Characterization of a high capacity calcium transport system in mitochondria of the yeast Endomyces magnusii.

The Ca2+ transport system of Endomyces magnusii mitochondria has been shown previously to be activated by spermine. Here we report it to be regulated also by low, physiological ADP concentrations, by the intramitochondrial NADH/NAD+ ratio, and by Ca2+ ions. The combination of all these physiological modulators induced high initial rates of Ca2+ uptake and high Ca2+-buffering capacity of yeast mitochondria, enabling them to lower the medium [Ca2+] to approximately 0.2 microM. The mechanisms of stimulation by these agents are discussed.

In animal cells, Ca 2ϩ 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 [Ca 2ϩ ] from the resting level of ϳ100 nM to a few hundred nM or more in calcium waves (3). An increased matrix [Ca 2ϩ ] i 1 then activates Ca 2ϩsensitive 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 Ca 2ϩ -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 Ca 2ϩ -sequestering organelle (6). In the yeast Saccharomyces cerevisiae, [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i in signal transduction also in yeast is thus gaining acceptance.
Mitochondria from S. cerevisiae and Candida utilis yeast cells were able to accumulate Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ -transporting system for Ca 2ϩ ions was rather low, with an apparent K m value of 150 -180 M (13,14). The Ca 2ϩ 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. Ca 2ϩ 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 Ca 2ϩ uptake is driven electrophoretically by the membrane potential, negative on the matrix side. Evidence was subsequently presented that the affinity of the Ca 2ϩ transport system was increased in the presence of low concentrations of polyamines below 25 M spermine or 100 M spermidine (15,16). This prompted us to search more systematically for naturally occurring modulators of the Ca 2ϩ -transporting system in these mitochondria. Here we report that ADP, Ca 2ϩ 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).

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 ϫ 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 ϫ g, and the supernatant centrifugated at 6000 ϫ 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 ϫ g for 18 min, and resuspended in a minimal volume of washing medium. The mitochondria thus obtained met all known cri-* This work was supported by Russian Foundation for Basic Research Grant 94-04-12717, by International Science Foundation Grants N17000 and N17300, and by Grant 35848 of the Research Council of Ecology and Natural Resources, Academy of Finland, for visits of Russian scientists for cooperation with Finnish research groups. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ teria of physiological intactness, as inferred from high respiratory rates upon oxidation of pyruvate ϩ 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.
Ca 2ϩ uptake was assayed with murexide as an Ca 2ϩ 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 K D for Ca 2ϩ in the sugar-based medium was found by a Scatchard plot to be 1.5 M. The kinetic parameters were obtained by curve fitting by Easy Plot, version 2.22-5, using the Hill equation (21 at V1 ⁄2 , and n ϭ the Hill coefficient. The protein content was determined by the method of Bradford (22) with bovine serum albumin as standard. Fig. 1 shows energydependent Ca 2ϩ uptake by E. magnusii mitochondria respiring on pyruvate ϩ malate. The addition of 100 M Ca 2ϩ caused an abrupt decrease in absorbance, reflecting the formation of the Ca 2ϩ -murexide complex, followed by an increase in absorbance due to Ca 2ϩ accumulation by mitochondria until a steady state for extramitochondrial free [Ca 2ϩ ] was reestablished. The net efflux rate of Ca 2ϩ even after accumulation of 300 M of Ca 2ϩ in the presence of inorganic phosphate or acetate on inhibition of uptake by anaerobiosis was very low, 17-18 nmol⅐min Ϫ1 ⅐mg Ϫ1 , the efflux being inhibitable by addition of Mg 2ϩ , Mn 2ϩ , or La 3ϩ (data not shown), indicating that changes in the initial rates of net Ca 2ϩ uptake were due to changes in influx rates. All Ca 2ϩ accumulated was released in response to addition of the specific Ca 2ϩ ionophore A23187, indicating the intramitochondrial localization of the Ca 2ϩ accumulated.

Uptake of Ca 2ϩ by Yeast Mitochondria-
The Stimulation of Ca 2ϩ Uptake in Yeast Mitochondria by ADP-The Ca 2ϩ accumulation by yeast mitochondria was enhanced by addition of 25 M 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 M) (Fig. 3). Addition of 100 mM KCl slightly increased the affinity for ADP (data not shown). The ADP-enhanced Ca 2ϩ uptake by yeast mitochondria was strongly prevented by low concentra-tions of atractyloside (Fig. 4), a specific inhibitor of adenine nucleotide translocator (23), but not by CSA, a potent and seemingly specific inhibitor of the Ca 2ϩ -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 Ca 2ϩ 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 V max being substantially increased. ADP thus moderately improved the Ca 2ϩ -buffering capacity of mitochondria, i.e. their ability to maintain a low extramitochondrial [Ca 2ϩ ] (Fig. 1, traces a and b; Fig. 2B).
The Stimulation of Ca 2ϩ 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 ϩ 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 Ca 2ϩ uptake (Fig. 5A) and a slightly improved Ca 2ϩ -buffering capacity (Fig. 5B). The Ca 2ϩ affinity of the transporter was increased, and half-maximal rate of uptake was obtained at 100 M, while in the control 155 M 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.
The Stimulation of Ca 2ϩ Uptake in Yeast Mitochondria by Ca 2ϩ -Ca 2ϩ ions themselves were found to modulate the yeast mitochondrial Ca 2ϩ transporter. Moderate amounts of Ca 2ϩ , added sequentially, progressively increased the uptake rates and lowered the external steady state Ca 2ϩ concentrations (Fig. 6, A and B, left traces). Similarly, preincubation of mitochondria for 1 min with 20 M Ca 2ϩ accelerated the uptake of moderate (50 and 100 M) Ca 2ϩ concentrations (Fig. 1, trace c,  and Fig. 7A), the [Ca 2ϩ ] at V1 ⁄2 being lowered to 63 M without changing the Hill coefficient. Remarkably, such a pretreatment of mitochondria enabled them to take up virtually all Ca 2ϩ from the incubation medium, lowering the steady state [Ca 2ϩ ] to a few micromolar at most (Fig. 1, trace c, and Fig. 7B).

The Stimulation of Ca 2ϩ Uptake in Yeast Mitochondria by the Combined Action of the Various Modulators-
The stimulatory effect of ADP was further potentiated by 25 M spermine (Fig. 1, traces a and b; Fig. 2, A and B). Added together, these two physiological modulators provided high rates of Ca 2ϩ uptake ( Fig. 2A) and an improved Ca 2ϩ -buffering capacity (Fig.  2B). Exceptionally high rates of Ca 2ϩ uptake were obtained with a combination of all these modulators, i.e. ADP, spermine, NADH and Ca 2ϩ (Fig. 8A and 9). The yeast mitochondria were then able, when exposed to 50 -300 M Ca 2ϩ , to maintain a constant steady state extramitochondrial [Ca 2ϩ ] close to the detection limit of the murexide technique (1-3 M; Fig. 8B), and even that of Arsenazo III (Fig. 9), i.e. ϳ0.2 M. 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 Ca 2ϩ transport system even in the presence of 0.5 mM Mg 2ϩ , 5 mM NaCl, and 100 mM KCl, mimicking the ionic composition of the cytosol (Fig. 8, A and B), the [Ca 2ϩ ] at V1 ⁄2 being 110, 96 M, and the Hill coefficients 2.15.

DISCUSSION
The main finding in this study is a potent stimulation of mitochondrial Ca 2ϩ transport in E. magnusii by ADP, Ca 2ϩ itself, and NADH in addition to the previously known stimulation by polyamines (15,16).
The Mechanism of Stimulation of Ca 2ϩ 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 Ca 2ϩ , 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 Ca 2ϩ efflux that is negligible, Ͻ20 nmol of Ca 2ϩ /min/mg of protein. Rather, these data indicate that the stimulatory effect of ADP on the rate of Ca 2ϩ accumulation was due to stimulation of the uptake mechanism itself. Rottenberg and Marbach (33,34) have interpreted the lowering of the steady state [Ca 2ϩ ] 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 Ca 2ϩ 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 Ca 2ϩ and stay coupled for a comparatively long time.
The Mechanism of Stimulation of Ca 2ϩ Transport by NADH-Yeast mitochondria offer a fertile field for evaluating Ca 2ϩ 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 Ca 2ϩ transport in E. magnusii yeast is unknown; NADH may influence the trans-port 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 ϩ that could enhance the Ca 2ϩ uptake mechanism.
The Similarities between E. magnusii and Animal Mitochondrial Ca 2ϩ Uptake-The Ca 2ϩ 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 Ca 2ϩ itself (36 -38). It may therefore be justified to call the E. magnusii yeast Ca 2ϩ transporter a mitochondrial calcium uniporter as well. The concerted action of spermine, ADP, NADH, and small concentrations of Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ up-FIG. 6. The effect of sequential additions of Ca 2؉ on Ca 2؉ transport by yeast mitochondria. In A, the addition of Ca 2ϩ was 50 M, and in B, 100 M. The traces at the right correspond to control experiments in which the Ca 2ϩ addition was the sum of Ca 2ϩ taken up after the first addition at left plus the amount added in the second addition of Ca 2ϩ . The initial rates of Ca 2ϩ uptake in nanomoles ϫ min Ϫ1 ϫ mg Ϫ1 protein were in A, after the first addition of Ca 2ϩ 43, after the second 113, and in the control 86, while in B the corresponding rates were 90, 184, and 118, respectively. take by Ruthenium Red in E. magnusii yeast mitochondria (42). Recently, stimulation of the fast mode of Ca 2ϩ 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 Ca 2ϩ uptake system has been demonstrated. One may speculate that there is a close correlation between the capacity of mitochondria for Ca 2ϩ 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 ϩ ratio. The sensitivity of the yeast mitochondrial Ca 2ϩ transporter to modulation by physiological effectors makes it a promising model in searching for other potential physiological modulators of Ca 2ϩ transport.