Journal of' General Microbiology (1986), 132, 1 153-1 158. Printed in Great Britain
1153
Deficiency of Uncoupler-stimulated Adenosine Triphosphatase Activity in
Yeast Mitochondria
By Z O H R A E Z Z A H I D , M I C H E L R I G O U L E T
AND B E R N A R D G U E R I N *
lnstitut de Biochimie Cellulaire et Neurochimie du CNRS, I rue Camiile Saint-Saens,
33077 Bordeaux Cedex, France
(Received 23 July 1985 ; revised I I November 1985)
Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The
protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced
only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was
dependent upon phosphate (P,) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing
potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only
observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions,
external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP
exchange experiments. These results are discussed in terms of a limitation of the translocasemediated ATP/ADP exchange in uncoupled mitochondria.
INTRODUCTION
Uncouplers stimulate the respiration of isolated yeast mitochondria and inhibit some energy
linked reactions (Ohnishi et al., 1967; Kovac et al., 1972). However, the ATPase activity,
assayed with added ATP, is only observed at alkaline pH ; uncouplers do not stimulate ATPase
activity at neutral pH (Kovac et al., 1968; Somlo, 1968). These results clearly indicate that a
collapse of ApH+ by uncoupler is not sufficient to unmask ATPase activity. Similar observations
have been reported with bacteria (Ferguson ez a/., 1976), and with plant (Jung & Hanson, 1975)
and tumour cell mitochondria (Kolarov et al., 1973; Pedersen & Morris, 1974; Hayashi et al.,
1980a,b; Knowles, 1982).
We report here the results of a study to determine whether the ATP-synthase works in a
reversible manner, as part of our continuing investigations of the control of the oxidative
phosphorylation system in yeast mitochondria (Rigoulet et al., 1983; Jean-Bart et al., 1984).
METHODS
The diploid Succharomyces cerecisiue strain Foam was grown aerobically with lactate as carbon source:
mitochondria were prepared from protoplasts, as described previously (Guerin et al., 1979). Protein concentration
was measured by the biuret method using bovine serum albumin as a standard.
ATP synthesis and hydrolysis were assayed at 20 "C in the following basal medium: 10 mhl-Tris/maleate,
0-65 M-mannitol, 0.3% (w/v) bovine serum albumin, 0.36 mM-EGTA, pH 6.7, containing 0.15 mg mitochondrial
protein ml-I. The particular conditions for synthesis and hydrolysis are given in the legends to the Figures and
Table. At defined times, 0.25 ml of the suspension was pipetted into vials containing 0-17 ml cold 0.75 M-TCAand
was immediately centrifuged. TCA was removed from the supernatant by extraction with diethyl ether. ATP,
Abbreijiations : AdN : adenine nucleotide ; P, : inorganic phosphate CCCP : carbonyl cyanide rnchlorophenylhydrazone ; F , : hydrosoluble factor of the mitochondrial ATP-synthase; AFH+ : transmembrane
electrochemical gradient for protons; ApH : transmembrane pH gradient ; A* : transmembrane electrical
potential.
0001-2841 0 1986 SGM
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Z . E Z Z A H I D A N D OTHERS
ADP and AMP were assayed by the bioluminescence method (Lundin & Baltscheffsky, 1978) using a luminometer
1250 (LKB-Wallac). For the ATP-32P,exchange, mitochondria were suspended in basal medium containing 0.6%
ethanol and 4 mM-Tris/phosphate. ATP synthesis was started by addition of 400 nmol ADP. When ATP synthesis
was over (after 10 rnin incubation), the exchange experiment was started by 3'P, addition, such that the total
external phosphate concentration was not significantly increased ; exchange was stopped at defined times by
addition of TCA (see above). After centrifugation, 0.4 ml of the supernatant was pipetted into vials containing
0.5 ml Norit suspension (100 mg mi-'). The suspension was thoroughly mixed, filtered through a paper disc
(Whatman no. l), then washed with 0.2 M-potassium phosphate and finally washed with distilled water. Filters
were dried and their radioactivity was counted. 32P,was purchased from CEA-Saclay (France).
RESULTS
Efect of CCCP on A TP synthesis and hydrolysis. Initial experiments were done to compare the
effect of the uncoupler CCCP on the synthesis and hydrolysis of ATP under similar
experimental conditions. Fig. 1 (a) shows that 5 PM-CCCP largely inhibited ATP synthesis.
Without uncoupler, maximal ATP synthesis was achieved 5 min after ADP addition. The effect
of CCCP added 10 min after ADP depended on the initial concentration of external Pi. For
0.2 mM-P,, CCCP induced only a very slow hydrolysis of ATP (Fig. 1a). For 4 mM-Pi, about 40%
of the ATP was rapidly hydrolysed and then no further hydrolysis was observed (Fig. lb).
Mersalyl, an inhibitor of the Pi-carrier, fully inhibited the CCCP-induced ATPase activity when
added just before the uncoupler (Fig. lb).
The sum of the amounts of ATP and ADP was constant during the course of the experiments,
indicating that the increase in ATP was due to added ADP phosphorylation and vice versa (Fig.
1b). The ATPase activity was inhibited by oligomycin (Fig. 1a ) and only slowly stimulated by
Mg2+, suggesting a low contamination of the mitochondrial preparation either by nonmitochondrial fractions or by disrupted mitochondria (not shown). This low Mg2+stimulation
appeared to be largely oligomycin-insensitive.
Efect of CCCP on the endogenouspool of adenine nucleotides. Phosphorylation and hydrolysis
of the internal AdN pool was studied without ADP addition (Table 1). In the absence of added
respiratory substrate, approximately 60% of the internal AdN pool was AMP. However, this
value varied and probably depended on the extent to which the mitochondria were depleted of
internal respiratory substrate. Ethanol addition induced a large increase in ATP and a decrease
in AMP ;ADP content increased slightly. CCCP induced ATP hydrolysis, suggesting complete
reversibility of the ATP synthase. Pi addition did not increase the amount of internal ATP
hydrolysed (not shown).
400
E 200 d
9
E:
W
300
CCCP
300
/
5
5
*
10
Time (min)
15
1001
5
15
10
Time (min)
Fig. 1. Effect of CCCP on ATP synthesis and hydrolysis. Mitochondria (0.15 mg protein ml-l) were
incubated in 4 ml basal medium containing 0.6% ethanol and 0.2 mM (a) or 4 mM (6) Tris/phosphate.
) added
ATP synthesis was started by addition of ADP (400 nmol). As indicated, CCCP (5 p ~ was
either before or 10 rnin after ADP addition. The Figure shows the amount of ATP in the absence (*)or
in the presence ( 0 )of 5 PM-CCCP,the amount of ATP in the presence of 4 pg oligomycin
or 50 PMmersalyl (m) added 8 rnin after ADP addition and 2 rnin before CCCP, and the amount of ADP in the
or in the presence (A)
of 5 PM-CCCP.
absence
(v)
(n
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1155
A TPase activity in yeast mitochondria
5
Time (min)
5
10
Fig. 2
10
Time (min)
15
Fig. 3
Fig. 2. ATP hydrolysis induced by valinomycin in a medium containing potassium phosphate.
Mitochondria were incubated in basal medium containing 4 mM-potassium phosphate, 0.2 pg
antimycin ml-I and 0.1 pg valinomycin ml-I. Reaction was started by addition of 420 nmol ATP.
50 pM-mersalyl added 1 min before ATP addition; a, no mersalyl added.
*,
Fig. 3. ATP hydrolysis induced by valinomycin in a medium containing potassium acetate or
potassium chloride. Experimental conditions were identical to those described in the legend to Fig. 2
except that potassium phosphate was replaced by 4 mM-potassium acetate (e)or 4 mM-potassium
chloride (*).
Table 1. Endogenous adenine nucleotide pool
Mitochondria (8 mg protein ml-I) were incubated in basal medium without any addition (A); in the
presence of 1 % ethanol for 2 rnin (B); or in the presence of ethanol for 2 min and then 5 p ~ - C C C Pfor
2 min (C). Means were calculated from 10 experiments. Nucleotide contents were assayed by the
bioluminescence method and are given as nmol AdN (mg protein)-' f SD.
A
B
C
ATP
ADP
AMP
Total AdN
0.7 & 0.1
2.7 f 0.3
0-6 f 0.2
0.8 f 0-2
1.0 f 0-3
1.5
0.3
2.4 f 0.6
0.2 2 0.1
2.0 f 0.4
3.9 f 0.9
3.9 2 0.7
4.1 f 0.9
Valinomycin-induced A TP hydrolysis. When added to medium containing K+, valinomycin
collapsed A+, but not ApH. Valinomycin induced a rapid and nearly total hydrolysis of added
ATP by mitochondria incubated in a medium containing 4 mM-potassium phosphate (Fig. 2).
Mersalyl, added before ATP, completely inhibited ATP hydrolysis.
ATP hydrolysis in a medium containing potassium acetate instead of potassium phosphate
was significant but lower than that observed in the presence of phosphate (Fig. 3). ATPase
activity was, however, very low in a medium containing potassium chloride instead of potassium
phosphate (Fig. 3).
Disruption of the permeability barrier by the use of detergent or by alkalinepH treatment. Kovic et
a f . (1968, 1972) reported that the oligomycin-sensitive ATPase activity in yeast mitochondria
was maximal at alkaline pH and low at neutral pH, and that this activity was only slightly
inhibited by atractyloside, an inhibitor of AdN translocation across the internal membrane,
thus suggesting that the exposure of mitochondria to alkaline pH destroyed the permeability
barrier. In our experiments, the ATPase activity was effectively higher at pH 8 than at pH 6-7
[Om8 and 0.1 pmol ATP hydrolysed min-' (mg protein)-', respectively]. Triton X-100 [0.75 mg
(mg protein)-' ] increased the ATPase activity fourfold at pH 6.7. However, Triton inhibited the
activity at pH 8. Addition of 10 mM-Tris/phosphate did not stimulate the ATPase activity either
at alkaline pH or in the presence of Triton.
ATP-32Pi exchange in state 4 . The experiments described above were done in order to define
the conditions which permit stimulation of the ATPase activity. To determine whether the ATP
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Z . E Z Z A H I D A N D OTHERS
-4
400
0 0 ~
% 300
-
300
:200
-
200
E
2
-B3
5
Y
fi
- 100 .c3
100
d-
ci
10
20
Time (min)
30
40
Fig. 4. ATP-32Pi exchange. Mitochondria were suspended in basal medium containing 0.6%ethanol
and 4 mM-Tris/phosphate. ATP synthesis was started by adding 400 nmol ADP and was monitored by
the bioluminescence method ( 0 )In
. parallel experiments 32Piwas added after 10 min incubation such
that the total external phosphate concentration was not significantly increased.
Incorporation of 3 2 P
into nucleotides measured as described in Methods; A,exchange reaction in the presence of 10 pg
oligomycin ml-' added 2 min before 32Piaddition.
*,
synthase worked in a reversible manner under conditions used for ATP synthesis, we compared
the ATP synthesis and the ATP-32P, exchange (Fig. 4). This experiment clearly showed that all
the ATP synthesized can be labelled by 32P1when the latter is added after the phosphorylation of
ADP is achieved (state 4). It was verified that 32Plwas incorporated only in the y-phosphate
position of ATP by using the glucose phosphate/hexokinase test (not shown). The ATP-32P,
exchange reaction was completely inhibited by oligomycin.
DISCUSSION
The ATP synthase in yeast mitochondria worked in a reversible manner under the
experimental oonditions that were used in the ATP synthesis experiments, since all the ATP
pool was available for ATP-32 Pi exchange.
The lack of the CCCP-induced ATPase activity may be interpreted a priori in two ways:
either as an inactivation of the ATPase complex under these conditions, or as an inaccessibility
of external ATP to the hydrolysis site on the ATPase. The results of some experiments presented
in this paper suggest that the ATPase is functional: (i) endogenous nucleotides were hydrolysed
upon addition of CCCP; (ii) hydrolysis of exogenous ATP was greatly stimulated when the
permeability barrier was disrupted either by alkaline pH or by detergent; (iii) ATPase activity
was observed when the protonophore was replaced by potassium phosphate plus valinomycin.
Similar observations have been reported with plant mitochondria and with mammalian
tumour mitochondria (see Introduction). Some studies have demonstrated that this anomalous
behaviour was due, in part, to leakage of endogenous Mg2+(Jung & Hanson, 1975; Barbour &
Chan, 1978; Hayashi et al., 1980b; Knowles & Kaplan, 1980). It has also been proposed that
factors other than Mg2+ leakage must be responsible for the lack of uncoupler-stimulated
ATPase (Knowles, 1982). From the present work, it appears that such an effect cannot explain
the low activity in yeast mitochondria since the endogenous ATP was hydrolysed and Mg2+
addition did not stimulate the oligomycin-sensitive hydrolysis of exogenous ATP.
More probably, the low CCCP-induced ATPase activity reflects the presence of a
permeability barrier to ATP. In energized mitochondria, for which A$ is negative inside, influx
of ADP3- and efflux of ATP4-, catalysed by translocase, are favoured (Vignais, 1976;
Klingenberg & Heldt, 1982). Thus, in hydrolysis (or ATP-32Pl exchange) experiments, the ATP
uptake can be limited either by A$ or by the endogenous ADP pool size, or by both.
Table 1 shows that the internal AdN pool size was low when mitochondria were incubated in
the absence of external ADP. Without external substrate or in the presence of uncoupler the
percentage of the total AdN pool which was present as AMP, an unexchangeable nucleotide,
was large. The low content of ADP may be responsible, in part, for a limitation of the ATPase
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A TPase actiuity in yeast mitochondria
CCCP
Hf
t
4
4
1
ATP
H+
PI
Pi
c
ADP
4
VAL
K+
H+
1157
t
t
1
ATP
m
ADP
4
ATP
ATP
+
(4
pi
(b)
Fig. 5 . ( a ) Electrogenic AdN exchange is compensated by a functional coupling between the CCCPmediated electrogenic H+ influx and the P,-carrier-mediated electroneutral H+ efflux. CCCP also
equilibrates the ATPase mediated electrogenic H+ efflux. (b)Valinomycin-mediated K + influx charge
compensates both the AdN exchange and the ATPase mediated H+ efflux. The internal H+
concentration is maintained by P, transport (or acetate diffusion). 1, P,-carrier; 2, translocase; 3 , H+ATPase.
activity by ATP influx, as already demonstrated in new-born rabbit liver mitochondria (Rulfs &
Aprille, 1982).
However, another important fact is the role of PI in stimulating the ATPase activity. There are
several possible explanations for this effect.
(i) Internal PI stimulated the ATPase itself. It has been reported that some anions including
phosphate can stimulate F, from mammalian mitochondria (Mitchell & Moyle, 1971 ; Ebel &
Lardy, 1975). However, Fig. 1 (b) shows that when PI was maintained inside mitochondria by
addition of mersalyl, no stimulation of ATPase activity by uncoupler was observed. Moreover,
10 mM-P, did not stimulate the activity of the isolated oligomycin-sensitive ATPase complex
(not shown).
(ii) The phosphate flux may contribute to charge compensation during the ATP/ADP
exchange. When the PIconcentration was sufficiently high (Fig. 1b) the CCCP-induced ATPase
activity was high but it stopped 1 min after uncoupler addition. The effect of the addition of
mersalyl before CCCP indicates the role of PI efflux in promoting hydrolytic activity; P, efflux
may be necessary to balance charge due to ATP4-/ADP3- exchange. This explanation does not
implicate a mechanistic electrogenic PIefflux via a P,-carrier different from the H+/Plsymporter
but rather a charge equilibration between anions and H+ upon CCCP addition as shown in Fig.
5(a). This minimal interpretation is in agreement with the fact that the activation was timedependent, since 1 min after uncoupler addition, mitochondria were PI-depleted (Rigoulet &
Guerin, 1979).
Maximal ATP hydrolysis was obtained in the presence of valinomycin, K+ and an H+donating anion (as PIor acetate). In this system, the total anion movement is charge-balanced by
K + influx as shown in Fig. 5 (b). K+ influx in yeast mitochondria has been shown to occur under
these conditions (Kovac et al., 1972). The requirement for a H+-donating anion is indicated by
mersalyl inhibition and by the lack of ATPase activity in the presence of KC1, in contrast to the
observation with tumour cell mitochondria (Knowles, 1982).
In conclusion, it appears that ATP synthase can work in a reversible manner in yeast
mitochondria but that CCCP-induced ATPase activity is limited by ATP influx. One apparent
reason for this limitation is the electrogenic nature of the ATP/ADP exchange. It is also likely
that the low ADP pool size compared to that in mammalian mitochondria is a rate-limiting
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1158
Z . EZZAHID A N D OTHERS
factor for this exchange. Physiologically, this observation is relevant, since yeast cells are able to
grow under conditions where mitochondria are poorly active compared to the glycolytic system.
The limitation of the ATP influx may prevent hydrolysis of the ATP synthesized in the cytosol.
The authors wish to thank Dr L. Kovic for helpful discussions. This work was supported by grants from
Universite de Bordeaux I1 and from Centre National de la Recherche Scientifique (ATP: Conversion de 1’Energie
dans les Membranes Biologiques).
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