for the Ca2+_binding site, but it is unlikely to be transported by the same Ca24porter since Mg2+ did not collapse A( (13).Comparison ofHmT and Pm Toxin, and Toxin Analogs on A4

Collapse in T Mitochondria. HmT toxin dissipated the AO gen-erated by either redox substrates (malate or succinate) or ATPin susceptible (T) corn mitochondria (Fig. 5, a, b, c). The rate ofdissipation was dependent on toxin concentration and completecoRapse of AO (within 1 min), could be achieved with 50 ng/mlof purified HmT toxin. The range of effective toxin concentra-tions was independent of the substrate used to generate the A4+.

In contrast, HmT toxin had no effect on As formed inmitochondria even at concentrations 20 to 40 times higher thanthat effective on T corn (Fig. 6). Extremely high concentrations(40-60 Ag/ml) caused a partial decrease in Asp (13).Analogs ofHimT toxin were also effective in dissipating Alp in

T, but not N, mitochondria. Figure 7 compares the activities ofvarious HmT analogs (RT2C, Cpd XIII, Cpd IV) and showsstriking differences in the effective concentrations required todissipate A4' in T corn mitochondria. Cpd XIII (Fig. 7a) is 41carbon atoms in length and has a nearly identical arrangementand placement of keto and hydroxy groups along the hydrocar-bon chain and thus most closely resembles native HmT toxin(Fig. 5; Ref. 30). Comparison ofFigure 5 with Figure 7a indicatesthat the concentration of Cpd XIII required for immediatedissipation of A4/ was very similar to that ofthe native toxin (50ng/ml). However, reduced native toxin, RT2C (Fig. 7b), and thesynthetic analog, Cpd IV (Fig. 7c), dissipated AO at considerablyhigher concentrations (>1000 ng/ml). Cpd IV has a chain lengthof25 carbons and resembles the midportion ofHmT toxin band1 (30, 31).Pm toxin, produced by an unrelated fungus (Phyllosticta may-

dis), also dissipated A4( in T mitochondria (Fig. 7d). This toxinpreparation consists of a series of compounds (7) closely relatedto each other and similar to the HmT toxin series (I19-21). Theprimary difference lies in the arrangement ofpolar groups, whichoccur in groups of two for Pm toxin and three for HmT toxin.Pm toxin was more potent than HImT toxin in its ability todissipate Alp; 5 ng/ml of Pm toxin was as effective (Fig. 7d) as50 ng/ml ofHmT toxin.The effect of the toxin and analogs on Ca2" uptake into T and

N mitochondria was also tested. As shown with the native toxin(17), the analogs decreased Ca2" uptake in T (Fig. 8), but not N,mitochondria (Table I). The relative effectiveness of the analogsand toxins was compared by estimating the concentration (innm) required to inhibit 50% of the activity (Table II). When themost potent toxin was set to 100%, the sequence of decreasingeffectiveness was:

Pm >HmT> Cpd XHI > RT2C> Cpd IVInhibitionofCa2+ uptake 100 30 > 11 1.8 > 1.0Dissipation ofAA 100 > 23 = 25 > 0.4 > 0.2

These results age with the relative effectiveness of the variouscompounds in collapsing AO (Table II).

In spite of apparent differences, the concentrations of HmTtoxin needed to dissipate the membrane potential or Ca2" gra-

672 KlIff-j"'LDEN AND SZE

DISSIPATION OF MITOCHONDRIAL MEMBRANE POTENTIAL BY HmT TOXIN

HmT

| 2000

a-.

Succinate

.03 O.D.

HimT4'

ATP

.0410.D.

2 rrlinFIG. 5. HmT toxin collapsed membrane potentials in T corn mito-

chondria. Each panel shows a senes of assays conducted in a similarmanner with HmT toxin (concentration expressed in ng/iml) added afterthe steady state potential was reached. Each reaction mixture contained2.5 mmiHepes-BTP (pH 7.4) 250 mm sorbitol I mM K2HOP4 plus thefollowing: (a) 650 ug mitochondrial protein, 25 Mm safranine, I mmmalate-BTP; (b) 500 ug mitochondrial protein, 5 FM rotenone, 20 Mm

safranine, 5 mm succinate-BTP; or (c) 450 Ag mitochondrial protein, 20Mm safranine 1 mm MgSO4, I mm KCN, and 1 mM ATP-BTP. FinalCCCP (a) concentration was 5 Mm. The structure of band 1, one of twomajor components of purified HmT toxin, is shown.

dients (13, 14) as well as those needed to inhibit Ca2" uptake arecomparable. Consistent with the hydrophobic nature ofthe toxin,the effect of toxin on A4( or Ca2" uptake activities was dependenton the toxin:protein ratio and the time of incubation (13).Membrane potential was measured with an average proteinconcentration of 500 iAg/ml, while the Ca2" uptake was assayed

.04 0.D.

Succinate

2 min

FIG. 6. HmT toxin did not collapse the membrane potential of Ncorn mitochondria. Two assays are drawn in parallel showing twosequential additions (10 ul each) ofHmT toxin (or 10 41 of the solvent,DMSO) to mitochondria after generation of a potential. Toxin concen-

tration is expressed in ng/ml. Final DMSO concentration was 2%. Thereaction mixture contained 2.5 mM Hepes-BTP (pH 7.4), 250 mM

sorbitol, 410 ;tg mitochondrial protein, 20 Mm safranine, 5 mM M9gCI2, 2mM K2HPO4, 5 gM rotenone, and 5 mm Na-succinate. Final CCCPconcentration was 5 MM.

with 50 to 100 jg protein/ml. Furthermore, the toxin concentra-tion required to dissipate Ai, by 50% was estimated from activityat 2 min after toxin addition. In contrast, Ca2" uptake wasroutinely measured after a 10 min incubation period. Since therelative inhibition is dependent on the time of incubation andthe membrane protein, the concentration ofHmT toxin requiredto dissipate A/ by 50% is estimated to be at least 20-fold higherthan that needed to inhibit Ca2" uptake. In general, the valuesdetermined experimentally (Table II) support the idea that sim-ilar concentrations of toxin are required to inhibit Ca2+ uptakeand dissipate the Al.Toxin Stimulates Mitochondrial ATPase Activity. If the toxin

collapses the membrane potential in T mitochondria just likeuncouplers, then the hydrolysis ofATP should be stimulated bythe toxins. Table III shows that both HmT and Pm toxinsstimulated ATP hydrolysis in T, but not in N, mitochondria.The stimulation of ATPase activity by HmT toxin was similarto that induced by gramicidin. These results agree with thosereported earlier by Bednarski et al. (2) and JM Daly and SJDanko (personal communication).

In order to demonstrate toxin stimulation of the ATPaseactivity, it is crucial to obtain a relatively high population oftight mitochondria that will hold a proton electrochemical gra-dient. In a previous study, we were unable to demonstrate anytoxin effect on mitochondria ATPase activity (A Kimber, un-published data), probably because the mitochondria were sub-jected to osmotic shock. This could be prevented by includingsucrose as an osmoticum in the ATPase reaction mixture.

DISCUSSIONWe have shown directly that HmT toxin, Pm toxin, and

various toxin analogs dissipated AA in susceptible (T), but notresistant (N), corn mitochondria. The A4v as detected by thespectrl shifts of safranine could be generated by either redoxsubstrates or ATP. While our studies were in progress, Bervilleet at. (3) reported that HmT toxin inhibited A4t formation incorn mitochondria. They found that mitochondria (3 mg protein)treated with 91 nM HmT toxin were unable to accumulate 3H-or '4C-tetraphenyl phosphonium, a lipid permeant cation. How-

a

CCCP

\,t~, i

673

HOLDEN AND SZE Plant Physiol. Vol. 84, 1987

ATP

a)

I.C

co

a)

>

a)

RT2C

b

1000

0.04f0.D.

ATP HQHOQH QHQHOH QHQHQH QHQHQH

c

FIG. 7. Collapse of membrane potential in T corn mitochondriainduced by Cpd XIII (a), reduced native toxin (b), Cpd IV (c), and Pmtoxin (d). All reaction mixtures contained 250 mm sorbitol in 2.5 mMHepes-BTP at pH 7.4, 500 Mg T mitochondrial protein, 30 uM safranine,I mm KCN, 1 to 2 mM K2HPO4, and 1 mM MgSO4 (5 mM MgCl2 forCmpd IV assays). Each panel shows several assays where the toxin or

analog was added 3 min after potential generation with 1 mm ATP.Toxin concentrations are expressed in ng/ml. Final CCCP conc was 5Mm. Inset shows the chemical structures of the toxins.

ever, they did not show the time-course of the toxin effect on

A0, or the dependence on toxin concentration. A major advan-tage of using an optical probe, like safranine, versus a radiola-beled cation, is that A# can be monitored continuously as afunction of time and thus early events (few seconds) can bedetected. Although the toxins dissipated AO very rapidly, we wereable to differentiate between the concentration dependence ofthe different toxins and the analogs.

Careful comparison of toxin analogs, Pm toxin and HmTtoxin, on both A4 dissipation and inhibition ofcalcium transport(Table II), yielded the following ranking in terms of effectivenesson T corn mitochondria: Pm >> HmT > Cpd XIII >> RT2C >

10Toxin (ng/ml)

FIG. 8. Effect oftoxins and analogs on malate-dependent Ca2l uptakeinto T mitochondria. The reaction mixtures were as described in TableI. Results are from the average of two to four experiments. The meanactivities in the absence of Pm, HmT, Cpd XIII, IV, and RT2C were1007, 1007, 875, 1367, and 1833 nmol/mg protein, respectively. Solidbars represent standard deviation; dotted bars represent range of twoexperiments.

Table I. Effect of Toxins and Analogs on Malate-Dependent Ca2+Uptake in N Mitochondria

The reaction mixture (0.5 ml) consisted of 175 mm sorbitol, 4 mMmalate-BTP, 25 mM Hepes-BTP (pH 7.4),5 mm K2HPO4,200 MM 45CaCI2(0.25-0.5 ,Ci/ml) and various concentrations of toxin in DMSO (finalDMSO concentration was 1%). The reaction was started with the addi-tion of mitochondria (50-100 Mg protein) and the incubation continuedfor 10 min at 22 to 24C. Results from average ofthree to five experiments(±SD).

Malate-dependent Ca2" UptakeCompound

Pm HmT XIII RT2C IV

Mg/ml % (nmol/mg protein)0 100 (403) 100 (1147) 100 (610) 100 (917) 100 (1030)0.1 92 ± 14 100 ± 10 103 ± 5 103 ± 10 98 ± 90.3 87±9 111± 19 97± 11 104±6 106±81.0 91 ±7 85±9 100± 10 105± 10 103±65.0 97 ± 12

Table II. Concentration of Toxins Required to Inhibit Ca2" Uptakeand to Dissipate A4t by 50% in T Mitochondriafrom Corn RootsThe 150 for inhibition of Ca2" uptake by the toxin and analogs was

estimated from the dose-response results of Figure 8. The I"o for Afdissipation was estimated from the data in Figures 5 and 7 (using the %inhibition at 2 min after toxin addition). The toxin concentrations areexpressed either as ng per 0.1 or 0.5 mg protein in a 1 ml reactionmixture or as nm (the mol mass of band 1 and band A were used forHmT and Pm toxin, respectively).

Toxin Concentration Required toMol Inhibit 50% ActivityToxmWt ATP-dependent Malate-dependent

i^,& ca2+ uptakeng/0.5 mg (nM) ng/0.J mg (nM)

HmT-C4, 768 20 (26) 0.8 (1.0)Pm-C33 584 3.5 (6) 0.2 (0.3)Cpd XIII-C41 752 18 (24) 2 (2.7)RT2C-C4, 784 900 (1148) 13 (16.6)Cpd IV-C25 440 900 (2045) 13 (29.5)

674

DISSIPATION OF MITOCHONDRIAL MEMBRANE POTENTIAL BY HmT TOXIN

Table III. Effect ofHmiT, Pm Toxin, and Gramicidin on ATPaseActivity ofMitochondria Isolatedfrom T andN Corn

Mitochondria was washed in resuspension buffer (without DTT andBSA) and repelleted. ATPase activity was measured by the release of Pi(9). The reaction mixture (0.5 ml) consisted of 30 mm BTP-Hepes at pH8.5, 50 mm KCI, 3 mm MgSO4, 3 mm ATP-BTP (pH 8.0), 124 mmsucrose, 25 mm sorbitol, and 58 ug (N) or 48 sg (T) mitochondrialprotein. After incubation for 15 min at 27C, the reaction was stoppedwith 1% (NH4)6Mo7024 in 2 N H2SO4. Activity of nonspecific phospha-tases was measured in the absence of MgSO4 and KCI and subtractedfrom the total. Data from one experiment representative of several.

ATPase ActivityTreatment

T N

MAmol Pilmg protein*h (%)Control 2.4 (100) 3.8 (100)HmT toxin (ng/ml)

10 4.4 (183)100 3.6 (150)

1000 3.8(100)Pm toxin (ng/ml)

10 3.5 (146)1000 2.9 (76)

Gramicidin (5 Mg/ml) 4.4 (183)Oligomycin (5 Mg/ml) 0.2 (8)

Cpd IV. None of these compounds had any significant effect onCa2+ uptake (Table I) or A4t generation in N corn mitochondria(Fig. 6). The strikingly similar effects (quantitative and qualita-tive) of the synthetic Cpd XIII and the native toxin on Tmitochondrial activities provide compelling evidence that thepolyketol structure assigned to the HmT toxin is correct (19-21,30). Our results not only agree with those of Suzuki et al. (30,31) and Danko et al. (7), but the relative effectiveness of thevarious compounds determined by in vitro assays here are quan-titatively similar to their results using an in vivo assay (dark CO2fixation by leaf slices). Since the reduced native toxin was lesseffective than the native toxin, we conclude that keto groupspresent in the native toxin are important for optimal activity butnot absolutely required. Cpd IV (C25) was significantly less effec-tive than the native toxin (C39-C41), suggesting an apparentimportance of the chain length for maximum activity. However,the greater activity of the Pm toxin with chain lengths of onlyC33-35 would suggest the spacing of side groups is also important.We have not compared the effectiveness of the HmT toxin

with methomyl, a carbamate insecticide. Although methomyl(CH3C[S-CH3] = N-0-CO-NH-CH3) appears structurally unre-lated to the HmT or Pm toxins, it also uncouples mitochondriaisolated from Texas male-sterile corn (18). Klein and Koeppe(18) showed that 4 mm methomyl stimulates oxygen uptake atrates comparable to that induced by 40 ng HmT toxin/mgmitochondrial protein (or about 13 nM). Since the effectiveconcentration of methomyl is considerably greater than those ofthe toxins, it is possible the primary mode of action of these twocompounds are different. Alternatively, both methomyl and theHmT toxin may act at different parts of a functional 'receptor,'and the interaction results in similar changes and therefore acommon biological response.

Dissipation of A4 by the HmT toxin (Fig. 5) is consistent withits ability to increase membrane permeability to ions. Whilechanges in A4p reflect movement of charge rather than a specificion, it is generally accepted that formation of a large membranepotential in energized mitochondria is caused by the electrogenicextrusion ofHI via the electron transport chain (12). Dissipationof the membrane potential by HmT toxin therefore supports theidea that toxin increases permeability to HI. Since HmT toxin

increases mitochondrial membrane permeability to Ca2" (14) aswell, the toxin may increase membrane permeability to ions ingeneral in T mitochondria.The specificity ofHmT toxin for cms-T corn strongly suggests

the presence of a target site unique to T mitochondria. Althoughthe results in this study do not provide obvious clues to itsidentity, it is reasonable to propose that an interaction of thetoxin with the site is a prerequisite for the increase in membranepermeability to ions. There is indirect evidence for a proteina-ceous receptor. Pham and Gregory (29) noted that cms-T cornmitochondria lost sensitivity to HmT toxin with time, but thatsensitivity was maintained by DTT, a thiol reagent. Furthermore,Bouthyette et al. (4) found that HmT toxin-induced swelling ofmitochondria could be blocked by DCCD. We have also foundthat DCCD will partially prevent the inhibition ofCa2+ transportand Al formation by HmT toxin (16). Although Bouthyette etal. (4) reported that ['4CJDCCD preferentially labeled a 9 kDprotein which appears to be part of the mitochondrial ATPase,we doubt that the DCCD-binding proteolipid of the ATPase isthe primary target site of the toxin. As reported by others (10,11, 23, 27, 28), we (13, 16) have also found that HmT toxininhibited malate-dependent, but not succinate-dependent, elec-tron transport. Whether this is caused by NAD+ leakage alone(3), or by a direct inhibition of electron transport via complex I(NADH-UQ oxidoreductase), or both has yet to be established.

Acknowledgments-We are indebted to Dr. Walter Bonn; (University of Penn-sylvania) and Dr. Thomas Elthon for stimulating discussions and their help withmeasuring membrane potential, and to Dr. W. VanderWouce (Photobiology Lab-oratory, United States Department ofAgriculture, Beltsville) for making an AmincoDW-2 spectrophotometer available. We are especially grateful to Drs. JM Daly(University of Nebraska), SJ Danko, Y Suzuki, and K Frantzen for providing thetoxins and analogs.

LITERATURE CITED

1. AKERMAN KEo, AL MOORE 1983 Phosphate dependent ruthenium red insen-sitive Ca2" uptake in Mung bean mitochondria. Biochem Biophys ResCommun 114:1176-1181

2. BEDNARSKI MA, S IZAWA, RP SCHEFFER 1977 Reversible effects of toxin fromHelminthosporium maydis race T on oxidative phosphorylation by mito-chondria from maize. Plant Physiol 59: 540-545

3. BERVILLE A, A GHAZI, M CHARBONNIER, J-F BONAVENT 1984 Effects ofmethomyl and Helminthosporium maydis toxin on matrix volume, protonmotive force, and NAD accumulation in maize (Zea mays L.) mitochondria.Plant Physiol 76: 508-517

4. BOUTHYETTE P-Y, V SPITSBERG, P GREGORY 1985 Mitochondrial interactionswith Helminthosporium maydis race T toxin: blocking by dicyclohexylcar-bodiimide. J Exp Bot 36: 511-528

5. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye bind-ing. Anal Biochem 72: 248-254

6. DALY JM 1981 Mechanisms of action. In RD Durbin, ed, Toxins in PlantDisease. Academic Press, New York, pp 331-383

7. DANKO SJ, Y KONO, JM DALY, Y SUZUKI, S TAKEUCHI, DA MCCRERY 1984Structure and biological activity of a host-specific toxin produced by thefungal corn pathogen Phyllosticta maydis. Biochemistry 23: 759-766

8. DAY DA, BL BERTAGNOLLI, JB HANSON 1978 The effect of calcium on therespiratory responses of corn mitochondria. Biochim Biophys Acta 502:289-297

9. FISKE CH, Y SUBBAROW 1925 The colorimetric determination of phosphorus.J Biol Chem 66: 375-400

10. FLAVELL R 1975 Inhibition of electron transport in maize mitochondria byHelminthosporium maydis race T pathotoxin. Physiol Plant Pathol 6: 107-116

11. GREGORY P, ED EARLE, VE GRACEN 1980 Effects of purified Helminthospo-rium maydis race T toxin on the structure and function ofcorn mitochondriaand protoplasts. Plant Physiol 66: 477-481

12. HANSON JB, DA DAY 1980 Plant mitochondria. In PK Stumpf, EE Conn, eds,The Biochemistry of Plants, Vol 1. Academic Press, New York, pp 315-358

13. HOLDEN M 1985 Helminthosporium maydis, race T (HmT) toxin effects onmitochondrial membranes from susceptible (cms T) corn. PhD thesis. Uni-versity of Maryland, College Park

14. HOLDEN MJ, H SZE 1984 Helminthosporium maydis T toxin increased mem-brane permeability to Ca2+ in susceptible corn mitochondria. Plant Physiol75: 235-237

15. HOLDEN MJ, H SzE 1985 Helminthosporium maydis race T toxin collapsesthe membrane potential of susceptible corn mitochondria. Plant Physiol 77:

675

676 HOLDEN

S-14816. HOLDEN MJ, H SzE Dicylohexylcarbodiimide protects against Helmintho-

sporiwn maydis race T toxin action on susceptible corn mitochondria. InAL Moore, B Beechey, eds, Proceedings ofthe Second International Meetingon Plant Mitochondria. Academic Press, New York. In press

17. KImBER A, H SzE 1984 Helminthosporiwn maydis T toxin decreased calciumtransport into mitochondria of susceptible corn. Plant Physiol 74: 804-809

18. KLEiN RR, DE KOEPPE 1985 Mode of methomyl and Bipolaris maydis (raceT) toxin in uncoupling Texas male-sterile cytoplasm corn mitochondria.Plant Physiol 77: 912-916

19. KoNo Y, JM DALY 1979 Characterization of the host-specific pathotoxinproduced by Helminthosporium maydis, race T, affecfing corn with Texasmale sterile cytoplasm. Biooranic Chem 8: 391-397

20. KONO Y, S TAKEUCHI, A KAWARADA, JM DALY, HW KNOCHE 1980 Structureofthe host-specific pathotoxins produced by Helminthosporium maydis, raceT. Tetra Lett 21: 1537-1540

21. KoNo Y, S TAKEUCHI, A KAWARADA, JM DALY, HW KNOCHE 1981 Studieson the host-spcific pathotoxins produced in minor amounts by Helmin-thosporium maydis, race T. Bioorg Chem 10: 206-218

22. LEAVER CJ, MW GRAY 1982 Mitochondrial genome oranization and expres-sion in higher plants. Annu Rev Plant Physiol 33: 373-402

23. MILLER RJ, DE KOEPPE 1971 Southern corn leaf blight: susceptible andresistant mitochondria. Science 173: 67-69

AND SZE Plant Physiol. Vol. 84, 1987

24. MooRE AL, WD BONNER JR 1982 Measurements of membrane potentials inplant mitochondria with the Safranine method. Plant Physiol 70: 1271-1276

25. NEUBURGER M, R DoucE 1980 Effect of bicarbonate and oxaloacetate onmalate oxidation by spinach leaf mitochondria. Biochim Biophys Acta 589:176-189

26. NICHOLLS D, K AKERMAN 1982 Mitochondrial calcium transport. BiochimBiophys Acta 683: 57-88

27. PAYNE G, Y KoNo, JM DALY 1980 A comparison of purified host specifictoxin from Helminthosporium maydis, race T, and its acetate derivative onoxidation by mitochondria from susceptible and resistant plants. PlantPhysiol 65: 785-791

28. PETERSON PA, RB FLAVELL, DHP BARRATT 1975 Altered mitochondrialmembrane activities associated with cytoplasmically-inherited disease sensi-tivity in maize. Theor Appl Genet 45: 309-314

29. PHAM NH, P GREGORY 1980 Loss of sensitivity to Helminthosporium maydisrace T toxin during aging of mitochondria isolated from Texas cytoplasmcorn. Plant Physiol 65: 1173-1175

30. SUZUKI Y, SJ DANKO, JM DALY, Y KONo, HW KNOCHE, S TAKEUCHI 1983Comparison of activities of the host-specific toxin of Helminthosporummaydis, race T and a synthetic C-41 analog. Plant Physiol 73: 440-444

31. SUZUKI Y, KJ TEGTMEIER, JM DALY, HW KNOCHE 1982 Analogs of host-specific phytotoxin produced by Helminthosporium maydis, race T. II Bio-logical activities. Bioorg Chem 11: 313-321

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