Ca2+ Efflux in Mitochondria from the YeastEndomyces magnusii

Calcium release pathways in Ca2+-preloaded mitochondria from the yeastEndomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca2+ release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca2+ and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca2+ efflux set in after uptake of ∼150 nmol of Ca2+/mg of protein. The rate of this efflux was proportional to the Ca2+ load and insensitive to aeration, protonophorous uncouplers, and Na+ions. Ca2+ efflux was inhibited by La3+, Mn2+, Mg2+, tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca2+efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca2+ efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na+-independent pathway, in many ways similar to the one in mammalian mitochondria. Calcium release pathways in Ca2+-preloaded mitochondria from the yeastEndomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca2+ release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca2+ and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca2+ efflux set in after uptake of ∼150 nmol of Ca2+/mg of protein. The rate of this efflux was proportional to the Ca2+ load and insensitive to aeration, protonophorous uncouplers, and Na+ions. Ca2+ efflux was inhibited by La3+, Mn2+, Mg2+, tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca2+efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca2+ efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na+-independent pathway, in many ways similar to the one in mammalian mitochondria. inner mitochondrial membrane transmembrane potential carbonyl cyanidem-chlorophenylhydrazone cyclosporin A mitochondrial permeability transition tetraphenylphosphonium Calcium plays an important role as an intracellular messenger in signal transduction (1Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar, 2Mooren F.C. Kinne R.K.H. Biochim. Biophys. Acta. 1998; 1406: 127-151Crossref PubMed Scopus (51) Google Scholar). Mitochondrial Ca2+ uptake is important in relaying a signal for stimulation of respiration and oxidative phosphorylation (3McCormack J.G. Denton R.M. Biochem. Soc. Trans. 1993; 21: 793-799Crossref PubMed Scopus (72) Google Scholar, 4Kavanagh N.I. Ainscow E.K. Brand M.D. Biochim. Biophys. Acta. 2000; 1457: 57-70Crossref PubMed Scopus (70) Google Scholar), which has encouraged study of mitochondrial Ca2+ handling (5Pozzan T. Rizzuto R. Eur. J. Biochem. 2000; 267: 5267-5273Crossref PubMed Scopus (47) Google Scholar). Mitochondria are receiving increasing attention also due to their central role in the mechanism of cell death (6Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (659) Google Scholar). Animal mitochondria take up Ca2+ by a uniport mechanism and release it by a number of different mechanisms, including Na+ antiport, a sodium-independent pathway, and via opening of a cyclosporin A-sensitive pore (for review, see Ref.7Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). Yeast mitochondria generally lack a Ca2+ uptake pathway of any significant physiological relevance (8Balcavage W.X. Lloyd J.L. Mattoon J.R. Ohnishi T. Scarpa A. Biochim. Biophys. Acta. 1973; 305: 41-51Crossref PubMed Scopus (30) Google Scholar, 9Tanida I. Hasegawa A. Iida H. Ohya Y. Anraku Y. J. Biol. Chem. 1995; 270: 10113-10119Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Instead, a vacuolar V-ATPase and a Ca2+/H+ antiporter regulate cytosolic [Ca2+] (10Förster C. Kane P.M. J. Biol. Chem. 2000; 275: 38245-38253Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, we have found that tightly coupled mitochondria from the yeast Endomyces magnusii are able to take up Ca2+ by a uniport mechanism, although the apparent Km is rather high, 150–180 μm (11Zvyagilskaya R.A. Leikin Y.N. Kozhokaru N.L. Kotelnikova A.V. Dokl. Akad. NAUK SSSR. 1983; 269: 1233-1240Google Scholar, 12Leikin Y.N. Votyakova T.V. Bazhenova E.N. Zvyagilskaya R.A. Kotelnikova A.V. Biochemistry (Moscow). 1987; 52: 676-682Google Scholar). Ca2+ uptake at low concentrations can, however, as in animal mitochondria, be substantially increased by micromolar concentrations of polyamines (13Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. FEBS Lett. 1990; 261: 139-141Crossref PubMed Scopus (18) Google Scholar, 14Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. J. Bioenerg. Biomembr. 1993; 25: 569-574Crossref PubMed Scopus (19) Google Scholar, 15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Also ADP, Ca2+ itself, and a high intramitochondrial NADH/NAD+ ratio stimulate mitochondrial Ca2+ uptake (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). In the presence of all these physiological modulators, the initial rate of Ca2+ uptake is quite high (up to 2 μmol/min/mg of protein), and the mitochondrial Ca2+-buffering capacity is remarkably high. Ca2+ uptake by E. magnusii mitochondria is thus potentially important in Ca2+ homeostasis and signal transduction (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). The Ca2+ efflux pathways are also of interest in this context. In this study, we have characterized the mitochondrial Ca2+ efflux mechanism, which shares many of its characteristics with the sodium-independent Ca2+ efflux mechanism of mitochondria from non-excitable mammalian cells (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar). The yeast E. magnusii strain VKM Y261 was grown in glycerol-containing semisynthetic medium as described previously (20Zvyagilskaya R.A. Selenshchikova V.A. Uralskaya L.A. Kotelnikova A.V. Biochemistry (Moscow). 1981; 46: 3-10Google Scholar). Cells were harvested at the late exponential growth phase (10–13 g (wet weight)/liter). Mitochondria were isolated by the method developed in our laboratory (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The oxygen consumption in mitochondrial suspensions was monitored polarographically with a Clark-type electrode in medium containing 0.6 m mannitol, 1 mm Tris phosphate (pH 7.4), 1 mm EDTA, 20 mm pyruvate, 5 mm malate, and mitochondria corresponding to 0.5 mg/ml protein. The mitochondrial preparations were well coupled, showing respiratory control ratios of ∼4 under these conditions. ADP/oxygen ratios were close to the theoretical maximum. Respiratory control and ADP/oxygen ratios were calculated as described by Chance and Williams (21Chance B. Williams G.R. Nature. 1955; 175: 1120-1121Crossref PubMed Scopus (268) Google Scholar). Mitochondria were fully active for at least 4 h after preparation. Ca2+ uptake was assayed by the murexide method employing dual wavelength photometry at 507–540 nm with a Hitachi 557 spectrophotometer. Unless otherwise specified in the figure legends, the incubation medium contained 0.6 m mannitol, 2 mm Tris phosphate or 20 mm Tris acetate (pH 7.4), 20 mm Tris pyruvate, 5 mm malate, 50 μm murexide, and mitochondria corresponding to 0.5 mg/ml protein. The inner mitochondrial membrane transmembrane potential (ΔΨ)1 was measured at the wavelength pair 523/555 nm with 7 μm safranin (22Åkerman K.E.O. Wikström M.K.F. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). Mitochondrial swelling was monitored by recording changes in absorbance at 540 nm. Protein was assayed by the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216357) Google Scholar) with bovine serum albumin as the standard. Mannitol, sorbitol, EDTA, EGTA, bovine serum albumin, ADP, spermine, valinomycin, nigericin, atractyloside, CCCP, Tris, pyruvate, malate, murexide, and CaCl2 were purchased from Sigma. Coomassie G-250, NADH, and safranin were from Serva. A23187 was from Roche Molecular Biochemicals. Dithiothreitol was from Reanal. Cyclosporin A (CsA) was a kind gift of Novartis. In the presence of Pi, yeast mitochondria were able to take up almost all of the Ca2+ added in successive aliquots (up to 600 nmol of Ca2+/mg of protein) and to retain it until the oxygen was exhausted after 5–10 min of incubation (Fig. 1, trace a). Anaerobiosis induced a rapid collapse of ΔΨ (Fig. 1,trace c), the driving force of Ca2+uptake by the calcium uniporter. The rate of Ca2+ uptake was much faster than that of efflux induced by anaerobiosis (Fig. 1), which is in accordance with the ability of the mitochondria to reduce the [Ca2+] to very low levels at steady state. On the other hand, upon collapse of ΔΨ, efflux could be by reversal of the uptake mechanism, the calcium uniporter. Intensive aeration of the incubation medium fully reestablished ΔΨ, prevented spontaneous Ca2+ release, and elicited re-uptake of the Ca2+ released (Fig. 1, trace a). Aeration before each Ca2+ addition also prevented Ca2+ release (Fig. 1, trace b) and a drop in ΔΨ (trace d), thus increasing the Ca2+-buffering capacity of the yeast mitochondria. The efflux rate was not affected by the addition of CsA (Fig. 1,trace a). Since no Ca2+ efflux was observed in respiring yeast mitochondria in the presence of Pi, acetate was used instead as a permeant anion. The Ca2+ efflux pathways in animal mitochondria are not influenced by acetate and thus can be studied in the presence of this anion, the calcium salt of which is soluble (24Rizzuto R. Bernardi P. Favoron M. Azzone G.F. Biochem. J. 1987; 246: 271-277Crossref PubMed Scopus (50) Google Scholar). In yeast mitochondria, Ca2+ efflux ensued after uptake of ∼75 μm Ca2+ (Fig.2, trace a). Ca2+ release was not due to a decline in ΔΨ (Fig. 2,trace b), and it was insensitive to micromolar concentrations of CsA (data not shown). The efflux was thus spontaneous and not due to anaerobiosis or induction of the mitochondrial permeability transition (MPT). The rate of Ca2+ efflux was proportional to the Ca2+ load (Fig.3 A). It was inhibited half-maximally by Pi at 0.3 mm (Fig.3 B).Figure 3Effect of Ca2+ load (A) and of Pi (B) on the rate of Ca2+ efflux in yeast mitochondria. The medium was the same as that described in the legend to Fig. 2, but without murexide. In A and B, 100 and 500 μm Ca2+ were added, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In acetate medium, Ca2+ uptake would cause mitochondrial swelling due to accumulation of calcium acetate in the matrix, whereas efflux would result in contraction. Fig.4 shows the effect of various agents on the contraction due to spontaneous Ca2+ efflux in mitochondria respiring on pyruvate + malate. In mitochondria to which 100 μm Ca2+ had been added, the addition ofN-ethylmaleimide only slightly slowed contraction, whereast-butyl hydroperoxide, oxalacetate, atractyloside, and the uncoupling agent CCCP in the presence of EGTA were without any effect (Fig. 4). In animal mitochondria, efflux of Ca2+ can be studied by inhibiting the uniporter with ruthenium red (25Moore C.L. Biochem. Biophys. Res. Commun. 1971; 42: 298-305Crossref PubMed Scopus (471) Google Scholar, 26Igbavboa U. Pfeiffer D.R. J. Biol. Chem. 1988; 263: 1405-1412Abstract Full Text PDF PubMed Google Scholar). However, theE. magnusii mitochondrial calcium uniporter is not inhibited by ruthenium red (12Leikin Y.N. Votyakova T.V. Bazhenova E.N. Zvyagilskaya R.A. Kotelnikova A.V. Biochemistry (Moscow). 1987; 52: 676-682Google Scholar) and may even be stimulated by it (16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar). In the acetate medium, there was a spontaneous net efflux of Ca2+ (Fig. but this not a We La3+, a inhibitor of mitochondrial Ca2+ Biochem. J. PubMed Scopus Google Scholar), could be used as an inhibitor of the uniporter. However, although Ca2+ uptake was to low concentrations of (Fig. 5 b), efflux was also slightly at concentrations (trace a). of Ca2+ uptake was at μm The of were thus different for uptake and efflux of The rate of sodium-independent Ca2+ efflux in animal mitochondria can be by a number of P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar, J. Physiol. 267: PubMed Google Scholar), including H. Marbach M. Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar), A. Bernardi P. Saris N.-E.L. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, 1999; Google Scholar), A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), N.-E.L. Bernardi P. Biochim. Biophys. Acta. 1983; PubMed Scopus (21) Google Scholar), polyamines (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar, A. Bernardi P. Saris N.-E.L. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), and tetraphenylphosphonium J. Physiol. 267: PubMed Google Scholar) as well as by (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar) and L.A. FEBS Lett. PubMed Scopus Google Scholar). We the effect of of these agents on Ca2+ efflux in E. magnusii mitochondria. Ca2+ efflux was inhibited by B, trace and by 5 trace a). also affected the rate of Ca2+ uptake and efflux with a different (Fig. and μm inhibited Ca2+ efflux by (Fig. 5 B, trace whereas Ca2+ uptake was inhibited by only (trace at mm almost Ca2+ efflux trace and inhibited Ca2+ uptake by only (trace In the of (Fig. 5 the of of Ca2+ uptake and efflux was even is a and inhibitor of the sodium-independent Ca2+ efflux pathway in animal mitochondria J. Physiol. 267: PubMed Google Scholar). We also found that in yeast mitochondria, inhibited Ca2+efflux (Fig. 5 with a effect at μm and a effect at in which is close to for mitochondria J. Physiol. 267: PubMed Google Scholar). of Ca2+ was inhibited only slightly by μm (data not shown). Ca2+efflux in yeast mitochondria was almost inhibited by mm Pi (Fig. a that was found to be in Ca2+ uptake (11Zvyagilskaya R.A. Leikin Y.N. Kozhokaru N.L. Kotelnikova A.V. Dokl. Akad. NAUK SSSR. 1983; 269: 1233-1240Google Scholar). increased the rate of Ca2+ efflux added before (Fig. trace and after (trace with no effect upon ΔΨ a and The Ca2+ release was and similar to that after the addition of the Ca2+ A23187 (Fig. The addition of A23187 after induced (Fig. trace a). The rate of Ca2+ efflux in yeast mitochondria was not affected by the of and was only slightly inhibited by micromolar concentrations of (data not shown). with 4 mm to reduce intramitochondrial had no effect (data not whereas these have been to stimulate Ca2+uptake (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). of mammalian mitochondria in medium was found to stimulate the sodium-independent Ca2+ efflux pathway, of of the inner mitochondrial membrane (24Rizzuto R. Bernardi P. Favoron M. Azzone G.F. Biochem. J. 1987; 246: 271-277Crossref PubMed Scopus (50) Google Scholar, L.A. FEBS Lett. PubMed Scopus Google Scholar, P. Azzone G.F. Eur. J. Biochem. 1983; PubMed Scopus Google Scholar). in yeast mitochondria, a Ca2+ efflux was observed mitochondria were in medium A, trace the mechanism of the efflux pathway, we the of CCCP protonophorous nigericin via and valinomycin Ca2+ efflux was only inhibited by nm CCCP (data not whereas effect was on the associated contraction of mitochondria (Fig. 4). Ca2+ efflux was inhibited by 100 nm nigericin medium was with 0.5 mm (Fig. 7 B, trace a). which and the inner mitochondrial membrane and is able to ΔΨ, slightly increased ΔΨ at The addition of 100 nm valinomycin medium was with 4 mm of ΔΨ and increased the rate of Ca2+ efflux (Fig. 7 In the presence of Pi, E. magnusii mitochondria are of massive uptake of Ca2+ Fig. In animal mitochondria, Pi has been to the release of Pi N.-E.L. Soc. Scholar), which is to be due to induction of P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). accumulation was observed in animal mitochondria in the presence of due to of calcium (for review, see E. Google Scholar) and of P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). In the of Ca2+ uptake by mitochondria is also stimulated by Pi, and the sodium-independent efflux is which was as due to of [Ca2+] by its to Pi (19Rottenberg H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar). In mitochondria, the sodium-independent Ca2+ efflux rate is stimulated that in the presence of Pi and is inhibited by the addition of Pi F. Eur. J. Biochem. PubMed Scopus Google Scholar). of Ca2+ uptake by Pi and of efflux have also been described in and were also as due to of Ca2+ by Pi R. A. A. A. Biochem. J. 1993; PubMed Scopus Google Scholar). The same mechanism may for the effect of Pi on Ca2+ efflux in E. magnusii mitochondria. The efflux of Ca2+ in acetate medium could by a reversal of the calcium uniporter upon of ΔΨ, by efflux as the antiporter M. M. E. Eur. J. Biochem. 79: PubMed Scopus Google Scholar) and the antiporter P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar, H. Marbach M. FEBS Lett. 1990; 274: 65-68Crossref PubMed Scopus (21) Google Scholar, J. Physiol. 267: PubMed Google Scholar) in animal mitochondria, or by opening of the However, the efflux rate was not influenced by uncoupling of CCCP or Na+ (data not which the uniporter and the antiporter is by showing that the uptake and efflux pathways in their to (Fig. 5 and as (Fig. 5 (Fig. 5 and (Fig. was only slightly inhibited by (data not whereas uptake was stimulated a effect at μm and at a effect at (15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Bazhenova E.N. Deryabina Y.I. Zvyagilskaya R.A. Dokl. Akad. NAUK SSSR. 1997; 353: 1-3Google Scholar). In the efflux rate was not influenced by of the mitochondrial suspension with 4 mm NADH, which a high of the intramitochondrial in yeast mitochondria R.A. V.A. Dokl. Akad. NAUK SSSR. 263: Scholar), although Ca2+ uptake is stimulated under these (17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In animal mitochondria, and Ca2+ release by the by the of the P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). magnusii mitochondria, Ca2+ efflux was only slightly affected by (data not shown). The addition of atractyloside, or of CCCP or of by had effect on the rate of mitochondrial contraction associated with Ca2+ efflux (Fig. 4). these that there is no opening of a pore in these mitochondria. is by the of by CsA and by the massive accumulation of Ca2+ in the presence of that would in animal mitochondria P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). of this has been found in mitochondria Pfeiffer D.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). are to the inner mitochondrial membrane by and inhibiting the and N.-E.L. E. H. A. Acta. 2000; PubMed Scopus Google Scholar), the not of relevance in the of yeast mitochondria as The mechanism of the of on Ca2+ efflux has not been It that in E. magnusii mitochondria, and their from the cytosolic these mitochondria have only a for R.A. Votyakova T.V. Bazhenova E.N. 1987; Google Scholar), and the calcium uniporter of Mn2+, or only at high concentrations (data not shown). these their without any is well as an important of the Ca2+ in animal mitochondria, Ca2+ uptake N.-E.L. Wikström M.K.F. FEBS Scholar, J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1988; Google Scholar, I. H. Biochem. Biol. 1996; Google Scholar, J.R. M. J. 1987; PubMed Scopus Google Scholar) and inhibiting Ca2+ efflux H. Marbach M. Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar, J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.R. M. J. 1987; PubMed Scopus Google Scholar). We have also a effect of low physiological concentrations of on Ca2+ uptake by E. magnusii mitochondria (13Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. FEBS Lett. 1990; 261: 139-141Crossref PubMed Scopus (18) Google Scholar, 14Votyakova T.V. Bazhenova E.N. Zvjagilskaya R.A. J. Bioenerg. Biomembr. 1993; 25: 569-574Crossref PubMed Scopus (19) Google Scholar, 15Deryabina Y.I. Bazhenova E.N. Zvyagilskaya R.A. Biochemistry (Moscow). 1996; 61: 1704-1713Google Scholar, 16Bazhenova E.N. Saris N.-E.L. Zvyagilskaya R.A. Biochim. Biophys. Acta. 1998; 1371: 96-100Crossref PubMed Scopus (16) Google Scholar, 17Bazhenova E.N. Deryabina Y.I. Eriksson O. Zvyagilskaya R.A. Saris N.-E.L. J. Biol. Chem. 1998; 273: 4372-4377Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The mechanism of the stimulation of the efflux rate by may be an in the of the an effect of of to membrane thus the and of Ca2+ from the It is also that Ca2+ the by the same effect on the which would the stimulation of efflux after uptake a inhibited Ca2+ efflux in the presence of 0.5 mm under that not ΔΨ, whereas valinomycin stimulated efflux in the presence of 4 mm (Fig. 7 these valinomycin a in ΔΨ and swelling Since there be no in the presence of and a drop in ΔΨ induced by CCCP not the efflux rate (Fig. it that the is due to of the mitochondrial The stimulation of efflux could be due to swelling (Fig. induced by accumulation of acetate in the these that Ca2+ efflux magnusii mitochondria is by a sodium-independent could be a with as a driving as in mitochondria. these are at the The to our for the that mitochondria from a yeast are with for and efflux of the of Ca2+ uptake and efflux pathways in E. magnusii mitochondria and their physiological in the cytosolic Ca2+ would Ca2+ uptake by respiring mitochondria via the Ca2+ with ΔΨ the inner mitochondrial membrane as the driving force P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). would a of Ca2+ for the of of to the respiratory (3McCormack J.G. Denton R.M. Biochem. Soc. Trans. 1993; 21: 793-799Crossref PubMed Scopus (72) Google Scholar). In our we found that the pyruvate magnusii mitochondria was by The Ca2+ that in the would be by to Pi and other Ca2+ uptake the as in the of added Pi, an in [Ca2+] would Ca2+ efflux by a sodium-independent mechanism, antiport, or by of a P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). The result would be an in medium but the increased rate of Ca2+ uptake in the Ca2+ would not be to ΔΨ (Fig. In these that E. magnusii mitochondria have a Ca2+ efflux pathway that is by In many it is similar to the sodium-independent Ca2+efflux pathway in mitochondria from non-excitable mammalian in of mitochondria, mitochondrial Ca2+efflux is by the same The efflux in E. magnusii was spontaneous and not due a decline in ΔΨ or induction of We are to A. for the in this