Phosphate transport in rat liver mitochondria. Properties of a Ca2+-activated uptake process in inverted inner membrane vesicles.

The mechanisms by which Pi and Ca2’ leave the mitochondrial matrix and re-enter the cytoplasm have been studied in inverted vesicles of inner membrane. Such vesicles have the matrix face of the membrane exposed to the external medium and an internal medium of known composition, and so provide a well defined system for the study of transport in the direction of matrix to cytoplasm. Energy-dependent uptake of Pi into such inverted inner membrane vesicles (IMV) from rat liver mitochondria is stimulated by the presence of either endogenous or added Ca2+. During Ca’+-stimulated Pi uptake, Ca2+ is taken up into an ethylene glycol bis(&aminoethyl ether)N,N,N’,N’-tetraacetic acid-inaccessible internal space. Ca2+ uptake is absolutely dependent on the uptake of Pi. In the absence of Pi, Ca2+ added to respiring IMV is not taken up and produces no H’ ejection or respiratory stimulation. Other anions such as acetate, thiocyanate, nitrate, or bicarbonate cannot replace Pi, even at &fold higher concentrations. Inhibition of Pi transport with N-ethylmaleimide completely iihibits Ca2+ uptake. Uptake of Ca2+ by inverted IMV is completely inhibited by externally added ruthenium red under the conditions used, even after passage of IMV over a cytochrome c-Sepharose affinity column to remove any remaining right-side-out vesicles. Uptake is greatly increased by addition of ATP or ADP in the presence of M&‘, but not by the ATP analog adenyl-5’-yl imidodiphosphate. This stimulation is not prevented by either oligomycin or carboxyatractyloside. Prior uptake of Pi will support uptake of Ca2+ at least as well as simultaneous uptake, but under these conditions no extra Pi uptake is observed. This makes an obligatory symport mechanism unlikely. A model which is consistent with the data presented depicts the driving force for Ca2+ uptake as being depression of the interior-positive membrane potential by prior electrophoretic uptake of Pi, coupled with precipitation of complexes of calcium phosphate.

The mechanisms by which Pi and Ca2' leave the mitochondrial matrix and re-enter the cytoplasm have been studied in inverted vesicles of inner membrane. Such vesicles have the matrix face of the membrane exposed to the external medium and an internal medium of known composition, and so provide a well defined system for the study of transport in the direction of matrix to cytoplasm.
Energy-dependent uptake of Pi into such inverted inner membrane vesicles (IMV) from rat liver mitochondria is stimulated by the presence of either endogenous or added Ca2+. During Ca'+-stimulated Pi uptake, Ca2+ is taken up into an ethylene glycol bis(&aminoethyl ether)N,N,N',N'-tetraacetic acid-inaccessible internal space. Ca2+ uptake is absolutely dependent on the uptake of Pi. In the absence of Pi, Ca2+ added to respiring IMV is not taken up and produces no H' ejection or respiratory stimulation.
Other anions such as acetate, thiocyanate, nitrate, or bicarbonate cannot replace Pi, even at &fold higher concentrations.
Inhibition of Pi transport with N-ethylmaleimide completely iihibits Ca2+ uptake. Uptake of Ca2+ by inverted IMV is completely inhibited by externally added ruthenium red under the conditions used, even after passage of IMV over a cytochrome c-Sepharose affinity column to remove any remaining right-side-out vesicles. Uptake is greatly increased by addition of ATP or ADP in the presence of M&', but not by the ATP analog adenyl-5'-yl imidodiphosphate.
This stimulation is not prevented by either oligomycin or carboxyatractyloside. Prior uptake of Pi will support uptake of Ca2+ at least as well as simultaneous uptake, but under these conditions no extra Pi uptake is observed.
This makes an obligatory symport mechanism unlikely. A model which is consistent with the data presented depicts the driving force for Ca2+ uptake as being depression of the interior-positive membrane potential by prior electrophoretic uptake of Pi, coupled with precipitation of complexes of calcium phosphate.
Since the first observations of mitochondrial ion transport, a link has been apparent between accumulation of Pi and Ca" (1, 2). The minimum possible interaction is defined by the equilibrium constant for complexation and precipitation of * This work was supported by Grant PCM 7813249 from the National Science Foundation and by funds from the Biomedical Research Support Grant, The Johns Hopkins University School of Medicine. 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. the various forms of calcium Pi. Whether there is, in addition, direct or indirect coupling of Ca'+ and Pi transport has been under study for more than a decade.
Uptake of Pi independent of Ca'+ movement has been characterized extensively in mitochondria.
All but a small fraction of the total uptake in liver appears to be coupled to influx of protons via an N-ethylmaleimide-and mercurialsensitive protein carrier (3). In contrast, uptake of Ca'+ appeared at first to be completely dependent on P, (1,2). This substantial energy-dependent uptake has been designated "massive loading" (4). The ability of mitochondria to take up a smaller amount of Ca'+ in the absence of added Pi, "limiting loading" (4, 5), has since received extensive study. This is the classical ruthenium red-and La"'-sensitive electrophoretic carrier-mediated Ca'+ uptake (6)(7)(8). A controversy currently exists over the net charge of the mobile Ca'+, with certain authors supporting a fully electrical Ca'+ movement accompanied by ejection of an equivalent amount of charge as H' (9)(10)(11)(12)(13). In contrast, Moyle and Mitchell have suggested that CaL' moves only together with P, (14,15) or certain monocarboxylate anions (16), accounting for their observation of an apparent Cal+ transfer.
Until recently the previously characterized pathways for Ca" and Pi uptake were considered to be totally responsible for controlling matrix concentrations of these ions (17,18). However, with the observation that the Ca"' (or Mn'+) distribution between matrix and cytosol may not be in equilibrium with the membrane potential (19,20), the movement of Ca"+ exclusively on a single, reversible, electrophoretic carrier has been questioned. Instead it has been suggested that separate influx and efflux pathways may exist (21-25), which maintain Ca'+ distribution at a steady-state, displaced from thermodynamic equilibrium, but necessary for proper cell function. Recently it has been shown by direct measurement in inverted inner membrane vesicles that in the absence of Ca'+ or ionophores P, can be driven at the expense of respiratory energy from the matrix to the cytosolic surface of the inner mitochondrial membrane (26). The objective of the current studies was to examine the interaction of Ca'+ with this P, transport mechanism and to gain information on matrix-to-cytosol Ca'+ movement as well.
Uptake of Ca2+ together with Pi into vesicles of mitochondrial inner membrane has been reported previously by Loyter et al. (27,28) and by Pedersen and Coty (29), but these results have been criticized because of the possibility of contamination of inverted with noninverted vesicles (30). However, since the existence of a second Casi transport system specialized for efflux is now being considered (21-25), it seemed important to re-evaluate whether Ca'+ can, in fact, be taken up by inverted inner membrane vesicles. A preliminary report by Gunter et al. (31) has described uptake of Ca'+ under conditions where the authors indicate that only inverted vesicles 7270 Phosphate and Ca2' Uptake by Inverted Mitochondrial Vesicles would be energized. To attack this problem we have used an inner membrane vesicle preparation from rat liver mitochondria which has been shown previously in this laboratory to be essentially completely inverted, using six different criteria (26). We report here that in these vesicles Ca"+ stimulates the energy-dependent uptake of Pi. Further, we demonstrate transport of Ca" from the matrix to the cytosolic surface and describe the properties of this uptake system. In an experiment shown the number of replicates is indicated. In all cases error is approximately &l nmol/mg.

RESULTS
As shown in Fig. 1, the preparation of IMV used for these studies contains a population of vesicles which are at least 95% inverted. Fig. 1A demonstrates, following the method of Wehrle et al. (26), that 96% of the total FCCP-stimulated ATPase activity of IMV can be inhibited by the peptide inhibitor from rat liver, which has a molecular weight of 12,300 (36). Fig. 1B demonstrates, following the method of Hackenbrock and Hammon (39), that respiration with succinate is not stimulated by addition of cytochrome c to IMV. In contrast, the mitoplasts from which the IMV were prepared are clearly deficient. Respiration is susceptible to stimulation by cytochrome c. The binding sites for cytochrome c, which are well established as being at the cytoplasmic surface of the inner membrane, are partially vacant but are inaccessible in these vesicles. Five other independent methods have also indicated that the inner membrane vesicles used for this study are essentially completely inverted (26).
In the course of studies on the uptake of Pi by inverted IMV, it became clear that Pi uptake was substantially increased in the presence of added Ca2+ (Table I). In fact, part of the P, uptake observed in the absence of added cation appears to depend on endogenous Ca2', as indicated by decreased uptake in the presence of EGTA. Experiments were performed to determine whether Ca'+ was actually entering the vesicles, or whether Ca2+ was only acting from the outside, either by precipitating Pi externally, or by causing some   Fig. 2 shows that Ca"+ is removed from the medium by IMV in response to respiration and that this occurs only in the presence of P,. Uptake is relatively slow (-30 to 40 nmol of Ca'+/min/mg) and linear for several minutes until anaerobiosis, which initiates efflux. As will be demonstrated again later, the Ca'+ uptake observed under these conditions is sensitive to ruthenium red in the external medium. That Ca'+ uptake by IMV is different from uptake by intact noninverted mitochondria can be seen in Fig. 3. In the absence of added P,, rat liver mitochondria take up Ca'+, with a characteristic H' ejection and transient stimulation of respiration (13). In contrast, inverted IMV show neither of these characteristics, and in the absence of Pi take up no Ca" (Fig. 2, Table II).
The dependence of Ca"' uptake on the concentrations of added Ca" and P, are shown in Fig. 4  what the rate-limiting step is in this complex reaction. Halfmaximal Ca2+ uptake appears to require about 50 PM calcium added, but the concentration of uncomplexed Ca2+ in the presence of the P, is undoubtedly much less. The P, concentration curve shows a distinct optimum at around 0.1 mM P,. This was found in Ca"+ electrode studies as well (not shown). Slightly higher P, concentrations (0.5 m) were routinely used, because of the greater reproducibility observed in the "plateau" region. Under the assay conditions of Table II net Ca2+ uptake is specific for P, among the anions tested. Neither permeant anions (NOs-, SCN-) nor permeant weak acids (OAc-, HCOZ-) can replace Pi, at least at similar concentrations.
Identical results were obtained using the Ca2+ electrode to avoid EGTA and centrifugation.
ATP and ADP, even in the presence of high levels of oligomycin, substantially increase the amount of Ca2+ taken up by inverted IMV (Table III). In contrast, the ATP analog AMP-PNP does not stimulate Ca2' uptake. The stimulation by adenine nucleotides appears to be completely dependent on added Mg'+, and it is insensitive to carboxyatractyloside, a potent inhibitor of the adenine nucleotide translocator.
The effect of oligomycin itself is seen in Table IV. For an equal time (5 min) the control and oligomycin-containing assays demonstrate the same amount of energy-dependent Ca*' uptake (in this case 16 to 17 nmol/mg), but this does not take into account the inhibition of vesicle respiration by oligomycin (26). While using the same amount of oxygen as in the oligomycin-containing incubation (in only 2.2 min), IMV without oligomycin could accumulate only 6 nmol of Ca*'/mg. Thus oligomycin does have a significant coupling effect even though no increase in net Ca2+ uptake is observed in the 5min assay.
As can be seen in Table IV, Ca*+ uptake is completely sensitive to inhibition by N-ethylmaleimide, an inhibitor of energy-dependent Pi transport in both mitochondria and inverted IMV (26). In addition, the uptake described here is sensitive to ruthenium red ( Fig. 2 and Table IV). This is true even if the IMV are eluted from an affinity column of cytochrome c bound to Sepharose. Any right-side-out vesicles will stick to the column (as verified with mitoplasts; see "Methods"). However, the vesicles which elute in the void volume of the column under low salt conditions still take up Ca'+. Although the uptake capacity is somewhat diminished, per-Phosphate and Ca2+ Uptake by Inverted Mitochondrial Vesicles 7273  IV   Effects of inhibitors  on Ca2+ uptake by IMV  IMV (different  preparations  for each experiment) were assayed for Ca'+ uptake as described in Table II, with the substrates  and additions  indicated. In Experiment 1: oligomycin added was 5 pg, 2.2.min incubation in the absence of oligomycin was accompanied by oxygen consumption equal to that in a 5-min incubation with oligomycin. In Experiment 2: IMV were preincubated with 50 nmol of N-ethylmaleimide/mg of protein for 2 min at 0°C before aliquots were assayed for Ca'+ uptake.
In Experiment 3: IMV were passed over an affinity column of cytochrome c bound to Sepharose 4B (see "Methods" for details) to remove any right-side-out vesicles before assay for Ca"' uptake in the presence or absence of ruthenium red (1 nmol/mg). Values corrected for nonenergy-dependent uptake and are averages of auadruulicates.

Incubation conditions
Respiration-dependent increase in Ca"+ content Pi. IMV were incubated as described in Table II with 5 mM K' succinate as substrate, except that potassium P, was 2 rnM and CaCb (0.5 mrvr) was added initially or after 5 min. In A, P, uptake is measured with initial (-0-) or delayed (-X-) Ca'+ addition. In B, Ca'+ uptake is measured with initial (-A-) or delayed (cl-) Ca2+ addition.
haps due to mechanical damage or fatty acid uncoupling, the ruthenium red inhibition is completely retained. The data in Table IV suggested that the previously described Pi transport mechanism (26) rather than a novel Ca2+ plus P, translocator is responsible for the Ca'+-stimulated P, movement observed here. In the experiments shown in Fig. 5 the movements of Ca2+ and P, are measured in identical incubations. It is obvious (Fig. 5A) that Cast added at the beginning of the incubation stimulates P, uptake. In Fig. 5B it can be seen that this is accompanied by uptake of Ca"+ into the EGTA-inaccessible intravesicular space. In contrast, Ca2+ added after a substantial amount of Pi has already been taken up does not stimulate a sudden burst of P, uptake even though the Ca2+ itself is taken up rapidly. This is not due simply to the order of addition. In experiments not shown, it was observed that, in a similar incubation under conditions where little net Pi had been accumulated in the first phase of the incubation (lower Pi concentration, shorter time), addition of Ca" induces a rapid Pi uptake whether added initially or later, and in either case Ca'+ was taken up as well. From this it appears clear that there is no requirement for simultaneous movement of the two ions. Ca'+ uptake can occur whether Pi has been taken up previous to Ca"' addition or is taken up together with Ca'+. The stimulation of P, uptake by Ca2', in contrast, occurs only if the vesicles have not previously accumulated a substantial amount of P,.

DISCUSSION
The experiments described here show that energy-dependent uptake of Pi by inverted inner membrane vesicles can be stimulated by the addition of Ca'+. Under these conditions Ca"+ is also taken up. It leaves the medium (Fig. 2) and enters an EGTA-inaccessible space (Table II) within the vesicle. This is not uptake by vesicles retaining the right-side-out orientation of intact mitochondria. In this paper (Fig. 1) and previously (26) the IMV used here have been shown by a number of independent criteria to be at least 95% inverted. IMV which are not retained on a cytochrome c affinity column still demonstrate Ca"+ uptake (Table IV). Furthermore, the Ca"' uptake in IMV differs in several respects from that in intact mitochondria. First, the requirement for P, is absolute ( Fig. 2, Table II). There is no limited loading (4) of Ca2+ in the absence of Pi. Uptake of Ca2+ is completely abolished by the Pi transport inhibitor N-ethylmaleimide, in contrast to the results in mitochondria (13). Second, the Ht ejection and transient stimulation of respiration typical of mitochondrial Ca2' uptake is entirely absent in IMV (Fig. 3).
The fact that the Ca2' uptake observed in inverted IMV is sensitive to the inhibitor ruthenium red (Fig. 2, Table IV) may indicate that under the conditions used (the precise internal milieu, external medium) the classical electrogenic carrier is responsible for Ca'+ movement. This carrier does appear to promote a ruthenium red-sensitive efflux of Ca'+ from mitochondria under a variety of conditions, including addition of EGTA (19) or phosphoenolpyruvate (40). However, it is important to note that in the present studies ruthenium red is added to the matrix surface of the membrane, a situation that never occurs in studies of Ca2+ efflux from intact mitochondria.
Efflux of Ca2+ under other conditions does appear insensitive to ruthenium red added at the cytosolic surface (8,41). This has suggested to some authors (21-25, 40) the involvement of a second, and somewhat different, Ca2+ carrier in efflux. However, it is impossible to say czpriori what effect ruthenium red may have on any carrier or carriers when added to the matrix surface. It might also be significant to note that in these studies (as opposed to studies of efflux from mitochondria) that Cazt and ruthenium red are added at the same surface, or that ruthenium red is added to a membrane surface at which a negative potential is found (see, for instance, 7274 Phosphate and Ca" Uptake by Inverted Mitochondrial Vesicles these concentrations could be attained easily within the matrix space depending on the degree to which Ca"+ is bound. 14 RLM Given the small internal volume of the IMV (1 pl/mg), internal complexation process undoubtedly also is occurring and probably provides the main driving force for net accumulation (uptake minus release) of Ca"' and of Ca'+-stimulated extra Pi. Although complexation of calcium and phosphate may not be a physiological driving force for CaZt or Pi extrusion from the mitochondrion, it is a convenient aid for revealing the existence of a potential efflux pathway. The inability of SCNor NO:%-to support Ca" accumulation emphasizes the importance of Ca'+ complexation in the overall Ca" uptake process measured here, as anions have been shown to enter inverted IMV in response to respiration (26). The inability of HC03to support uptake indicates again that a process different from g IMV mitochondrial uptake is occurring in inverted IMV. Stimulation of Pi-dependent Ca'+ uptake by ATP and ADP is substantial (Table III). Although the similar effect on mitochondrial calcium plus Pi uptake has been ascribed to the ability of ATP to stabilize calcium:Pi granules (42), such an effect can be ruled out in the present studies. ADP cannot be phosphorylated to ATP in the presence of high levels of oligomycin. Carboxyatractyloside, a powerful inhibitor of the adenine nucleotide translocator, has no effect on the stimulation. The site involved is clearly on the matrix surface of the membrane. Because it is the Mg"' complexes which are active, nonspecific permeability increase due to chelation of necessary Mg'+ by nucleotide can also be ruled out. That Ca'+ uptake by inverted IMV requires transported Pi as opposed to Pi in the external medium or externally bound P, is indicated by the complete abolition of Ca2+ uptake by Nethylmaleimide (Table IV). Although a direct effect of Nethylmaleimide on Cal+ transport cannot be ruled out, inhibition of P, transport both by Pi plus H+ electroneutral symport (3) and by electrophoretic Pi uniport (26) are known to be sensitive to this SH group reagent. That Ca*' transport is not directly coupled to cotransport of Pi is suggested by Fig.  5. Pi accumulated in the absence of Ca2' can support Ca2+ uptake at least as well as Pi taken up at the same time as Ca'+. Uptake of Ca" mto P,-preloaded vesicles does not stimulate further Pi uptake. These data, together with the classical inhibitor sensitivities of Ca"' uptake to ruthenium red and N-ethylmaleimide, permit construction of a tentative model to describe the matrix-to-cytoplasm directed transport observed in IMV.
internal proton, which would simultaneously provide a chemical gradient for CaZt accumulation and partial charge compensation.
A completely electroneutral Ca/2 H' exchange might be expected to support some Ca** uptake in the absence of Pi, but other constraints might apply to prevent observable levels of uptake, so the data here cannot be taken to rule out such exchange. In any case precipitation of calcium:Pi is clearly necessary to pull Ca2+ uptake. Other anions are ineffective. The simplest possible model for the observed uptake of Ca'+ and Pi which is consistent with the data is shown in Fig.  6, which also includes the classical model of Ca'+ and Pi uptake by mitochondria (43). The present results in no way rule out the presence of other transport systems or more complex regulation or both. In the intact mitochondrion ( Fig. 6A) electron transport generates an interior-negative membrane potential. Ca2+ is taken up electrophoretically, probably with two positive charges. The resulting decrease in the membrane potential causes increased respiration and protons are ejected, compensating for the positive charge introduced by Ca*', until the matrix becomes excessively alkaline. Endogenous COZ, as HC!G'-, may provide some internal acidification (44). Added P,, taken up by electroneutral proton symport, neutralizes interior alkalinity and supports the uptake of large additional amounts of Ca2+ (5).
In contrast, in inverted IMV (Fig. 6B) respiration induces an interior-positive membrane potential and Ca2+ cannot enter at all in the absence of anion. Pi is free to enter electrophoretically (26). This causes a decline in the membrane potential and Ca'+ may enter, fully charged, or perhaps in exchange for The presence of two independent Ca2+ transport mechanisms, considered necessary to explain the apparent displacement of the matrix/cytosol Ca2+ gradient from equilibrium (23,45) is not inconsistent with the present data. The carrier functioning under the conditions used here is sensitive to ruthenium red at its matrix face. Whether this represents the action of the classical electrophoretic carrier or a novel carrier has not been determined.
What carrier is involved in physiological efflux is equally unclear. The possible involvement of an N-ethylmaleimide-insensitive Ca2+ plus Pi symporter (14,15) can, however, be ruled out by the data (Table IV and Fig.  5). A Na+/Ca2+ exchange such as that described for mitochondria from heart and certain other tissues (46,47) appears not to be present in liver (47) and would therefore appear to have no role in the Ca*+ and P, movements described here. (Also, Na' was specifically excluded from all solutions.) The possibility of an electroneutral Ca2+/2 H+ exchanger has been discussed in connection with Ca2+ efflux from rat liver and Ehrlich ascites cell mitochondria (41). Akerman (48) has reported an acid-pulse-induced Ca*+ efflux from rat liver mitochondria.
Tsuchiya and Rosen (49, 50) have described a Ca'+/H+ exchange system in inverted bacterial vesicles. The need for more study of the mechanism of Pi and Ca2+ influx and efflux, and the regulation of steady-state ion levels in mitochondria and whole cells is apparent. Since the completion of the work reported here, a brief communication has appeared in which the uptake of Ca2+ by IMV is described (51). As in earlier studies (27-29) and in the present work, such uptake requires the uptake of Pi. The IMV