Vol. 77, No. 4, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FACTORS AFFECTING OLIGOMYCIN INHIBITION OF YEAST MITOCHONDRIAL ATPase
Richard Johnston, Stephen Scharf and Richard 8. Criddle Department of Biochemistry and Biophysics
University of California Davis, California 95616
Received July 6,1977
SUMMARY
The level of inhibition of yeast mitochondrial ATPase by oligomycin has been altered by the addition of several reagents. Potassium chloride greatly enhances oligomycin inhibition while sodium chloride has no effect. Both dihydrolipoic acid and lipoic acid enhance inhibition while lipoamide has no effect. As pH is varied from 9.5 down to 5.5, inhibition by oligomycin is decreased. Addition of low levels of either mercuric ion or iodoacetic acid blocks oligomycin inhibition but not enzyme activity. These results are interpreted in terms of a model in which the oligomycin binding site of ATPase can exist in two forms with relative amounts of these forms governed by the added reagents.
INTRODUCTION
Oxidative phosphorylation in mitochondria is inhibited by low levels of
the antibiotic oligomycin. The site of inhibition is at the ATPase (ATP syn-
thase) enzyme complex in the mitochondrial inner membrane (i). Oligomycin
sensitive ATPase activity can be assayed in submitochondrial particle prepara-
tions or in detergent solubilized preparations of ATPase which contain soluble
F 1 ATPase bound to a "membrane sector" (2,3,4,5). The level of oligomycin
sensitivity in these preparations has been somewhat variable. This has been
attributed to such factors as differences in source of enzyme, lipids associated
with the complex, methods of purification, and alterations in the complex due
to mutation (6,7,8,9). In this communication, the effects of reagents or con-
ditions altering ATPase sensitivity to oligomycin are analyzed to determine
what these may imply about the functioning of the ATPase complex.
Several important findings regarding oligomycin inhibition of ATPase aid
in the interpretation of these studies. It is generally agreed that oligomycin
inhibits ATP synthesis or hydrolysis in membrane systems by blocking proton
translocation (i0). It does this by reversibly binding to the "membrane sector"
Vol. 77, No. 4, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the ATPase complex at, or in the near vicinity of the small hydrophohic
protein, subunit 9 (Domenclature of Tzagoloff, 6) (11,122. Correspondingly
yeast mitochondrial mutants both resistant to oligomycin inhibition and altered
in subunit 9 have been prepared in different laboratories (13,14). These re-
sults imply a relationship among proton transport by the ATPase, oligomycin
binding, and protein subnnit 9. Studies on reagent induced changes in oligo-
mycin inhibition of ATPase may then be considered in relation to the functional
role of subunit 9 in the enzyme complex.
MATERIALS AND METHODS
Saccharomyces cerevisiae strains D243-4A (a,ade,lys) and two oligomycin resistant mutants OR4 and OR8 prepared from this strain were used in all studies reported (9). Cells were grown in batch cultures on 1% Difco Peptone, 1% Yeast Extract and 2% glucose media to late log phase. Cells were har- vested and mitochondria prepared as described by Enns and Criddle (12). Sub- mitochondrial particles and Triton X-100 solubilized oligomycin sensitive ATPase were prepared by the method of Tzagoloff and Meagher (6). Oligomycin sensitive ATPase was then purified by chromatography as described by Enns and Criddle (12). The specific activities of purified oligomycin sensitive ATPase were in the range of 14 to 20 ~moles ATP hydrolyzed per minute per mg protein.
ATPase assays were performed using the coupled spectrophotometric method of Monroy and Pullman (16) in pH 7.4 Tris HCI buffer containing 0.006 M MgCI 2. For studies of activity at other pHs the following buffers were used: pH 5.5, 0.01 M sodium acetate, pH 6 to 7.5, 0.01 M sodium citrate, pH 8.0 and above, 0.01 M Tris hydrochloride. Oligomycin and other inhibitors were added as methanolic or aqueous solutions.
Oligomycinwas obtained from Calbiochem, dinitrophenol from Matheson Coleman and Bell, and antimycina from Calbiochem. Other reagents used were all reagent grade.
at different levels of oligomycin are shown in Figure i. Inhibition by oligo-
mycin is completely reversible and appears to be an equilibrium binding phe-
nomenon. ATPases from oligomycin resistant mutant yeasts, such as OR4 and OR8
shown in Figure i, are also inhibited by oligomycin, but higher levels of anti-
biotic are required. At high levels of oligomycin, inhibition of ATPase acti-
vities of these mutants is essentially complete; thus the inhibitor binding
sites on such mutants are not lost but are altered either directly or indirectly
so that a decreased affinity results.
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I 0 0
80 A
60 > s
40
~ 2o D 2 4 5
I I I I I I I I I0 2 0 5 0 4 0 5 0 6 0 70 8 0
p.g e l i g o m y c i n / m g p r o t e i n
Fig. i. Oligomycin inhibition of ATPase enzymes isolated from different strains of Saccharomyces cerevisiae. Triton solubilized OSATPases from sub- mitochondrial particles prepared from the oligomycin sensitive strain, D243-4A and two resistant mutants of this strain, OR4 and OR8, were assayed at the oligomycin levels shown. The specific activities of these preparations were 7.2, ii and 6 pmoles ATP hydrolyzed per minute per mg protein, respectively.
2 o ~ / ~ ~ °
i 5 0 I 0 0
P.g ol igomycin/mg protein
B 0
20
._~ 4 0
.a 6o
8o
lOG
3°I I0
] 1 i ~ t 6 7 8 9
I I I I / 6 7 8 9 I o
pH
Fig. 2. Effect of pH on oligomycin inhibition of ATPase. Oligomycin sensitivity of OSATPase purified by chromatography on Sepharose 6B was assayed at the pHs indicated. To direct attention just to oligomycin inhibition profiles, the data is normalized in part A to show activity at 100% at each pH in the absence of oligomycin. The inset in part B shows the pH-activity profile for the enzyme preparation used in the normalization procedure. Part B also shows the inhibi- tion of ATPase activity caused by i0 ~g oligomycin per mg protein at each of the pH values tested. Assay procedures and buffers.used for pH adjustments are de- tailed in Materials and Methods.
136'3
Vol. 77, No. 4, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
]00
:~ 40
20
" + K C t " ' "-
Oligo + KCI
mM Salt
Fig. 3. Enhancement of oligomycin inhibition by potassium chloride. The effect of added KCI on the measured ATPase activity in submitochondrial particle prep- arations is shown in the top curve (+ KCI). Oligomycin was then added to iden- tical enzyme reaction mixtures to a final concentration of i0 ~g per mg protein. This caused 55% inhibition of enzyme activity. The effect of KCI on the activ- ities of these oligomycin inhibited ATPase samples is shown in the lower curve (Oligo. + KCI). Similar measurements were made using added NaCI (Oligo. + NaCI). The experiment was repeated using OSATPase prepared from mutant OR4 except that 45 ~g oligomycin/mg protein was used (OR40ligo. + KCI).
Changes in solution pH greatly alter oligomcyin inhibition of ATPase ac-
tivity. In Figure 2a, the inhibition of ATPase activity by oligomycin is shown
at various pH conditions. Combining this data with ATPase activity versus pH
measurements (inset of Fig. 2b) gives the relation between extent of inhibition
by oligomycin and solution pH shown in Figure 2b. The curve resembles a simple
titration curve with mid-point near pH 7.2. At low pH, virtually no inhibition
is observed with i0 pg oligomycin per mg protein, while at higher pH nearly
100% inhibition is noted. These results indicate that at low pH, either the
binding of oligomycin is decreased or that bound oligomycin is not inhibitory.
Consideration of Figure 2 favors the conclusion that a decreased oligomycin
binding is the predominant factor since, at any given pH, higher levels of
added oligomycin still cause some inhibition.
Low levels of potassium chloride added to ATPase reaction mixtures do
not have a measurable effect on enzyme activity, but potassium chloride does
greatly increase the effectiveness of oligomycin as an inhibitor of the enzyme
in submitochondrial particles. Figure 3 illustrates that when sufficient oligo-
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Vol. 77, No. 4, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mycin was present to cause approximately 55% inhibition of ATPase activity,
addition of potassium chloride made inhibition essentially complete. Sodium
chloride, even at much higher concentrations, had no effect on activity or on
the degree of oligomycin inhibition. Studies with the oligomycin resistant
mutant OR4 indicated that this mutation resulted in nearly a 20-fold increase
in the concentration of potassium chloride required to affect oligomycin inhibi-
tion. The effect of potassium chloride is reversible by dialysis to remove
added salt. Potassium nitrate also enhanced oligomycin inhibition, however,
these measurements were complicated by the inability to get levels of inhibition
approaching i00% in potassium nitrate solutions. Rubidium chloride had no
effect on activity or oligomycin inhibition in this system. The KCI effect is
not observed with Triton solubilized ATPase preparations.
Again, potassium chloride either enchances binding of oligomycin to the
complex or alters the sensitivity of the complex to bound oligomycin. The
curves showing oligomycin inhibition of various levels of added potassium
chloride and the changes noted with OR4 again suggest a change in binding
rather than a direct effect on sensitivity of enzyme to bound oligomycin. This
conclusion is further supported by the apparent rival relationship between
potassium and hydrogen ions for enhancement or reduction of oligomycin sensi-
tivity. In the pH range below 8 and with KC1 concentrations less than 20 mM,
the pH versus inhibition profile of Figure 2b is shifted to a higher or lower
pH range by addition of correspondingly lower or higher concentrations of potas-
sium chloride.
Two reagents which react with sulfhydryl groups at low concentrations also
alter oligomycin inhibition. Figure 4 illustrates that adding i0 ~M HgCI 2 or
20 ~M iodoacetate prevents oligomycin inhibition of ATPase. These reagents
have no measurable effect on ATPase activity at the very low concentration
used. In contrast to these results, both dihydrolipoate and oxidized lipoic
acid enhance oligomycin inhibition while lipoamide has no effect (Fig. 5).
Dithiothreotol had no effect and 20 ~g/ml oleic acid did not alter oligomycin
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Vol. 77, No. 4 ,1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I00
"~- 6C
20
I I
2'5 50 7'5 I00 oligo gg/mg protein
+Hg ++ A ~ - - - " " Z,~' IOO
- +IAA 210 ~ 90 L 1,8 80
1.4 5 60 '~ l.Z 50
1,0 ~2 40
.8 30 I I I
.6 2 4. 5 6 I @ Minutes 125
I I I / 5 I0 15 2 o
/~g Lipoic Acid Compound
Fig. 4. Prevention of oligomycin inhibition of ATPase by mercuric ion and by iodoacetic acid. Oligomycin sensitivity of ATPase in submitochondrial particles was determined in the absence of added reagents (lower curve) and in the presence of added i0 ~M HgCI 2 or 20 pM iodoacetic acid (IAA). These two reagents had no effect on the measured activity of ATPase in the absence of oligomycin at the levels employed.
Fig. 5. The effects of lipoic acid on oligomycin inhibition of ATPase in sub- mitochondrial particles. In part A, the time course of the reactions are shown for i) ATPase alone and 2) ATPase plus i0 Dg/ml oligomycin. The reactions illustrated by curves 3), 4) and 5) were the same as in 2) except that 20 ~g/ml of lipoamide, oxidized lipoic acid and dihydrolipoic acid, respectively, were added immediately prior to addition of oligomycin. In part B of the figure, the relative effects of lipoic acid compounds on oligomycin inhibition are shown. In each experiment, the reaction conditions were the same as used for curve 2) in part A of this figure except that varying amounts of the different lipoic acid derivative were added. Percent activity was calculated relative to the reaction rate of curve 2) to indicate the change in oligomycin inhibition caused by added lipoie acid compounds. Again, curve 3) represents addition of lipoamide, 4) oxidized lipoicacid, and 5) dihydrolipoic acid. Addition of these three reagents to ATPase assays in the absence of oligomycin had no measurable effect
on enzyme activity.
sensitivity of these preparations. Several other reagents which inhibit oxi-
dative phosphorylation were tested for their effects on oligomycin inhibition.
Dinitrophenol, azide, arsenate CCCP and antimycin A had no observable effects.
The ionophoric molecules valinomycin and nigericin do reverse oligomycin
inhibition as reported previously (16).
DISCUSSION
Inhibition of ATPase activity by oligomycin results from the reversible
binding of the antibiotic to a component located in the '~embrane sector" of
the enzyme complex. Previous studies have shown that this component is probably
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Vol. 77, No. 4,1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enzyme subunit 9 (12). The equilihrium nature of the binding suggests that the
level of inhibition of enzyme activity may be related to the amount of oligo-
mycin bound. Several seemingly unrelated reagents influence o!igomycin inhibi-
tion. Consideration of these reagents suggests that their effects on activity
result from reaction with enzyme rather than with the antibiotic. All prob-
ably alter the properties of the oligomycin binding site on the enzyme.
A mechanism for this is suggested from studies of protein subunit 9 in
oligomycin sensitive (OS) and resistant mutant strains (OR) of yeast. Subunit
9 from OS strains is isolated largely in oligomeric form (~70%) with molecular
weight near 45,000 (11,13,14). Subunit 9 isolated from class B OR-mutants (14)
is predominantly a monomer (>90%). Thus, OR-mutation caused a change in this
protein that is seen in vitro as an oligomer to monomer conversion. The OS-
protein has a high affinity for the antibiotic and the OR-protein has a low-
ered affinity. The above data indicates that the various added reagents may
mediate conversion of enzyme from an oligomyein binding to a non-binding form.
This may be represented by the equilibrium postulated below:
IAA, or Hg ++, or OR-mutants H+ + Form A.K + ~ --~ Form B.H+ + K +
oligomycin, or lipoic acid
Form A is represented as the oligomycin binding form while Form B has
little affinity for the inhibitor. Increased K + or lipoic acid favors Form A
and enhanced inhibitor binding. Lower pH favors Form B and decreased inhi-
bition. Iodoacetic acid and Hg ++ both cause formation of Form B. This hy-
pothesis implies that addition of iodoacetate or Hg++would also cause an up-
take of protons and shift the equilibrium towards Form B. Such pH changes
have been reported in studies with membrane bound ATPases (17).
The K + effect on oligemycin inhibition was quite specific and cannot be
explained by an ionic strength change. Since K + and H+appear to compete in
their effects on oligomycin inhibition, our postulated equilibrium was writ-
ten to include these species directly. This recalls equilibrium observations of
reciprocal transport of H ÷ and K + in mitochondria [18) and suggests subunit 9may
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Vol. 77, No. 4,1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mediate the i:i exchange of H + and K +. Our previous studies showing reversal
of oligomycin inhibition of ATPase by ionophoric compounds which are capable
of carrying both K + and H + through membranes but not by ionophores which trans-
port only H +, support the proposal that these ions are linked in some aspect
of ATPase function (16). The proposed changes in subunit 9 recall the "con-
formational" model for coupling proton translocation to ATP synthesis proposed
by Boyer in which a proton mediated energy-linked change in protein structure
is an intermediate step (19). Recently Griffiths has described reactions
based on dihydrolipoate that give rise to ATP synthesis (20,21). Our demon-
stration that lipoate alters oligomycin inhibition supports his suggestion
that these reactions may reflect in vivo ATP synthesis. It appears that pro-
ton translocation, conformational change and chemical coupling may all be
brought together at the site of oligomycin inhibition.
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3. Hatefi, Y., Stigall, D.L., Galante, Y. and Hanstein, W. (1974) Biochem. Biophys. Res. Comm. 61, 313-321.
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Spithill, T.W. and Trembath, M.K. (1976) in The Genetic Function of Mito- ahondrial DNA (A.M. Kroon and C. Sarcone, eds.), pp. 163-173, North Holland Publ. Co., Amsterdam.
15. Monroy, G.C. and Pullman, M.E. (1967) in Methods in Enzymology (Estabrook, R. and Pullman, M.E., eds.), Vol. X, pp. 500-512.
16. Johnston, R., Scharf, S. and Criddle, R.S. (1977) FEBS Letters 75, 213-216. 17. Green, D.E. (1974) Biochim. Biophys. Aeta 346, 27-78. 18. Southard, J.H., Penniston, J.T. and Green, D.E. (1973) J. Biol. Chem. 248,
3546-3550. 19. Boyer, P. (1975) FE~S Letters 58, 1-6. 20. Griffiths, D.E. C1977) Biochem. J. 16Q, 809-812. 21. Griffiths, D.E. (1976) in Genetics and Biosenesis of Chloroplasts and
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