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J. Biol. Chem., Vol. 276, Issue 44, 40502-40509, November 2, 2001

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Free Fatty Acids Activate a Vigorous Ca²⁺:2H⁺ Antiport Activity in Yeast Mitochondria^*

Patrick C. Bradshaw, Dennis W. Jung, and Douglas R. Pfeiffer

From the Department of Molecular and Cellular Biochemistry, Ohio State University Medical Center, Columbus, Ohio 43210

Received for publication, June 1, 2001, and in revised form, July 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The accumulation and retention of Ca²⁺ by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca²⁺ transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca²⁺ or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependant manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca²⁺ and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca²⁺:2H⁺ antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca²⁺ accumulation and release when exogenous Ca²⁺ and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca²⁺-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca²⁺ plus ETH 129.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ plays important roles in the cell signaling systems of eucaryotic organisms (1). In mammalian cells, mitochondria participate in these systems through the Ca²⁺ uptake and release activities that they contain (2). These include a Ca²⁺ uniporter for accumulation, an Na⁺/Ca²⁺ exchange activity for release, a second release activity that is poorly characterized, and a general diffusion pore that is referred to as the permeability transition pore (3). Physiological roles of the latter structure are not known with certainty but are thought to include the propagation of cytoplasmic Ca²⁺ waves through repetitive opening and closing of a low conductance state (4, 5). In addition to influencing extramitochondrial Ca²⁺-regulated processes, mitochondrial transport activities adjust the free Ca²⁺ concentration in the matrix space and thereby regulate the production of reducing equivalents and the synthesis of ATP (6).

In simpler eucaryotes such as yeast, the cell signaling roles of Ca²⁺ are less developed, and there is a corresponding reduction in the role of mitochondria in maintaining Ca²⁺ homeostasis. Yeast mitochondria do not contain a calcium uniporter (7-9) and are thought to lack high activity release carriers that oppose the uniporter in other mitochondria (10). They do contain a general diffusion pore in the inner membrane and are subject to a permeability transition; however, Ca²⁺ does not regulate the transition as it does in mammalian mitochondria (8). In addition, the production of reducing equivalents and ATP synthesis are not regulated by Ca²⁺ in yeast mitochondria, in so far as is known (11). On the other hand, matrix space pyrophosphatase activity is Ca²⁺-regulated in mitochondria from both yeast (12) and mammalian (13) sources, and there may be other roles for Ca²⁺ that are unidentified at present.

A study that employed yeast mitochondria loaded with a Ca²⁺ indicator (fluo-3) showed that the matrix space Ca²⁺ concentration is established by a simple equilibration with the extramitochondrial concentration (14). The activity responsible appeared to be slow, and it was not clear whether the range of adjustment would encompass that which is attained in yeast cytoplasm through normal processes. In a related study we utilized a strain of yeast that expresses aequorin in the mitochondria to further investigate the mechanism of equilibration. The presence of a slow Ca²⁺ transport activity was confirmed, it was found to be reversible, and it was seen that a relatively broad range of matrix space Ca²⁺ concentrations can be attained via the activity, similar to the physiological range of variation in cytoplasm.¹ Our study also showed that yeast mitochondria in vivo accumulate and retain Ca²⁺ through the action of ionophore ETH 129, whereas accumulation was transient in the case of isolated mitochondria. The latter observation conflicted with another report in which it was seen that isolated yeast mitochondria retain Ca²⁺ accumulated via the ionophore under a particular set of conditions (8).

In the present report we describe further factors that influence interactions between Ca²⁺ and yeast mitochondria, particularly as they relate to Ca²⁺ transport. The results show that mitochondria from the yeast Saccharomyces cerevisiae contain a vigorous Ca²⁺ transport activity that has long eluded detection. The activity is regulated by free fatty acids that arise by degradation of mitochondrial phospholipids, and the phospholipase responsible for that process is also newly discovered. Aspects of the data have been described in abstract form (15).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Yeast Mitochondria-- The yeast strain W303-1A (S. cerevisiae) was grown aerobically at 30 °C in a medium containing 2% lactate, 1% yeast extract, 2% peptone, 0.05% dextrose, and 0.01% adenine at pH 5.0, and cells were harvested during the logarithmic phase (A₆₀₀ = 1.8-2.2). Mitochondria were isolated from spheroplasts as described previously (8), except that 0.6 M sucrose was used in the homogenization medium instead of the 0.6 M mannitol that is usually employed (16). The resulting preparations were maintained on ice and were suspended in 0.6 M mannitol, 20 mM HEPES (K⁺) (pH 6.8), containing 0.75 mg/ml BSA² and 0.1 mM EGTA. Protein concentration was determined by a reduced volume Biuret method in which 50 µl of the final preparation were solubilized by adding an equal volume of 10% deoxycholate (Na⁺), with 0.4 ml of Biuret reagent added to the resulting mixture. BSA was employed as the standard.

Determination of Ion Transport, , and Related Parameters-- Unless noted otherwise, mitochondria were incubated at 1 mg protein/ml and at ~25 °C in a medium that contained 0.6 M mannitol, 10 mM P_i (K⁺), 10 mM HEPES (TEA⁺), pH 7.2, plus 1 mM ethanol as an oxidizable substrate. Compounds having a low solubility in water were added from stock solutions prepared in methanol or Me₂SO. Ca²⁺ transport and membrane potential were monitored using the indicating dyes antipyrylazo III (A_720-790) and safranin O (A_511-533), respectively (17, 18). An SLM Aminco DW-2C spectrophotometer operated in the dual wavelength mode was employed for these purposes. The same instrument, operated in split beam mode, was used to monitor swelling as a decrease in apparent absorbance of mitochondrial suspensions at 540 nm. Oxygen consumption data were obtained using a Clark-type electrode in parallel experiments.

Atomic absorption spectroscopy was utilized to monitor the transport of other cations by yeast mitochondria. Briefly, samples were taken at appropriate times, and the mitochondria were sedimented rapidly in a microcentrifuge. After carefully removing supernatants, the pellets were solubilized with 2 N percholoric acid (overnight) and centrifuged a second time. The resulting supernatants were diluted with an appropriate volume of distilled water, and the concentration of the cation in question was subsequently determined.

Determination of Free Fatty Acids and Mitochondrial Phospholipids-- Samples containing 3 mg of mitochondrial protein were extracted by a modified Folch technique (19), following the addition of a known amount of heptadecanoic acid (17:0), which was employed as an internal standard (20). The lipid-containing phase was reacted with diazomethane to produce fatty acid methyl esters (21), and silica gel minicolumns were used to separate these from other fractions (19). The fatty acid methyl esters were quantitated by gas-liquid chromatography, using an instrument equipped with a capillary column and a computing integrator (19, 20). Peak areas were converted to units of nmol/mg of mitochondrial protein by considering the internal standard peak area and the amount of protein represented by the sample (19, 20). Total mitochondrial phospholipids were estimated from measurements of lipid phosphorous in an aliquot of the initial extract (22).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Maintenance of ETH 129-mediated Ca²⁺ Transport and Requires the Presence of BSA-- ETH 129 is an electrogenic ionophore for divalent cations (23-26) that allows yeast mitochondria to accumulate Ca²⁺ in vivo and in vitro (8).¹ Under in vitro conditions an oxidizable substrate must be present, together with inorganic phosphate, which is normally used in the concentration range of 5-10 mM. As in mammalian mitochondria, the substrate is required to support development of , which is the immediate driving force for Ca²⁺ accumulation. Phosphate is required to maintain the yeast permeability transition pore (yPTP) in a closed state, to reduce the transmembrane pH gradient and thereby to enhance and, presumably, to reduce the free concentration of Ca²⁺ in the matrix space by participating in the formation of insoluble complexes (8).

While investigating the above features we noticed that ETH 129-mediated Ca²⁺ accumulation by yeast mitochondria is subject to variation in terms of the rate of transport, the external Ca²⁺ concentration that is attained, and the tendency of previously accumulated Ca²⁺ to be released during an extended incubation (Fig. 1). This is despite the fact that ETH 129 is a well behaved ionophore for Ca²⁺ in model systems, displaying properties that are highly reproducible (26), and despite the fact that the Ca²⁺ transport activities of mammalian mitochondria do not show this degree of variability as long as the permeability transition is avoided. Accordingly, it appeared that variability reflects specific properties of yeast mitochondrial preparations and that it might relate to the differing capacity of these mitochondria to retain Ca²⁺ in vivo and in vitro, as described in the Introduction.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Variable efficiency of ETH 129-mediated Ca2+ transport in yeast mitochondria. Conditions for all traces were the same except that different preparations of mitochondria were employed. The mitochondria were incubated at 1.0 mg of protein per ml and at 25 °C in a medium that contained 0.6 M mannitol, 10 mM HEPES (TEA+, pH 7.2), 10 mM Pi (K+), 0.5 mM ethanol, 0.5 mg/ml BSA, 0.1 mM antipyrylazo, and 80 µM CaCl2. Were indicated, 2.4 µM ETH 129 was added to initiate Ca2+ accumulation, which was monitored via the antipyrylazo III signal as described under "Experimental Procedures." A downward deflection indicates accumulation, and the heavy bar associated with each trace shows the signal at an external Ca2+ concentration of ~0.

Insight into the underlying cause of variability is provided by Fig. 2, wherein it is shown that Ca²⁺ accumulation by freshly prepared yeast mitochondria is slow and limited in a medium that does not contain BSA. The addition of BSA after transport has begun improves both the rate and extent parameters in a concentration-dependent manner. For the particular preparation employed when obtaining that figure, a maximal effect was seen at a BSA concentration near 0.25 mg/ml (~4 µM). BSA is similarly effective when present from the beginning of incubations, whereas high molecular mass polymers (10 kDa polyethylene glycol, 100 kDa dextran) that have similar effects on medium colloid osmotic properties are ineffective (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   BSA stimulates ETH 129-mediated Ca2+ transport. Mitochondria were incubated at 1.0 mg of protein per ml and at 25 °C. The medium contained 0.6 M mannitol, 10 mM HEPES (TEA+, pH 7.2), 10 mM Pi (K+), 0.1 mM antipyrylazo III, 12 µM safranine O, and 10 µM EGTA (TEA+). Additions of 1 mM ethanol, 60 µM CaCl2, and 3.6 µM ETH 129 were made as indicated in the figure, and Ca2+ accumulation was monitored via the antipyrylazo III signal as described under "Experimental Procedures." A downward deflection indicates accumulation. When utilized, BSA was added at 0.0625, 0.125, or 0.25 mg/ml, as indicated by the number associated with the individual traces.

Effects of BSA on are examined in Fig. 3. These mitochondria develop and maintain a large during ethanol oxidation when BSA is absent, although BSA increases the magnitude to some degree (Fig. 3A). The addition of Ca²⁺ or ETH 129 alone has little effect on (Fig. 3B); however, both agents together produce a sustained reduction (Fig. 3C). This reduction persists for more time than would be required to complete Ca²⁺ uptake if BSA were present, based on Fig. 2, and is not duplicated when ionomycin is added with Ca²⁺ instead of ETH 129 (Fig. 3C). Ionomycin is an electroneutral ionophore for Ca²⁺ (27, 28) that equilibrates Ca²⁺ and H⁺ gradients across membranes. Accordingly, Fig. 3C shows that the simple presence of Ca²⁺ in the mitochondrial matrix space does not effect . Rather it is the entry of Ca²⁺ via an electrogenic process that is required.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Effects of ETH 129 and Ca2+ on . Mitochondria were incubated as described in the legend to Fig. 2, except that antipyrylazo III was not present. For all traces, 1 mM ethanol was added where indicated, and was monitored via the safranine signal as described under "Experimental Procedures." An upward deflection indicates increasing . For all traces, 4 µM of the uncoupler FCCP was added at ~700 s to determine the signal at  = 0. A, 0.5 mg/ml of BSA was present or absent from the beginning of the incubation, as indicated. B, BSA was absent for both traces, and 80 µM CaCl2 or 3.6 µM ETH 129 was added as indicated. C, same as B except both CaCl2 and an ionophore were added. The ionophore employed was ETH 129 or ionomycin, as indicated.

The sustained reduction of produced by ETH 129 plus Ca²⁺ can be reversed by the subsequent addition of BSA (Fig. 4). As in the case of enhanced Ca²⁺ accumulation (Fig. 2), the degree of reversal is a function of the BSA concentration such that a direct correlation is seen between the actions of BSA on and Ca²⁺ transport. When BSA is absent and transport is minimal, is low. Conversely, the efficient transport seen in the presence of BSA is associated with a large .

View larger version (16K): [in this window] [in a new window]   Fig. 4.   BSA restores when Ca2+ and ETH 129 are present. Conditions were the same as described for Fig. 3C, with ETH 129 utilized instead of ionomycin. was monitored via the safranine signal as described under "Experimental Procedures," with an upward deflection of the individual traces indicating an increasing value. When utilized, BSA was added at 0.0625, 0.125, 0.25, 0.50, or 1.0 mg/ml, as indicated by the number associated with the individual traces.

Additional data pertaining to the sustained reduction of are shown in Fig. 5. This reduction is apparently unrelated to the yeast mitochondrial permeability transition given that the large amplitude of swelling that accompanies the transition induced by respiration in the absence of phosphate (compare Fig. 5, A and B) is not seen when is depressed by ETH 129 plus Ca²⁺ (Fig. 5C). Furthermore, there is little effect of BSA on respiration when ETH 129 and Ca²⁺ are present (compare Fig. 5, C and D), and in neither case does respiration become inhibited as it does when the transition occurs (compare Fig. 5, A and B). Other features of the respiration data are also of interest and are considered under "Discussion."

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Effects of ETH 129 and Ca2+ on swelling and respiration. A, mitochondria were incubated at 1.0 mg of protein per ml and at 25 °C. The medium contained 0.6 M mannitol, 10 mM HEPES (TEA+, pH 7.2), 10 mM Pi (K+), and 10 µM EGTA (TEA+). 1 mM ethanol (ETOH) was added where indicated while swelling (solid line) and oxygen consumption (dotted line) were monitored as described under "Experimental Procedures." B, same as A except the medium did not contain Pi. C, same as A except that 80 µM CaCl2 and 3.6 µM ETH 129 were added where shown. D, same as C except that the medium contained BSA at 0.5 mg/ml. As a point of reference, the rate of oxygen consumption in A was 34.8 ng atoms O/min/mg protein.

Analogous Actions of Free Fatty Acids and EGTA-- BSA binds fatty acids with high affinity but also binds a range of molecules that are hydrophobic in nature (29, 30). To determine whether the fatty acid binding activity of BSA is involved in its effects on ETH 129-mediated Ca²⁺ transport we first investigated the actions of exogenous oleate (18:1) on the process. With BSA present initially at 0.5 mg/ml, the addition of increasing oleate levels during Ca²⁺ accumulation first inhibited further accumulation and then caused a reversal of the transport that had already occurred (Fig. 6). These findings indicate that free fatty acids do indeed diminish Ca²⁺ transport and that BSA acts by reducing the available fraction.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of exogenous oleate on ETH 129-mediated Ca2+ transport. Mitochondria were incubated, and Ca2+ transport was monitored as described in the legend to Fig. 2, with 1 mM ethanol and 0.5 mg/ml BSA present at 0 s. 80 µM CaCl2 and 3.6 µM ETH 129 were added where indicated, followed by an addition of oleate (Na+) dissolved in methanol. The number associated with the individual traces gives the amount added in units of nmol/mg protein.

Because fatty acids uncouple yeast mitochondria through a mechanism that requires participation of the adenine nucleotide translocase (31) it also seemed possible that the Ca²⁺-releasing activity of fatty acids and opposing effects of BSA might merely reflect uncoupling by that type of mechanism. However, we found no effect of carboxyatractyloside on the depression of produced by ETH 129 plus Ca²⁺ when BSA was absent (data not shown). As further considered under "Discussion," this argues against a significant role for fatty acid-derived uncoupling, because carboxyatractyloside is an effective inhibitor of the translocase activity in yeast mitochondria (32). Furthermore, the depression of is reversed when EGTA is added to the external medium. This is seen regardless of whether Ca²⁺ transport is occurring early or late during incubation (compare Fig. 7, A and B) and whether has been suppressed briefly or for an extended period before EGTA is added (compare Fig. 7, A and C). In addition, there is a graded response to multiple additions of EGTA when the concentration arising from a single addition is less than the total concentration of Ca²⁺ (Fig. 7C). Thus, a continuing presence of free Ca²⁺ is required to maintain the suppression of even when BSA is not present to bind fatty acids, and the extent of suppression is a function of the free Ca²⁺ concentration.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   EGTA restores when Ca2+ and ETH 129 are present. Mitochondria were incubated, and was monitored as described in the legend to Fig. 3. For all panels and traces, additions of ethanol (ETOH; 1 mM) and CaCl2 (80 µM) and ETH 129 (3.6 µM) were as shown in the figure. 4 µM of the uncoupler FCCP was added at ~900 s in all cases to determine the signal at  = 0. A, trace a, the medium contained 0.5 mg/ml of BSA from 0 s. Trace b, BSA was not present, and 2 mM EGTA (TEA+) was added where indicated. B, same as A except the addition times were altered as shown, to demonstrate that results do not depend on that variable. C, trace a, same as A, trace b, except that EGTA was added incrementally as shown. The first four additions were of 30 µM EGTA, whereas the final addition was 2 mM. Trace b, same as A, trace b except that addition of 2 mM EGTA was delayed as shown, again to demonstrate that results do not depend on the time of addition.

A further point relating to fatty acid-dependant uncoupling is derived from Fig. 8. There it is seen that suppression of begins immediately during titration with exogenous oleate when ETH 129, free Ca²⁺, and BSA are available (Fig. 8, dotted lines). This is in contrast to the titration preformed in the presence of excess EGTA where free Ca²⁺ is not available, but fatty acid-dependant uncoupling could still occur. Under those conditions exogenous oleate does not depress until the level added exceeds 15 nmol/mg protein. This shows that the levels of fatty acids required to produce an extensive uncoupling are higher than those that mediate the decrease in that arises in the presence of free Ca²⁺ and ETH 129. 

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Oleate differentially effects in the presence and absence of EGTA. Mitochondria were incubated, and was monitored as described in the legend to Fig. 3, except that CaCl2, ETH 129, and BSA were present from the beginning of the incubations. For the data shown as solid lines, 2 mM EGTA (TEA+) was also present from the beginning, whereas EGTA was not present for the data shown as dotted lines. 1 mM ethanol was added at 0 s, followed by repetitive additions of 5 nmol/mg protein of oleate (Na+) as shown. When utilized FCCP was added at 4 µM.

Free Fatty Acids Accumulate in Isolated Yeast Mitochondria-- To determine whether the free fatty acid levels in our yeast mitochondria are sufficient to alter Ca²⁺ transport, these values were determined as a function of incubation time. Indeed, large amounts of free fatty acids accumulate during incubation at 25 °C, at an initial rate of approximately of ~0.4 nmol/min/mg protein (Fig. 9). This rate was not altered substantially by the presence or absence of ethanol (Fig. 9, inset), by free Ca²⁺ and ETH 129, or by the presence of BSA. However, the rate is decreased to ~0.3 nmol/hr/mg protein when the mitochondria are maintained on ice (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Phospholipase activity in isolated yeast mitochondria. Mitochondria were incubated in 0.6 M mannitol, 10 mM HEPES (TEA+, pH 7.2), 10 mM Pi (K+), 100 µM EGTA (TEA+), and 1 mM ethanol. Samples were taken periodically for the determination of free fatty acids as described under "Experimental Procedures." The associated table shows the composition of free fatty acids that were present in the sample taken at 170 min. Inset, same as the main panel except that ethanol was present () or absent (), and a shortened time course was employed to conform to the other figures. FFA, free fatty acids.

16:0, 16:1, 18:0, and 18:1 account for ~90% of the accumulating fatty acids (Fig. 9). These compounds and their relative proportions are typical of the fatty acid composition found in yeast phospholipids (33). Parallel determinations of lipid phosphorus showed that the phospholipid content of the preparations decreased at ~50% of the rate at which the fatty acids accumulate in total (data not shown). Furthermore, there was little change in the rate or composition parameters when Percoll gradient-purified mitochondria (34) were employed instead of the standard preparation (data not shown). Accordingly, it appears that the free fatty acids that accumulate arise from mitochondrial phospholipids and that both sn-1 and sn-2 positions are hydrolyzed by the degradative activities.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Mitochondria Contain an Endogenous Ca²⁺ Transport Activity-- Mammalian mitochondria that are treated with Ca²⁺ plus A23187 or ionomycin behave in much the same way as yeast mitochondria that are treated with Ca²⁺ plus ETH 129. A23178 and ionomycin are electroneutral ionophores that exchange matrix space Ca²⁺ for two external protons. Accordingly they reverse uniporter-dependent Ca²⁺ accumulation and a futile cycle of accumulation, and release is established when the ionophore and the uniporter are active simultaneously (35). This cycle dissipates the proton motive force (suppresses ), inhibits Ca²⁺ accumulation, and releases Ca²⁺ that was accumulated previously, depending on the levels of Ca²⁺ and the ionophore employed (36, 37). Given the similarities to those findings, we interpret the present data to indicate that such a cycle is operating in yeast mitochondria that are treated with Ca²⁺ plus ETH 129.

As noted, with yeast mitochondria the cycle arises upon addition of an electrogenic ionophore whereas an electroneutral ionophore must be employed with mammalian mitochondria. Given that both an electrogenic and an electroneutral transporter are required to produce the cycle, the most straightforward explanation for the difference is that yeast mitochondria contain a high activity Ca²⁺ transport mechanism that is electroneutral, in contrast to mammalian mitochondria where the dominate mechanism is electrogenic (the Ca²⁺ uniporter). We adopt this interpretation and propose further that the yeast activity is regulated by free fatty acids, such that it is poorly active when free fatty acids are not available (e.g. in the presence of excess BSA), and progressively more active as the free fatty acid levels rise. These interpretations explain (i) why efficient Ca²⁺ accumulation and retention requires the presence of BSA (to limit activity of the endogenous transporter that opposes accumulation), (ii) why remains suppressed in the presence of ETH 129 plus Ca²⁺ when BSA is absent (because the ongoing futile cycle continuously expends the proton motive force), and (iii) why is restored by the addition of BSA or EGTA (both agents interrupt the cycle).

Potential Alternative Interpretations-- In considering the merits of the above interpretation it is worthwhile to evaluate other possibilities. For example, it might be maintained that the free fatty acids are acting as Ca²⁺ ionophores, to affect a direct Ca²⁺:2H⁺ exchange, given reports that this occurs in model systems (e.g. see Refs. 38 and 39), and that fatty acids facilitate monovalent cation transport in mitochondria (40). We do not favor this interpretation, because the ionophoretic activities of fatty acids are modest when they have not been partially oxidized (e.g. see Ref. 41), which is not the case in the present experiments. In addition, we were unable to establish an energy-dissipating cycle of Ca²⁺ uptake and release by adding exogenous fatty acids to liver mitochondria and then loading them with Ca²⁺. At the fatty acid levels of interest in this study (0-20 nmol/mg protein) an uncoupling effect of the fatty acid was observed, but this was eliminated substantially upon Ca²⁺ accumulation (data not shown). Were the fatty acids promoting a significant Ca²⁺:2H⁺ exchange, an apparent uncoupling effect would have remained.

It might also be maintained that the yPTP is involved in the actions of ETH 129 plus Ca²⁺, because data like those shown in Fig. 1 can be obtained with mammalian mitochondria that are approaching the permeability transition. In further similarity to the data presented, the permeability transition in mammalian mitochondria is stimulated by Ca²⁺ accumulation, membrane depolarization, and low levels of free fatty acids, whereas it is reversed by Ca²⁺ chelation and other maneuvers that counteract inducing conditions (3, 42-44). We do not favor this interpretation, because the present data were obtained in the presence of 10 mM P_i, which is fully effective as an inhibitor of the yPTP (8, 45). In addition, decavanadate has no effect on the apparent energy dissipating cycle of Ca²⁺ accumulation and release (data not shown), and minimal swelling is seen while the cycle is operating (Fig. 5). The lack of inhibition by decavanadate is significant, because this compound is another inhibitor of yPTP and is effective at low concentrations (46).³ The absence of swelling means that mannitol (molecular weight = 182) cannot permeate the inner membrane, whereas larger molecules are permeable when the yPTP is open (8).⁴

The respiration data shown in Fig. 5 are also pertinent to a possible involvement of yPTP. Yeast mitochondria oxidize ethanol at a rate that is controlled primarily by the kinetic properties of ethanol dehydrogenase, rather than by limitations reflecting proton pumping against the electrochemical proton gradient (47-49). Accordingly, there is usually only a modest effect on the rate of respiration when yeast mitochondria are uncoupled, provided with the substrates for ATP synthesis, etc. This can explain why there is little effect of ETH 129-mediated Ca²⁺ pumping on the rate on respiration, as seen in Fig. 5. More to the point, when the yPTP is opened respiration falls dramatically, because matrix space NAD⁺/NADH is released from the matrix.³ Because respiration is not impeded in the presence of Ca²⁺ and ETH 129, by this criterion also there is no indication that the yPTP has opened.

The possible involvement of yPTP substates and the recently identified "nonclassic permeability transition" (50, 51) should also be considered. yPTP substate involvement seems improbable, because endogenous Mg²⁺ is retained when ETH 129 and Ca²⁺ are present (see below) and because is only fractionally suppressed under those conditions (Fig. 3). Thus, any pore-like substate that might be involved would have the unlikely property of accepting Ca²⁺ while substantially rejecting Mg²⁺ and H⁺. These same factors argue against an involvement of the nonclassic permeability transition, as does the absence of swelling. In mammalian mitochondria occurrence of the nonclassic permeability transition reflects opening of a pore that is similar in size to the permeability transition pore and requires fatty acid levels that are substantially higher than those of interest here (50, 51).

A third type of potential interpretation relates to the uncoupling activity of fatty acids as described by Skulachev (52) and by others (53). In that mechanism, protonated free fatty acids rapidly flip-flop from the outer to the inner monolayer of the inner mitochondrial membrane and then ionize, delivering a proton to the matrix space. Thereafter, the uncoupling cycle is completed by transporting (flipping) the ionized carboxylate function back to the outer monolayer. The latter step requires catalysis by an anion transporter, with the adenine nucleotide translocase being primarily responsible in yeast mitochondria (31). It might be maintained that the efficiency of this cycle is somehow increased by matrix Ca²⁺, such that a given level of free fatty acids would produce more uncoupling following Ca²⁺ loading via ETH 129. Although this interpretation can explain aspects of the present data, it is at odds with the failure of carboxyatractyloside to restore when Ca²⁺, ETH 129, and free fatty acids are present (data not shown). It also conflicts with the inhibitory action of Ca²⁺ loading on fatty acid-dependent uncoupling in liver mitochondria, as mentioned above, and with the failure of ionomycin to substitute for ETH 129 in promoting the dissipation of (Fig. 3).

We note further that oleate does not form a tertiary complex with Ca²⁺ and ETH 129 that is competent to transport Ca²⁺ by an electroneutral mechanism, as shown in a recent investigation (26). This point is of interest, because such a complex could, in effect, facilitate the flip-flop of the ionized compound and improve the efficiency of fatty acid-derived uncoupling. With that possibility also eliminated, there is little prospect for alternative interpretations wherein the present results would reflect a changing efficiency of fatty acid-derived uncoupling.

Nature and Specificity of the Transporter-- With alternative interpretations discounted, it is of interest to consider the reaction catalyzed by the newly discovered transport activity and to examine its specificity for Ca²⁺. As noted above, a straightforward exchange of Ca²⁺ for 2H⁺ is sufficient to explain the present data, and in fact, the yeast vacuolar membrane is known to contain a Ca²⁺ antiporter of this type (54, 55). A preliminary examination of the yeast proteome data base (56) showed that additional proteins possessing this activity may be produced by yeast, and so there are candidates for a mitochondrial isoform. Nevertheless, it is necessary to consider a possible exchange of Ca²⁺ for K⁺, or for TEA⁺, because both were present in the external media at significant concentrations when most of the data were obtained. To do this we replaced either monovalent cation with the other and observed the effect on the rate of Ca²⁺ cycling (suppression of ). No effect was seen, which supports the proposed role of H⁺ (data not shown). We also examined the swelling of yeast mitochondria in media containing high concentrations of Ca²⁺, Mg²⁺, or K⁺ acetate. Swelling under those conditions typically reflects entry of the cation in exchange for H⁺, accompanied by an uncatalyzed entry of acetic acid (HA). We found that swelling occurs in Ca²⁺ or K⁺ acetate, but not in Mg²⁺ acetate, and that BSA inhibits only in the case of Ca²⁺ (Fig. 10A). Swelling in K⁺ acetate reflects activity of the well known K⁺/H⁺ antiporter in yeast mitochondria (57), whereas the failure to swell in Mg²⁺ acetate reflects the absence of an analogous activity that transports Mg²⁺ (see below). The data obtained in Ca²⁺ acetate are as expected if yeast mitochondria contain Ca²⁺:2H⁺ antiporter that requires free fatty acids for activity, supporting our interpretation.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   The Ca2+ release activity is a H+ antiporter that transports Sr2+ but not Mg2+. A, mitochondria were incubated at 1.0 mg of protein per ml and at 25 °C. The medium contained 0.3 M Mg2+, Ca2+, or K+ acetate, as indicated, 10 mM HEPES (TEA+), pH 7.2, and 4 µM FCCP. Where indicated, BSA was present at 1.0 mg/ml from 0 s, which corresponded to the time at which mitochondria were added. B, mitochondria were incubated as described in the legend to Fig. 2, with 0.5 mg/ml of BSA and 20 µM oleate present at 0 s. 1 mM ethanol, 80 µM of the indicated cation chloride, 3.6 µM ETH 129, and 4 µM FCCP were added as shown. At the arrow labeled sample, a 1-ml aliquot was taken from the 3-ml volume in the cuvette, and the mitochondria were rapidly sedimented in a microcentrifuge. The distribution of the exogenous cation between the supernatant and the pellet was then determined by atomic absorption spectroscopy, as described under "Experimental Procedures." The number associated with the individual traces gives the percent of added cation that was found in the mitochondria. These samples were also used to determine whether the endogenous content of Mg2+ had been released. About 85% of the endogenous Mg2+ was retained in all cases.

Preliminary data relating to the specificity of the transporter for Ca²⁺ are shown in Fig. 10B. In the presence of ETH 129, BSA, and an excess of oleate, Sr²⁺ and Ca²⁺ are similarly effective at depressing , but Mg²⁺ is ineffective. These data indicate that the Ca²⁺ release activity transports Sr²⁺ if free fatty acids are available and are in line with the effectiveness of ETH 129 as an ionophore for Sr²⁺ but not Mg²⁺ (26). Also shown are the fractions of the added cations that were found in the mitochondria during futile cycling. With Ca²⁺ and Sr²⁺ only ~1/3 was internal, consistent with a rapid release activity that opposes uptake of either cation mediated by the ionophore. Exogenous Mg²⁺ remained in the extramitochondrial volume as expected, but of greater interest, endogenous Mg²⁺ was largely retained whereas Ca²⁺ or Sr²⁺ cycling is ongoing (see the legend to Fig. 10B). The latter observation indicates that the release activity is selective against Mg²⁺.

Phospholipid Degradation and Biological Implications-- Despite the available genomic sequence, little is known about the function and regulation of phospholipases in yeast (33). Yost et al. (58) used an assay based on exogenous substrates to show that disrupted mitochondria from cells grown on a fermentable or nonfermentable carbon source display phospholipase A₁ and A₂ activities. To our knowledge, our report is the first description of endogenous phospholipid degradation within the intact organelle. As in the earlier study, the activity described here is Ca²⁺-independent and substantial. For comparison, the initial rate is sufficient to degrade about 5% of total mitochondrial phospholipids within 1 h, which is similar to the activities found in rat liver and heart mitochondria (43, 59). The activity described here was observed at pH 7.2, whereas Yost et al. (58) found high activity at pH 5 and 9 and minimal activity near pH 7. Differences reflecting the form of substrate presentation may explain the variation.

Based on the present data, it is clear that free fatty acids arise in isolated yeast mitochondria at a rate sufficient to strongly influence the Ca²⁺ transporter on a time scale of minutes. Nevertheless, it is not clear how the phospholipase activity and free fatty acid levels would be controlled in vivo, so it is difficult to project a physiological function for the Ca²⁺ transporter based upon regulation of this type.

The high specific activity and directionality of the transporter are additional factors to consider. From Figs. 1, 2, and 5, yeast mitochondria can accumulate Ca²⁺ at a rate of ~40 nmol/min/mg protein when the ETH 129 level is 2.4 nmol/mg protein. This is a minimal estimate of activity for the release carrier, because the accumulation rate can be entirely negated when sufficient free fatty acids are present (Fig. 5). This rate greatly exceeds the rate of Ca²⁺ release from rat liver mitochondria when the permeability transition is avoided and more modestly exceeds the maximal release rates that can be attained in heart and brain mitochondria (3). The true rate available in yeast mitochondria may, of course, be still higher. The rapid rate of Ca²⁺ release suggests that the activity responsible participates in regulating cell Ca²⁺ levels in yeast. However, if the activity indeed exchanges Ca²⁺ for 2H⁺, it would seem that the pH gradient would have to reverse its normal orientation for a net accumulation of Ca²⁺ to occur. It remains to be determined whether this is possible in vivo.

As a final point, it is interesting to note that mitochondria are thought to have arisen from a symbiotic bacterium and that antiport-type Ca²⁺ transport activities are common in the envelope membrane of bacteria, whereas gated Ca²⁺ channels are not common (60). Considering yeast to be a primitive eucaryote, the present data then suggest that the earliest mitochondria were able to transport Ca²⁺ by an antiport activity alone. The Ca²⁺ uniporter, which is thought to be a gated Ca²⁺ channel (3, 61), must have arisen later, at a point in evolution that remains to be determined.

	FOOTNOTES

^* This work was supported by The Wallace Research Foundation, by American Heart Association Grant 9650626N, and by a grant from MitoKor, Inc. (San Diego, CA).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, Ohio State University, 1645 Neil Ave., Hamilton Hall 310A, Columbus, Ohio 43210-1218. Tel.: 614-292-8774; Fax: 614-292-4118; E-mail: pfeiffer.17@osu.edu.

Published, JBC Papers in Press, July 16, 2001, DOI 10.1074/jbc.M105062200

¹ Submitted for publication.

³ Manuscript in preparation.

⁴ The actions of cyclosporin A, a potent inhibitor of the permeability transition in mammalian mitochondria, are not considered here, because this compound does not inhibit the yPTP (8).

	ABBREVIATIONS

The abbreviations used are: BSA, bovine serum albumin; , membrane potential, FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TEA⁺, tetraethylammonium cation; yPTP, yeast mitochondrial permeability transition pore.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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