Biochimica et Biophysica Acta 1456 (2000) 67^76

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Interactions of arsenate, sulfate and phosphate with yeast mitochondria

Paulina Cortës, Vicente Castrejön, Josë G. Sampedro, Salvador Uribe *
Department of Biochemistry, Instituto de Fisiolog|¨a Celular, Universidad Nacional Autönoma de Mëxico (UNAM),
Apdo Postal 70-242, 04510 Mexico City, Mexico

Received 15 July 1999; received in revised form 9 November 1999; accepted 15 November 1999

Abstract

In the presence of K‡ , addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane
potential (v8) and this is prevented by millimolar concentrations of phosphate (PO4 ). Different monovalent and polyvalent
anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO4 ) and
sulfate (SO4 ) were as efficient as PO4 to protect mitochondria against the K‡ mediated swelling, depletion of the v8, and
decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO4 , SO4 or AsO4 was inhibited by
mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO4 and AsO4
on the F1 F0 -ATPase were tested : both SO4 and AsO4 inhibited the synthesis of ATP following competitive kinetics against
PO4 and non-competitive kinetics against ADP. The mersalyl sensitive uptake of 32 PO4 was not inhibited by SO4 or AsO4 ,
suggesting that the synthesis of ATP was inhibited at the F1 F0 -ATPase. The hydrolysis of ATP was not inhibited, only a
stimulation was observed when AsO4 or sulfite (SO3 ) were added. It is suggested that the structure and charge similarities of
PO4 , AsO4 and SO4 result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site,
occupation by any of these three anions results in protection against uncoupling by K‡ ; at the second site, in the F1 F0 -
ATPase, AsO4 and SO4 compete for binding against PO4 leading to inhibition of the synthesis of ATP. ß 2000 Elsevier
Science B.V. All rights reserved.

Keywords: Arsenate; ATP synthesis; F1 F0 -ATPase; Phosphate; Permeability transition pore; Sulfate; Yeast mitochondria

1. Introduction gers the opening of a large, unspeci¢c conductance

In mammalian mitochondria, Ca2‡ overload trig-
Abbreviations: AsO4 , inorganic arsenate; CCCP, carbonyl cy-
anide m-chlorophenylhydrazone; MES, morpholinoethanesul-
fonic acid; NEM, N-ethylmaleimide ; PO4 , inorganic phosphate;
PT, permeability transition; PTP, permeability transition pore;
SO3 , inorganic sul¢te; SO4 , inorganic sulfate; U/4, uncoupled
state/state 4 ratio; YMUC, yeast mitochondria unselective chan-
nel; v8, transmembrane potential
* Corresponding author. Fax: +52 (5) 6225630;
E-mail: suribe@i¢siol.unam.mx
pathway termed the permeability transition pore
(PTP) and hence, to depletion of the transmembrane
potential (vi) [1^3]. The role of PTP in physiological
apoptosis or in pathological conditions such as cell
death due to ischemia-reperfusion has been discussed
[3]. In Saccharomyces cerevisiae, the yeast mitochon-
dria unselective channel (YMUC) is a counterpart of
the PTP [4]. However, the PTP and the YMUC are
regulated di¡erently: in mammalian mitochondria,
the PTP is inhibited by ATP while it is opened by
inorganic phosphate (PO4 ) [1^4], while in yeast the
opposite e¡ects are observed, i.e., the YMUC is in-

0005-2728 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.
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68 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76
duced by ATP and locked in the closed position by
Pi [4]. Furthermore, neither Ca2‡ nor cyclosporine,
which is a PTP inducer and inhibitor, respectively [3],
seems to have any e¡ects on YMUC [4].
Thus PO4 , the most abundant polyvalent anion in
the cell, plays at least two vital roles in the matrix of
mitochondria: (i) as a substrate in the phosphoryl-
ation of ADP by the F1 F0 -ATPase [5] and (ii) as a
mitochondrial membrane permeability modulator
during cation uptake [6^8]. In this regard, it has
been reported that the addition of PO4 results in
an increase in the uptake of Ca2‡ both in mamma-
lian [9,10] and in yeast mitochondria [11].
In diverse biological systems, arsenate (AsO4 ) and/
or sulfate (SO4 ) may compete against, or substitute
for, PO4 [12]. In many enzymes AsO4 substitutes for
PO4 leading to products which are rapidly hydro-
lyzed [13]. Among the best known cases is the one
catalyzed by D-glyceraldehyde-3PO4 dehydrogenase
[13,14]. In mitochondria, AsO4 reacts with ADP in
a F1 F0 -ATPase catalyzed arsenylation, yielding an
easily hydrolyzable product and uncoupling oxida-
tive phosphorylation [13,15^17]. AsO4 is also known
to compete for the PO4 transport system in Strepto-
myces granaticolor [18]. During the synthesis of cys-
teine in Euglena mitochondria, SO4 is activated to
yield adenosine 3P-phosphate 5P-phosphosulfate in a
PO4 sensitive process [19].
In S. cerevisiae, PO4 protects mitochondria during
exposure to K‡ [4]. In the absence of PO4 and in the
presence of high concentrations of K‡ , addition of
ATP or ethanol leads to massive mitochondrial swel-
ling and vi depletion [4,20,21]. Addition of PO4 in
the millimolar range prevents both swelling and vi
depletion [4,22]. In contrast to the mammalian or-
ganelle, yeast mitochondria are protected by millimo-
lar concentrations of PO4 without the need of further
additions [4,23].
Di¡erent anions were tested in yeast mitochondria
for their protection activity against the K‡ mediated
e¡ects on swelling or vi. Mersalyl sensitive protec-
tion by PO4 , SO4 and AsO4 was observed. In addi-
tion SO4 and AsO4 inhibited the ATP synthase. It is
suggested that the structural similarities between
PO4 , SO4 and AsO4 result in speci¢c interaction
with at least two di¡erent binding sites inside the
matrix of yeast mitochondria: occupation of a site
which controls YMUC conductance by PO4 , SO4 or
BBABIO 44805 24-12-99
AsO4 results in protection against K‡ ; in addition,
binding to the PO4 binding site in the F1 F0 -ATPase
results in competitive inhibition of the synthesis of
ATP by SO4 or AsO4 .
2. Materials and methods
2.1. Materials
All reagents were of the highest purity available
commercially. Glucose, mannitol, morpholinoethane-
sulfonic acid (MES) safranine-O, hexokinase and
L-nicotinamide adenine dinucleotide phosphate
(NADP‡ ) were from Sigma (St. Louis, MO). Ly-
ophilized glucose-6-phosphate dehydrogenase was
from Worthington Biochem (NJ). Ammonium sul-
fate, phosphoric acid and sulfuric acid were from
Baker (Mexico City). Mersalyl was a kind gift from
Dr. Edmundo Chävez (National Institute of Cardiol-
ogy, Mexico City). The radioactive isotope 32 PO4
was from New England Nuclear (Dupont, DE).
2.2. Biologicals
Baker's yeast S. cerevisiae was purchased from a
local yeast factory (La Azteca, SA). The cells were
grown by suspending 40 g of cells (w/w) in 1 l of a
rich liquid medium [24] and aerating (3 l/min) for 8 h
at 30³C. The cells were centrifuged twice in a refri-
gerated centrifuge (Sorvall, R5B) at 4000Ug for
5 min, resuspended in 1 l of water and starved for
16 h under aeration at 30³C [25].
2.3. Isolation of mitochondria
After incubation, yeast were centrifuged (4000Ug
for 5 min, twice) and resuspended in 0.6 M mannitol,
5 mM MES, 0.1% bovine serum albumin, pH 6.8
(TEA) at a ¢nal concentration of 0.5 g (w/w)/ml.
The cell suspension was poured into a Braun homog-
enizing glass and mixed with 50% (v/v) of 0.5 mm
diameter glass beads. Homogenization was per-
formed for 12 s and the resulting suspension was
decanted into centrifuge tubes. From the homoge-
nate, mitochondria were isolated by di¡erential cen-
trifugation as described before [25]. Mitochondrial
protein was determined by the Biuret method [26].
 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76
2.4. Transmembrane potential (vi)
The vi was determined as described by Akerman
and Wikstro«m [27], following the changes in absorb-
ance of safranine-O at 511^533 nm in a double beam
spectrophotometer (SLM-Aminco DW2000, Urbana,
IL) in dual mode.
2.5. Mitochondrial swelling
Experiments were performed by following the de-
crease in absorbance of a mitochondrial suspension
in a DW2000 spectrophotometer in split mode at a
wavelength of 550 nm.
2.6. Oxygen consumption
The rate of oxygen consumption was measured in
a closed water jacketed glass chamber equipped with
a Clark electrode connected to an oximeter (Ysi-
5300) and to a chart recorder. The temperature was
maintained at 30³C with a circulator bath (Haake).
Other additions were as indicated under each experi-
ment. Mitochondria 0.5 mg prot/ml were added and
oxygen consumption in the resting state (state 4) was
recorded. After approx. 2 min, 4 WM FCCP was
added and the rate of oxygen consumption in the
uncoupled state was measured.
2.7. ATP synthesis
The rate of ATP synthesis was measured using an
enzyme coupled assay following the reduction of
NADP‡ at 340^390 nm in a DW2000 spectropho-
tometer in the dual mode [28]. The lyophilized en-
zymes were resuspended each day as follows: hexo-
kinase was resuspended in water to 13 mg/ml.
Glucose-6-phosphate dehydrogenase was resus-
pended in 5 mM citrate pH 7.0 to a ¢nal concentra-
tion of 1000 U/ml. AMP was not added because in
yeast mitochondria the adenylate kinase activity is
minimal [22,29].
2.8. ATPase activity
ATP hydrolysis was determined by measuring the
production of orthophosphate as described by Fiske
and Subbarrow [30]. The reaction mixture contained
BBABIO 44805 24-12-99
69
20 mM Tris-HCl pH 8.5 plus 5 mM MgCl2 . After
temperature equilibration at 30³C, mitochondria
were added to a ¢nal concentration of 0.25 mg
prot/ml and incubated for 10 min under mild agita-
tion in a water bath (New Brunswick agitation
bath); then the reaction was stopped with 0.1 ml of
30% trichloroacetic acid.
32
2.9. PO4 uptake by mitochondria
The uptake of 32 PO4 was measured in the presence
of di¡erent anions [31]. Mitochondria were incubated
for 2 min at 25³C in a gyrstary water bath (New
Brunswick) and then a 100 Wl aliquot was withdrawn
and vacuum ¢ltered through a 0.6 Wm pore diameter
Millipore ¢lter. The sample was washed immediately
with 100 mM NaHPO4 , pH 6.8, three times. The
¢lters were allowed to dry and then they were trans-
ferred to scintillation vials and 5 ml of scintillation
liquid was added. Vials were introduced in a Beck-
man LS6500 multipurpose scintillation counter using
the phosphate program. The uptake of PO4 in nmol
(mg prot)31 is reported.
3. Results
In the presence of high K‡ concentrations, addi-
tion of ATP or ethanol to isolated yeast mitochon-
dria results in swelling and vi depletion [4,32]. In-
creasing concentrations of PO4 both prevent and
revert the K‡ mediated e¡ects [32]. It was decided
to analyze whether other anions protected yeast mi-
tochondria to a comparable extent. The vi protec-
tion e¡ects of diverse salts of acetate, nitrate, SO3 ,
SO4 , AsO4 and PO4 were measured at a ¢nal con-
centration of 4 mM (Table 1). As expected [22,32],
the vi reached the maximum detectable values either
in the absence of K‡ and 0^0.4 mM PO4 , or in the
presence of 4 mM PO4 , with or without K‡ . In con-
trast, in the presence of 40 mM KCl and low PO4 the
vi was minimal and varied with the addition of
di¡erent anions as follows: Na‡ -AsO4 and all the
SO4 salts tested generated a vi as high as the one
observed with 4 mM PO4 . The NH‡ 4 salts of acetate,
nitrate or chloride resulted in a small vi, up to
40 mV. Na‡ -acetate, Na‡ -nitrate and Na‡ -SO3 did
not protect the vi (Table 1).
70 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76

Fig. 2. E¡ect of PO4 on the K‡ promoted swelling of yeast mi-

tochondria. Modulation by mersalyl. Experimental conditions
as in Fig. 1 except no safranine O was added. (a) No additions;
Fig. 1. E¡ects of increasing concentrations of phosphate, arsen- (b) 4 mM PO4 plus 100 WM mersalyl; (c) 4 mM PO4 ; (d) 4 WM
ate or sulfate on the transmembrane potential of yeast mito- CCCP. Where indicated, 40 mM KCl was added from a 1 M
chondria. Modulation by mersalyl. Experimental conditions as stock solution. Measurements were performed at room tempera-
in Table 1, except 40 mM KCl. The indicated concentrations of ture, in a DW2000 Aminco Spectrophotometer in split mode at
anions were added from 1 M stock solutions. Mersalyl was al- 550 nm.
ways added at a ¢nal concentration of 100 nmol per mg pro-
tein. When mersalyl was added, mitochondria were incubated
30 s before adding the anion. Anions were: F, PO4 ; E, mersal-
yl-PO4 , R, SO4 ; O, mersalyl-SO4 ; b, AsO4 ; a, mersalyl-AsO4 .
Data are the means of three duplicate determinations and bars
represent standard deviations.
Table 1
E¡ect of di¡erent anions on the K‡ mediated transmembrane
The vi was protected by 4 mM PO4 , SO4 or AsO4 potential (vi) depletion in isolated yeast mitochondria
to the same extent. In order to de¢ne the concentra- Addition vi (mV)
tion dependence of the protection e¡ect, the same None 178 þ 2
parameter (vi) was measured at di¡erent concentra- K‡ 3þ6
tions of PO4 , SO4 and AsO4 . In addition, in order to K‡ +TEA-PO4 174 þ 11
determine the mitochondrial compartment where the K‡ +Na-PO4 151 þ 37
anion protected, experiments were conducted both in K‡ +NH4 -SO4 156 þ 13
K‡ +Tris-SO4 141 þ 6
the presence and in the absence of the PO4 transport
K‡ +Na-SO4 160 þ 8
inhibitor mersalyl (Fig. 1). In the absence of anions, K‡ +Na-AsO4 164 þ 20
the vi was very low and increased with the concen- K‡ +NH4 -NO3 43 þ 15
tration of each of the three anions. The increase in K‡ +NH4 -Ac 44 þ 11
vi was always slightly higher for PO4 than for com- K‡ +NH4 -Cl 38 þ 10
K‡ +Na-Ac 8þ3
parable concentrations of SO4 or AsO4 (Fig. 1).
K‡ +Na-SO3 8 þ 10
When the same experiments were performed in the K‡ +Na-CO3 0þ0
presence of mersalyl, the anion protection was elim-
Reaction mixture: 0.6 M mannitol, 5 mM MES, pH 6.8 (TEA).
inated, such that at 4 mM anion the vi was between 0.4 mM PO4 bu¡er, pH 6.8 (TEA), 0.2 mM MgCl2 , 1 Wl etha-
24 and 29 mV (Fig. 1). Two conclusions are sug- nol/ml, 10 WM safranine-O, 0.5 mg mitochondrial prot/ml. Ad-
gested by the results reported in Fig. 1: ¢rst, SO4 ditions: K‡ was 40 mM KCl. The indicated anion salt was al-
and AsO4 enter the matrix using a transport system ways 4 mM; depending of the salt, the pH was always adjusted
which is similar to the PO4 transporter, at least in its in the stock solutions to 6.8 using NaOH, NH4 OH, TEA, Tris
or HCl as needed. Final volume 2 ml, temperature 25³C. At
sensitivity to mersalyl; and second, PO4 , SO4 and
the end of each trace, 4 WM FCCP was added to determine ab-
AsO4 protect the vi from inside the mitochondrial sorbance at 0 mV. Absorbance changes were followed in a
matrix. DW2000 Aminco spectrophotometer in dual mode at 511^533
The above results suggested that the anions protect nm. Numbers are the means of three determinations þ S.D.

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 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76 71

The K‡ mediated mitochondrial swelling and vi

depletion were prevented by SO4 and AsO4 , suggest-
ing that these anions resemble PO4 in their mem-
brane protection properties. Thus, the e¡ects of in-
creasing concentrations of SO4 or AsO4 on the rate
of oxygen consumption in state 4 and in the un-
coupled state were explored in the presence of

Fig. 3. E¡ect of di¡erent concentrations of sulfate or arsenate

on the rate of oxygen consumption by yeast mitochondria. Ex-
perimental conditions as in Table 1 except 40 mM KCl. Tem-
perature was maintained at 30³C. (A) SO4 ; (B) AsO4 . a, state
4; b, uncoupled state, generated with 4 WM CCCP. Data are
the means of three duplicate determinations. Bars represent
standard deviation.

from the mitochondrial matrix. This was further ex-

plored by measuring the pattern of K‡ mediated
mitochondrial swelling in the absence and in the
presence of mersalyl. In the absence of anions, addi-
tion of 40 mM KCl resulted in rapid swelling (Fig.
2a). Swelling was prevented in the presence of 4 mM
AsO4 (Fig. 2d). Addition of mersalyl to the incuba-
tion mixture prevented the AsO4 mediated e¡ect
(Fig. 2b). In the presence of an uncoupler no swelling
was detected, demonstrating that the K‡ mediated
swelling is an energy dependent process (Fig. 2c).
The same pattern of swelling e¡ects was observed
when SO4 or PO4 (results not shown) [18,31] were
used instead of AsO4 .
Fig. 4. Inhibition kinetics of the rate of synthesis of ATP by
di¡erent concentrations of sulfate or arsenate in the presence of
increasing concentrations of phosphate. Experimental conditions
as in Table 1. The enzyme coupled assay for ATP required the
additions of 162.5 Wg/ml hexokinase, 2 U/ml glucose-6-phos-
phate dehydrogenase, 20 mM glucose and 1.4 mM NADP‡ .
The reaction was started by adding 40 mM ADP. The indicated
anion was added in di¡erent ¢nal concentrations as follows: a,
no addition; R, 2 mM; F, 4 mM; b, 8 mM. (A) Sulfate; in-
sert: double reciprocal plot of A; (B) arsenate; insert: double
reciprocal plot of B.

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72 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76

40 mM KCl (Fig. 3). In the control, without PO4 ,

SO4 or AsO4 , the rate of oxygen consumption in
state 4 was very high and it increased only slightly
after the addition of an uncoupler, resulting in an
uncoupled/state 4 ratio (U/4) of 1.1 þ 0.1 (Fig. 3).
As increasing concentrations of SO4 were added,
the rate of oxygen consumption in state 4 decreased
while the rate of oxygen consumption in the un-
coupled state was nearly constant; at 4 mM SO4 ,
U/4 was 1.8 þ 0.1 (Fig. 3A). When increasing concen-
trations of AsO4 were added, the rate of oxygen con-
sumption in state 4 decreased, reaching lower values
than those observed with SO4 and in this case the
uncoupled state decreased; at 4 mM AsO4 , U/4 =
2.0 þ 0.2 (Fig. 3B). These values were very near the
U/4 of 1.83 obtained in the presence of 4 mM PO4
here (results not shown) and elsewhere [22]. In the
absence of K‡ , neither AsO4 nor SO4 had any e¡ects
on the rate of oxygen consumption in state 4 or in
the uncoupled state (results not shown). None of the
other anions listed in Table 1 had any e¡ects on the
rate of oxygen consumption by yeast mitochondria
(results not shown).
The similarity in the e¡ects of SO4 , AsO4 and PO4
suggests that there is a binding site in the matrix face
of the internal membrane or in the matrix where
occupation by any of these anions results in protec-
tion. If this is the case, these anions should compete
for other PO4 -speci¢c binding sites. An ideal model
to test this possibility is the F1 F0 -ATPase, where PO4
binding at a speci¢c site in each K-L dimer is required
during the synthesis of ATP [5]. Thus, the rate of
ATP synthesis was measured in the presence of dif-
ferent concentrations of SO4 or AsO4 and in the
presence of increasing concentrations of PO4 (Fig. Fig. 5. Inhibition kinetics of the rate of synthesis of ATP by
4) or ADP (Fig. 5). di¡erent concentrations of sulfate or arsenate in the presence of
The synthesis of ATP was inhibited by SO4 (Fig. increasing concentrations of ADP. Conditions were as in Fig.
4A) following competitive kinetics against PO4 (Fig. 4, except PO4 was always 1 mM while ADP was varied as indi-
cated. Anion concentrations were: a, no addition; R, 2 mM;
4A, insert) such that the Vmax was maintained at, or
F, 4 mM; b, 8 mM. (A) Sulfate; insert: double reciprocal plot
very near, the value of 251 nmol ATP (minUmg of A; (B) arsenate; insert: double reciprocal plot of B.
prot)31 obtained in the absence of SO4 , while the
apparent Km increased with the SO4 concentration,
from 0.45 mM PO4 in the control up to 1.29 mM Km increased up to 1.55 mM PO4 at 8 mM AsO4
PO4 in the presence of 8 mM SO4 (Fig. 4A, insert). (Fig. 4B, insert).
In the presence of increasing concentrations of AsO4 The SO4 or AsO4 mediated inhibition of the syn-
the same pattern of inhibition was observed (Fig. 4B) thesis of ATP was assayed in the presence of increas-
and the Vmax also remained near the control value of ing concentrations of ADP (Fig. 5). In the presence
251 nmol ATP (minUmg prot)31 while the apparent of di¡erent concentrations of SO4 , the synthesis of

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 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76
Table 2
Uptake of 32 PO4 by yeast mitochondria; modulation by anions
or by mersalyl
Addition Phosphate uptake (nmol/mg protein)
None 1.84 þ 0.49
Na-AsO4 1.74 þ 0.25
NH4 -SO4 3.00 þ 0.16*
Na-SO4 2.11 þ 0.34
Tris-SO4 1.66 þ 0.06
Mersalyl 0.04 þ 0.06*
Reaction mixture: as in Table 1, except 1 mM PO4 , 20 Wl 10%
H2 O2 , 2 mg mitochondrial prot/ml. The indicated salt was al-
ways 4 mM. Where indicated, 100 WM mersalyl. Numbers are
the means of three determinations þ S.D.
*P 6 0.02 versus the control as estimated using a two-tailed
Student's t-test.
ATP was inhibited (Fig. 5A). Inhibition followed
non-competitive kinetics against ADP, such that at
8 mM SO4 the Vmax decreased more than two times,
from 182 nmol ATP (minUmg prot)31 in the con-
trol, to 74 nmol ATP (minUmg prot)31 while the
apparent Km remained almost unchanged, with a val-
ue of 9.13 WM ADP in the control and 9.23 WM ADP
in the presence of 8 mM SO4 (Fig. 5A, insert). Ad-
dition of AsO4 also inhibited the synthesis of ATP in
the presence of increasing concentrations of ADP
(Fig. 5B) following non-competitive kinetics (Fig.
5B, insert). In the presence of 8 mM AsO4 the
Vmax decreased to 60 nmol ATP (minUmg prot)31
while the apparent Km of 9.07 WM ADP was very
near the control value (Fig. 5B, insert).
In contrast to the SO4 or AsO4 mediated inhibi-
tion of the synthesis of ATP, the rate of ATP hydro-
lysis was not modi¢ed by any of the anions used in
this study (results not shown). Furthermore, a slight
acceleration in the ATPase activity by SO3 and by
AsO4 was detected. The control ATPase was 266
nmol PO4 (minUmg prot)31 , and it increased to
380 nmol PO4 (minUmg prot)31 in the presence of
SO3 and to 354 nmol PO4 (minUmg prot)31 in the
presence of AsO4 .
To test whether the e¡ects of SO4 and AsO4 on the
synthesis of ATP were due to a direct e¡ect on the
enzyme or to an inhibition of PO4 transport, the
e¡ects of SO4 and AsO4 on the uptake of 32 PO4
were measured (Table 2). Under our experimental
conditions, neither AsO4 nor SO4 inhibited the up-
take of PO4 . Furthermore, the NH‡ 4 salt of SO4 in-
BBABIO 44805 24-12-99
73
creased the uptake of PO4 to about two times the
uptake observed in the control. The uptake of PO4
was inhibited almost completely by mersalyl (Table
2).
4. Discussion
Among a number of anions tested, SO4 and AsO4
were as e¤cient as PO4 [22,32] to protect isolated
yeast mitochondria against the K‡ mediated uncou-
pling e¡ects. All three anions, PO4 , AsO4 and SO4 ,
protected mitochondria against the depletion of v8,
the decrease in the U/4 ratio of respiration rates and
the swelling of the matrix. Anion protection was pre-
vented by mersalyl, suggesting that the site of inter-
action for these anions was located in the mitochon-
drial matrix or in the inner face of the internal
membrane. Also tested were the polyvalent anions
nitrate and sul¢te and the monovalent anions acetate
and chloride, but none of these protected against the
deleterious e¡ects of K‡ .
The mechanism for the PO4 mediated preservation
of the v8 is still unde¢ned; nonetheless, the high
structural homology of PO4 , AsO4 and SO4 and
the lack of e¡ects of other anions suggest that the
spatial distribution of atoms and charges in these
anions is important for their interaction with a spe-
ci¢c binding site inside mitochondria. This putative
PO4 binding site might be in the inner side of a large
400 pS ion channel located in the internal membrane
which is closed by PO4 and opened by ATP [33].
Occupation of the anion binding site would result
in protection against the K‡ e¡ects, regardless of
whether the anion is PO4 , AsO4 or SO4 .
The proposal that the structural similarities be-
tween PO4 , AsO4 and SO4 resulted in undiscrimi-
nated binding to a speci¢c site was supported by
the interaction of these three anions with the well
known PO4 binding site in the ATP synthase. Of
all the anions tested here, only AsO4 and SO4 inhib-
ited the synthesis of ATP, following competitive ki-
netics against PO4 and non-competitive kinetics
against ADP.
In diverse biological systems, undiscriminated in-
teractions of PO4 , SO4 and/or AsO4 with speci¢c
binding sites have been documented. In glyceralde-
hyde-3-PO4 dehydrogenase AsO4 substitutes for PO4
74 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76
yielding 3PO4 ,glyceroyl-AsO4 , which rapidly hydro-
lyzes to 3PO4 -glycerate plus AsO4 eliminating a
phosphorylation site in glycolysis [13,14]. There are
many examples of soluble enzymes where AsO4 re-
acts in place of PO4 [13]. The F1 F0 -ATPase catalyzes
the arsenylation of ADP to yield ADP-AsO4 , which
is spontaneously hydrolyzed to ADP+AsO4 [13,15^
17] promoting an atractyloside resistant, oligomycin
sensitive uncoupling in mitochondria [15^17]. As a
consequence of the rapid hydrolysis, ADP-AsO4 for-
mation can be detected in submitochondrial particles
but not in intact mitochondria [16].
Competition between PO4 and SO4 is illustrated in
Euglena mitochondria, where during the synthesis of
cysteine, SO4 is activated on the outer surface of the
inner mitochondrial membrane in an ATP coupled
reaction to yield adenosine 3P-phosphate 5P-phospho-
sulfate; in this system, PO4 inhibits the activation of
SO4 following competitive kinetics [19]. In bacteria,
inhibition of PO4 uptake by AsO4 and SO4 has been
described [18]. In yeast mitochondria AsO4 inhibits
the binding of 32 PO4 to a proteolipid which is be-
lieved to be the PO4 carrier [34].
The AsO4 , SO4 and PO4 mediated protection of
mitochondria was inhibited by mersalyl, as observed
when measuring the v8 and the mitochondrial swel-
ling suggesting that (i) each of these anions needs to
be transported into the matrix in order to protect the
organelle and (ii) the uptake of AsO4 and SO4 re-
sembles the PO4 transporter, at least in its mersalyl
sensitivity. In this regard, there is a report indicating
that AsO4 and PO4 inhibit the uncoupled respiration
in yeast mitochondria in a mersalyl sensitive fashion
[35]. Furthermore, in S. granaticolor [18] and prob-
ably in Escherichia coli [36] PO4 transport is inhibited
by AsO4 or SO4 following competitive kinetics.
In regard to the inhibition of the synthesis of ATP
by AsO4 or SO4 , this was due to direct interaction
with the F1 F0 -ATPase and not to inhibition of the
PO4 transporter because under our conditions, AsO4
and SO4 did not inhibit the mersalyl sensitive uptake
of 32 PO4 . This is in contrast to results indicating that
AsO4 inhibits the binding of 32 PO4 to the putative
PO4 transporter protein [34]. It would be interesting
to test higher concentrations of AsO4 or SO4 to de-
¢ne whether PO4 transport inhibition may be ob-
served. Although the synthesis of ATP was inhibited,
BBABIO 44805 24-12-99
the backwards reaction, i.e. the hydrolysis of ATP,
was not a¡ected by SO4 and it was even slightly
accelerated by AsO4 . The other anions used in this
study did not modify the activity of the F1 F0 -
ATPase (results not shown) except for SO3 , which
stimulated the hydrolysis of ATP in agreement with
other reports [37^39].
For each anion, the NH‡ 4 salt always resulted in
higher v8 than the Na‡ or Tris salt. Furthermore,
NH4 -SO4 enhanced the uptake of PO4 with respect
to the control while the Na‡ or Tris salts had no
e¡ect. NH‡ 4 probably has more e¡ects on the vpH
than in the vi resulting in charge preservation [40].
Yeast mitochondria are a better model than mam-
malian mitochondria to study the e¡ects of PO4 . In
yeast mitochondria PO4 protects without the need to
add nucleotides or Mg2‡ [11]. In contrast, when
mammalian mitochondria are exposed to high exter-
nal Ca2‡ , the addition of PO4 leads to the PT unless
Mg2‡ and ATP are added [9,10,12,13], making it
di¤cult to evaluate the PO4 speci¢c e¡ects in mam-
mals.
In mammalian mitochondria, the generation of
large Ca2‡ -PO4 insoluble aggregates in the mito-
chondrial matrix has been proposed to result in bu¡-
ering of Ca2‡ and protection of the vi [9,10]. In
yeast mitochondria, the generation of K‡ -PO4 aggre-
gates is not likely, as K‡ is monovalent and in addi-
tion, 2^4 mM PO4 is su¤cient to protect against the
deleterious e¡ects of 40 mM K‡ [4,32]. Thus, at least
in yeast mitochondria the mechanism of protection
seems to involve association with a speci¢c binding
site in the organelle and not to anion-cation interac-
tions. The putative binding site where protection is
mediated has similar binding speci¢city to the PO4
binding site in the F1 F0 -ATPase.
Acknowledgements
This work was partially supported by grants
IN229798 from DGAPA-UNAM and 27568-N
from SEP-CONACYT. PC is a Fundaciön UNAM
fellow. The authors thank Dr. Edmundo Chävez for
helpful discussions and for the gift of the reagent
mersalyl. The technical assistance of Ramön Mëndez
is acknowledged.
 P. Cortës et al. / Biochimica et Biophysica Acta 1456 (2000) 67^76
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