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INTRODUCTION |
In animal cells, Ca2+ acts as an important second
messenger in signal transduction. The signal is relayed to the
mitochondrial matrix by the calcium uniporter (1, 2), which in the fast mode may respond to increases of [Ca2+] from the resting
level of ~100 nM to a few hundred nM or more in calcium waves (3). An increased matrix
[Ca2+]i1
then activates Ca2+-sensitive enzymes, i.e.
various dehydrogenases, pyrophosphatase, and ATP synthase. This
constitutes a mechanism for the short term regulation of cellular
respiration and oxidative phosphorylation (4).
Yeast and fungal cells also have elements of a Ca2+-based
signal transduction system that functions in a manner similar to that of animal cells, except that the vacuolar system seems to have replaced
the endoplasmic reticulum (5) as the major
Ca2+-sequestering organelle (6). In the yeast
Saccharomyces cerevisiae, [Ca2+]i was
found to be very low (100 nM) and tightly controlled by
transport processes across the plasma membrane and tonoplast membranes
(7). A transient increase in the [Ca2+]i was
found to be an indispensable response of S. cerevisiae cells
to mating pheromone (7, 8), and in initiating morphogenesis and
differentiation of fungi and dimorphic yeasts (9, 10). Participation of
[Ca2+]i in signal transduction also in yeast is
thus gaining acceptance.
Mitochondria from S. cerevisiae and Candida
utilis yeast cells were able to accumulate Ca2+ only
slowly, even from unphysiologically high (mM)
concentrations (11), indicating that the uptake lacked physiological
significance. In our laboratory, it was found that Endomyces
magnusii yeast cells had mitochondria capable of accumulating
Ca2+ from moderate concentrations. This yeast species has
large (as implied by the name magnusii) polynucleated cells,
with a fully competent oxidative phosphorylation system having three
conservation sites in the respiratory chain like animal mitochondria
and no alternative electron transfer pathways (12). The affinity of the
Ca2+-transporting system for Ca2+ ions was
rather low, with an apparent Km value of 150-180 µM (13, 14). The Ca2+ uptake process was
energy-dependent since it was strongly inhibited by
omission of oxidizable substrate or by the presence of a respiratory inhibitor or uncoupler. Ca2+ uptake was associated with a
transient depolarization of the inner mitochondrial membrane,
reversible oxidation of cytochrome b, and stimulation of
respiration. This implies an uniport mechanism, in which
Ca2+ uptake is driven electrophoretically by the membrane
potential, negative on the matrix side. Evidence was subsequently
presented that the affinity of the Ca2+ transport system
was increased in the presence of low concentrations of polyamines below
25 µM spermine or 100 µM spermidine (15, 16). This prompted us to search more systematically for naturally occurring modulators of the Ca2+-transporting system in
these mitochondria. Here we report that ADP, Ca2+ ions, and
exogenous NADH also may serve as potent activators of the E. magnusii mitochondrial calcium transporter. Some of these data
have been reported at meetings (17, 18).
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EXPERIMENTAL PROCEDURES |
E. magnusii yeast, strain VKM Y261, was cultivated in
agitated Erlenmeyer flasks at 28 °C in batches of 100 ml in a medium (18) containing glycerol (0.6-1.0%, v/v) as the source of carbon and
energy. Cells were harvested at the late exponential growth phase when
the cell density corresponded to 10-13 g wet weight/liter.
Mitochondria were isolated by the method designed in our laboratory.
Cells were incubated at room temperature for 30 min in 50 mM Tris-HCl buffer, pH 8.6, containing 10 mM
dithiothreitol, washed twice with distilled water, resuspended in 50 mM Tris-EDTA buffer, pH 5.8-5.9, containing 1.2 M sorbitol and 50 mg of helicase per g of original cell
(wet weight), and incubated at 30 °C under mild stirring for 15-20
min to form spheroplasts. After centrifugation at 400 × g for 10 min, the pellet was washed twice with 1.2 M sorbitol containing 0.4% (w/v) bovine serum albumin, and
pH was adjusted to 6.0. The pellet was further homogenized with a
Dounce homogenizer in a medium containing 10 mM Tris-HCl
buffer, pH 7.2, 0.4 M mannitol, 0.5 mM EDTA,
0.5 mM EGTA, and 0.4% bovine serum albumin. The homogenate
was mixed with an equal volume of the same buffer, except that 0.4 M mannitol was substituted for 0.6 M mannitol,
centrifugated for 10 min at 1200 × g, and the
supernatant centrifugated at 6000 × g for 18 min. The
pellets from the second spin were gently resuspended in approximately
20 ml of washing medium (EDTA and EGTA were excluded), recentrifugated
at 6000 × g for 18 min, and resuspended in a minimal
volume of washing medium. The mitochondria thus obtained met all known
criteria of physiological intactness, as inferred from high respiratory rates upon oxidation of pyruvate + malate with respiratory control values of 5-6 and ADP/O ratios close to their theoretically expected maxima, and were fully active for at least 7 h when kept on ice.
Oxygen consumption was monitored polarographically in a medium
containing 0.6 M mannitol, 2 mM Tris phosphate,
20 mM Tris pyruvate, 5 mM Tris malate, pH 7.4, and 0.5 mg/ml mitochondrial protein.
Ca2+ uptake was assayed with murexide as an
Ca2+ metallochromic indicator by dual-wavelength photometry
using the Hitachi spectrophotometer and the wavelength pair 504-570 nm
(19) in the medium above at pH 7.4. Some key experiments were verified
using the more sensitive metallochromic indicator Arsenazo III (20)
with the wavelength pair 665-685 nm. Its KD for
Ca2+ in the sugar-based medium was found by a Scatchard
plot to be 1.5 µM. The kinetic parameters were obtained
by curve fitting by Easy Plot, version 2.22-5, using the Hill equation
(21) Y = m/[1 + (k/x)n], where m = Vmax, k = [S] at
V1/2, and n = the Hill coefficient. The protein content was determined by the method of Bradford (22) with
bovine serum albumin as standard.
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RESULTS |
Uptake of Ca2+ by Yeast Mitochondria--
Fig.
1 shows energy-dependent
Ca2+ uptake by E. magnusii mitochondria
respiring on pyruvate + malate. The addition of 100 µM Ca2+ caused an abrupt decrease in absorbance, reflecting
the formation of the Ca2+-murexide complex, followed by an
increase in absorbance due to Ca2+ accumulation by
mitochondria until a steady state for extramitochondrial free
[Ca2+] was reestablished. The net efflux rate of
Ca2+ even after accumulation of 300 µM of
Ca2+ in the presence of inorganic phosphate or acetate on
inhibition of uptake by anaerobiosis was very low, 17-18
nmol·min
1·mg
1, the efflux being
inhibitable by addition of Mg2+, Mn2+, or
La3+ (data not shown), indicating that changes in the
initial rates of net Ca2+ uptake were due to changes in
influx rates. All Ca2+ accumulated was released in response
to addition of the specific Ca2+ ionophore A23187,
indicating the intramitochondrial localization of the Ca2+
accumulated.

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Fig. 1.
Ca2+ transport in yeast
mitochondria. Trace a, control; trace b, in the
presence of 25 µM ADP; trace c, mitochondrial suspension pretreated with 20 µM Ca2+ for 1 min. The medium contained 0.6 M mannitol, 2 mM
Tris phosphate, pH 7.4, 20 mM pyruvate, 5 mM
malate, 50 µM murexide, and the amount of mitochondria
corresponded to 0.5 mg of protein/ml. Where indicated, 100 µM Ca2+, 25 µM spermine,
A23187, 25 µM ADP, 1 µM A23187, or 1 mM EGTA were added.
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The Stimulation of Ca2+ Uptake in Yeast Mitochondria by
ADP--
The Ca2+ accumulation by yeast mitochondria was
enhanced by addition of 25 µM ADP (Fig. 1, traces
a and b, and Fig.
2A). The effect of ADP was
specific (other examined mono-, di-, and trinucleotides, including AMP,
cAMP, GMP, GDP, IDP, ATP, or GTP, were inactive; data not shown),
saturated at very low concentrations (with a half-maximal stimulation
at 2-3 µM) (Fig. 3).
Addition of 100 mM KCl slightly increased the affinity for
ADP (data not shown). The ADP-enhanced Ca2+ uptake by yeast
mitochondria was strongly prevented by low concentrations of
atractyloside (Fig. 4), a specific
inhibitor of adenine nucleotide translocator (23), but not by CSA, a
potent and seemingly specific inhibitor of the
Ca2+-induced, phosphate-dependent
permeabilization (MPT, pore) of the inner mitochondrial membrane (Fig.
4). These data appear to indicate that ADP acted from the matrix side
and stimulated the electrogenic Ca2+ uptake mechanism
itself. In the presence of ADP, the uptake kinetics were changed from
sigmoidal to more hyperbolic (Fig. 2A), the Hill coefficient
being lowered from 2.59 to 2.11, and the Vmax being substantially increased. ADP thus moderately improved the Ca2+-buffering capacity of mitochondria, i.e.
their ability to maintain a low extramitochondrial [Ca2+]
(Fig. 1, traces a and b; Fig. 2B).

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Fig. 2.
Effect of ADP and spermine on the initial
rate of Ca2+ uptake (A) and the steady state
medium free [Ca2+] (B). Closed
circles, control; open circles, in the presence of 25 µM ADP; semiclosed circles, in the presence of
25 µM ADP and 25 µM spermine.
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Fig. 3.
Dependence of the initial rate of
Ca2+ uptake on ADP concentration. The Ca2+
addition was 100 µM.
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Fig. 4.
Effect of atractyloside (Atr) and
CSA on the ADP-stimulated Ca2+ uptake by yeast
mitochondria. The concentrations of Ca2+,
atractyloside, and CSA added were 100, 33, and 0.25 µM,
respectively.
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The Stimulation of Ca2+ Uptake in Yeast Mitochondria by
NADH--
Normally, freshly isolated E. magnusii yeast
mitochondria showed a rather low intramitochondrial NADH/NAD ratio,
which, however, could be considerably increased by short term
incubation with NADH (data not shown). Yeast mitochondria, unlike many
animal mitochondria, readily oxidize exogenous NADH. The dehydrogenase responsible for its oxidation is located on the outer surface of the
inner mitochondrial membrane and donates electrons to the respiratory
chain at the level of coenzyme Q, bypassing the site I of energy
conservation (12). In mitochondria from glycerol-grown E. magnusii cells, NADH along with glycerol 1-phosphate is the most
rapidly oxidizable substrate "monopolizing" the respiratory chain
and potently inducing reversed electron flow (24, 25). To have a higher
NADH/NAD+ ratio in the matrix, the mitochondrial suspension
was incubated with 4 mM NADH for 0.5 min (this is
sufficient to effectively induce reversed electron flow; Ref. 25) and
then used a small aliquot of the suspension (50-fold dilution) to avoid
external NADH serving as an additional respiratory substrate.
Mitochondria treated in this way exhibited very high rates of
Ca2+ uptake (Fig.
5A) and a slightly improved
Ca2+-buffering capacity (Fig. 5B). The
Ca2+ affinity of the transporter was increased, and
half-maximal rate of uptake was obtained at 100 µM, while
in the control 155 µM was required, but the Hill
coefficient was not changed. The stimulatory effect of NADH was not due
to ADP contamination in the NADH preparation used.

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Fig. 5.
The effect of preincubation of yeast
mitochondria with NADH on the initial rate of Ca2+
transport (A) and the steady state medium free
Ca2+ (B). Closed circles, control;
open triangles, the mitochondrial suspension was
preincubated with 4 mM NADH in the incubation medium for
0.5 min and then a small aliquot (50-fold dilution) was used for the
registration of Ca2+ transport.
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The Stimulation of Ca2+ Uptake in Yeast Mitochondria by
Ca2+--
Ca2+ ions themselves were found to
modulate the yeast mitochondrial Ca2+ transporter. Moderate
amounts of Ca2+, added sequentially, progressively
increased the uptake rates and lowered the external steady state
Ca2+ concentrations (Fig. 6,
A and B, left traces). Similarly,
preincubation of mitochondria for 1 min with 20 µM
Ca2+ accelerated the uptake of moderate (50 and 100 µM) Ca2+ concentrations (Fig. 1, trace
c, and Fig. 7A), the
[Ca2+] at V1/2 being lowered to 63 µM without changing the Hill coefficient. Remarkably,
such a pretreatment of mitochondria enabled them to take up virtually
all Ca2+ from the incubation medium, lowering the steady
state [Ca2+] to a few micromolar at most (Fig. 1,
trace c, and Fig. 7B).

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Fig. 6.
The effect of sequential additions of
Ca2+ on Ca2+ transport by yeast
mitochondria. In A, the addition of Ca2+
was 50 µM, and in B, 100 µM. The
traces at the right correspond to control
experiments in which the Ca2+ addition was the sum of
Ca2+ taken up after the first addition at left plus the
amount added in the second addition of Ca2+. The initial
rates of Ca2+ uptake in nanomoles × min 1 × mg 1 protein were in A,
after the first addition of Ca2+ 43, after the second 113, and in the control 86, while in B the corresponding rates
were 90, 184, and 118, respectively.
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Fig. 7.
The effect of preincubation of yeast
mitochondria with 20 µM Ca2+ on the initial
rate of Ca2+ transport (A) and the steady state
medium free [Ca2+] (B). Preincubation
was carried out for 1 min in the incubation medium in the presence of
substrate and 20 µM Ca2+ at room temperature
with gentle stirring. Then varying amounts of Ca2+ were
added and its uptake recorded. Closed circles, control; open triangles, after preincubation.
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The Stimulation of Ca2+ Uptake in Yeast Mitochondria by
the Combined Action of the Various Modulators--
The stimulatory
effect of ADP was further potentiated by 25 µM spermine
(Fig. 1, traces a and b; Fig. 2, A and
B). Added together, these two physiological modulators
provided high rates of Ca2+ uptake (Fig. 2A) and
an improved Ca2+-buffering capacity (Fig. 2B).
Exceptionally high rates of Ca2+ uptake were obtained with
a combination of all these modulators, i.e. ADP, spermine,
NADH and Ca2+ (Fig.
8A and
9). The yeast mitochondria were then
able, when exposed to 50-300 µM Ca2+, to
maintain a constant steady state extramitochondrial
[Ca2+] close to the detection limit of the murexide
technique (1-3 µM; Fig. 8B), and even that of
Arsenazo III (Fig. 9), i.e. ~0.2 µM. The
level was maintained for 10-15 min, i.e. until the
experiment was terminated (data not shown). It is noteworthy that yeast
mitochondria supplemented with the physiological effectors preserved
the improved kinetic properties of the Ca2+ transport
system even in the presence of 0.5 mM Mg2+, 5 mM NaCl, and 100 mM KCl, mimicking the ionic
composition of the cytosol (Fig. 8, A and B), the
[Ca2+] at V1/2 being 110, 96 µM, and the Hill coefficients 2.15.

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Fig. 8.
Combined effect of modulators on the initial
rate of Ca2+ transport (A) and the steady state
medium free [Ca2+] (B). Closed
circles, control; open squares, in the presence of 25 µM ADP, 25 µM spermine, and 1-min
preincubation of yeast mitochondria with NADH plus 20 µM
Ca2+; semiclosed squares, in the presence of all
modulators and 0.5 mM Mg2+, 5 mM
NaCl, and 100 mM KCl.
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Fig. 9.
Effect of modulators on Ca2+
uptake measured by Arsenazo III. The standard medium was
supplemented with 25 µM Arsenazo III. The wavelength
couple 665-685 nm was used to monitor [Ca2+]. Additions
were made as indicated by the arrows in trace a; Ca2+, 100 µM; ADP, 20 µM;
spermine 25 µM. In trace b, mitochondria were
preincubated with 1 mM NADH and suspended in the standard medium containing 20 µM ADP and 25 µM
spermine from the beginning of the experiment. The free
[Ca2+] was calculated using a KD of
1.5 µM for the calcium-Arsenazo III complex.
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DISCUSSION |
The main finding in this study is a potent stimulation of
mitochondrial Ca2+ transport in E. magnusii by
ADP, Ca2+ itself, and NADH in addition to the previously
known stimulation by polyamines (15, 16).
The Mechanism of Stimulation of Ca2+ Transport by
ADP--
The protective action of adenine nucleotides on mammalian
mitochondria has long been known (26-28). The most likely explanation is that ADP counteracts the swelling induced by Ca2+,
i.e. MPT (29-31) and the opening of the CSA-sensitive pore
(32). We could not, however, find any evidence in favor of the
existence of MPT in E. magnusii mitochondria. Therefore, it
is unlikely that the effects of ADP and atractyloside are mediated by
the modulation of pore opening, i.e. due to inhibition of
Ca2+ efflux that is negligible, <20 nmol of
Ca2+/min/mg of protein. Rather, these data indicate that
the stimulatory effect of ADP on the rate of Ca2+
accumulation was due to stimulation of the uptake mechanism itself. Rottenberg and Marbach (33, 34) have interpreted the lowering of the
steady state [Ca2+] by ADP in brain mitochondria as a
stimulation of the uniporter via a conformation change of the adenine
nucleotide translocator since the efflux rate for Ca2+ was
low and CSA only slightly affected the steady state. The apparent lack
of MPT in these yeast mitochondria would readily explain their ability
to retain large amounts of Ca2+ and stay coupled for a
comparatively long time.
The Mechanism of Stimulation of Ca2+ Transport by
NADH--
Yeast mitochondria offer a fertile field for evaluating
Ca2+ transport as affected by gradual changes in the redox
state of mitochondrial pyridine nucleotides. In animal mitochondria,
the picture is complicated by MPT that is stimulated by oxidation of
pyridine nucleotides, presumably because of interrelations between the
redox states of pyridine nucleotides, glutathione, and pore vicinal SH
groups (35). The mechanism by which NADH modulates mitochondrial
Ca2+ transport in E. magnusii yeast is unknown;
NADH may influence the transport directly or via the redox state of
glutathione and SH groups. Oxidation of added NADH followed by reversed
electron flow could conceivably effect reduction of matrix
NAD+ that could enhance the Ca2+ uptake
mechanism.
The Similarities between E. magnusii and Animal Mitochondrial
Ca2+ Uptake--
The Ca2+ uptake system of
E. magnusii yeast mitochondria displays striking
similarities with the well characterized animal mitochondrial calcium
uniporter. In both systems, the driving force is the transmembrane potential, negative on the matrix side. Both transport systems are
stimulated by polyamines and by Ca2+ itself (36-38). It
may therefore be justified to call the E. magnusii yeast
Ca2+ transporter a mitochondrial calcium uniporter as well.
The concerted action of spermine, ADP, NADH, and small concentrations
of Ca2+ ions may confer to these yeast mitochondria a
similar role as for the animal uniporter (4), i.e. the short
term regulation of mitochondrial respiration and oxidative
phosphorylation. Yeast mitochondria may even be more important in the
overall Ca2+ homeostasis than animal mitochondria. However,
there is one striking difference in that the animal mitochondrial
calcium uniporter is very sensitive to Ruthenium Red (39), while the
E. magnusii one is less sensitive, if at all, and in this
respect resembles plant mitochondria (40). Saccharomyces
yeasts have a low activity, Ruthenium Red-sensitive cation transporter
(41). We have even seen stimulation of Ca2+ uptake by
Ruthenium Red in E. magnusii yeast
mitochondria (42). Recently, stimulation
of the fast mode of Ca2+ uptake by very low levels of
Ruthenium Red in liver mitochondria was reported (3).
It is remarkable that E. magnusii so far is the only yeast
species in which an efficient mitochondrial Ca2+ uptake
system has been demonstrated. One may speculate that there is a close
correlation between the capacity of mitochondria for Ca2+
transport and the structural organization of the respiratory chain,
i.e. the functioning of complex I of the respiratory chain as a coupling site in oxidative phosphorylation, and the absence of
alternative electron flow pathways bypassing this site (12). These two
features contribute to establishing a high matrix NADH/NAD+
ratio. The sensitivity of the yeast mitochondrial Ca2+
transporter to modulation by physiological effectors makes it a
promising model in searching for other potential physiological modulators of Ca2+ transport.