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Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria
AKCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 2, June, pp. 47’7-483, 1983
Isolation and Reconstitution of an A/-Ethylmaleimide-Sensitive Phosphate Transport Protein from Rat Liver Mitochondria’ JANNA
P. WEHRLE
AND
PETER L. PEDERSEN’
Laboratory for Molecular and Cellular Bioenergetics, Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received October 19, 1982, and in revised form February
1, 1983
An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of Fi-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent M,‘s of 30,000 and 35,000. Labeling of the 35,000 M, band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, pchloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H’ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F,-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 M, component.
the Pi/H+ symport system is inhibited by N-ethylmaleimide (2, 3). This differential inhibitor sensitivity was used earlier in this laboratory (4) and then in other laboratories (5, 6) to selectively label the Pi/ H+ transporter. The major protein labeled with radioactive NEM was shown to have a molecular weight slightly exceeding 30,000 M,. in both liver and heart mitochondria. (7, 8). More recently, a number of brief communications or preliminary reports have indicated that the Pi/H+ transport system can be solubilized and substantially purified from both bovine and porcine heart
Mitochondria contain two transport systems for inorganic phosphate, a Pi/H’ symport system and a Pi/dicarboxylate antiport system (For a recent review, see Ref. (1). Both systems are sensitive to mercurial SH reagents like pCMB3 but only ‘This work was supported by National Science Foundation Grant PCM 78-13249 to P.L.P. ‘To whom all correspondence should be addressed. 3 Abbreviations used: DABS, diazobenzenesulfonate; DTT, dithiothreitol; NEM, N-ethylmaleimide; pCMB, p-chloromercuribenzoate; IMV, inner membrane vesicles; SDS, sodium dodecyl sulfate; DATD, N,N’-diallyltartardiamide. 477
0003-9861/83 Copyright All rights
$3.00
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478
WEHRLE
AND
mitochondria (8-12) and from flight muscle mitochondria (8). The best preparations take advantage of the observation of Riccio et al. (13) that during purification of the ADP/ATP transporter some mitoafter solubilization chondrial proteins, with detergent, do not bind to hydroxylapatite powder. The ADP/ATP and Pi/H’ transport system appear to share this property (8, 12, 13). In this paper we describe a procedure developed for rat liver mitochondria for the isolation of a protein with both Pi transport activity and inhibitor sensitivities characteristic of the inner membrane Pi/H+ symport system. Unlike the procedures used for heart, and flight muscle (8), we found it necessary to commence the purification in the rat liver system with purified inner membrane vesicles (rather than intact mitochondria) and then to remove the Fi-ATPase with urea (14). Detergent treatment could then be employed to solubilize the Pi carrier and purification effected by using hydroxylapatite powder. The final preparation contains two major Coomassie blue-staining bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of the two bands exhibiting an M, of 35,000 has properties characteristic of Pi/H+ symport system. EXPERIMENTAL
PROCEDURES
Materials [%Y\r-ethylmaleimide and [35S]sulfanilic acid were purchased from New England Nuclear and used as provided. [‘*P]Orthophosphoric acid, also from New England Nuclear, was incubated for 3 h in 1 N HCl at lOO”C, diluted with KPI, and neutralized prior to use. Bio-Gel HTP hydroxylapatite powder was purchased from Bio-Rad Laboratories, as were all electrophoresis reagents except urea (ultrapure, Schwarz Mann) and ampholytes (LKB). Triton X-100 (scintillation grade) was purchased from Research Products International. Asolectin (soybean phosphatides) was purchased from Associated Concentrates. DABS was synthesized as described by Dilley et aL (15). All other chemicals were reagent grade and were used as provided.
Methods Preparation ofurea 1MV. Purified inner membrane vesicles (IMV) were prepared from fresh rat liver
PEDERSEN mitochondria exactly as described by Wehrle et al. (16). Urea IMV (depleted of F,-ATPase) were prepared as described by Pedersen and Hullihen (14). Urea IMV in volumes of 1 ml or less could be frozen in liquid Nz for several months or until needed. Isolation of phosphate transport protein. Urea IMV were washed twice by homogenization and centrifugation in 120 mM NaCl, 10 mM Nap,, 0.1 mM EDTA, pH 7.2, then resuspended in the same buffer to a protein concentration of 10 mg/ml. Sufficient 25% Triton X-100 (v/v) was added to give a final detergent concentration of 1.4%. The mixture was vortexed and allowed to stand for 5 min. Dry Bio-Gel HTP (0.5 g/ ml of solubilized membrane) was added, mixed thoroughly, and allowed to stand 5 min, then centrifuged at 27,OOOgfor 15 min. The supernatant was removed carefully and passed over a lo-ml (bed volume) column of G 25 Sephadex (1.5 X 10 cm) equilibrated with buffer C (10 mM NaPi, 10 mM Tris-Cl, 2 mM MgC&, pH 7.2), eluting with the same buffer. This preparation of phosphate transport protein could be stored for several days at 0-5’C. Freezing resulted in irreversible precipitation of protein. The entire isolation procedure was carried out at 0-5°C with the exception of the G 25 Sephadex step which was carried out at room temperature. Preparation of liposomes. Asolectin was purified before use and stored under Nz as the chloroform solution at 20°C. For preparations of liposomes, aliquots (7.5 pmol of lipid phosphorus) were dried under Nz in stainless-steel tubes, redissolved in ether, and dried under Nz followed by vacuum. Buffer C (2.0 ml) was added to one tube, the suspension sonicated at 0-5”C, 30 s/min for 15 min. This suspension was then transferred to a second tube of dry phospholipids and the additional phospholipid sonicated under the same conditions. The resulting suspension was saturated with N, and stored frozen for 1 week or less. Recmstitutim of P, transpmt. Phosphate transport protein in buffer C (lo-15 pg of protein) was mixed with preformed liposomes in buffer C and buffer C containing 32Pi. This mixture was quick frozen, partially thawed, then sonicated for 15 s with a sonicator microprobe. Assay of P, transpm-t. Pi efflux from phospholipid vesicles was assayed at 4°C essentially as described by Wohlrab (8). Vesicles reconstituted with buffer C containingZPi were diluted into nonradioactive buffer. At the times indicated, pCMB (250 nmol) was added and the entire reaction mixture applied to a lo-ml column (1 X 11 cm) of Dowex AG l-X8 (40- to 60mesh, Cl- form). Liposomes were eluted with 5% (v/ v) glycerol, 0.1 mM NaN,, and counted by liquid scintillation. Polyacllylamide gel electrophoresis. SDS slab gels (16% total acrylamide) were run using the procedure of O’Farrell (17), except that acrylamide was cross-
PHOSPHATE
TRANSPORT PROTEIN OF LIVER MITOCHONDRIA
479
linked with NJ’-diallyltartardiamide, DATD (1O:l) rather than methylene bisacrylamide. Gels were stained with Coomassie blue R-250. For determination of radioactivity unstained tracks were cut out, sliced in 2-mm pieces, incubated at 25°C for 30 min with 0.2 ml fresh 50% (w/v) NaI04, neutralized with HOAc, dark adapted, and counted by liquid scintillation in BudgetSolve. RESULTS
Isolation of a Phosphate Transport Fraction from Ra,t Liver Mitochondria Initial studies focussed on using the scheme devised by Wohlrab (8) for isolating a phosphate transport fraction from bovine heart and flight muscle mitochondria. In this scheme, whole mitochondria are solubilized with Triton X-100 and then treated with hydroxylapatite powder. The final scheme yields two protein bands upon polyacrylamide gel electrophoresis in SDS, one of 30,000 M,. which binds carboxyatractyloside and another of 34,000 M,. which appears to be (or to comprise) the Pi/H+ symport system (8) (Fig. 1). When applied to rat liver mitochondria, the Wohlrab procedure did in fact yield a substantially purified protein fraction containing peptides of molecular weights noted above (Compare Figs. 1A and 1B). However, the final fraction as shown in Fig. 1B was consistently contaminated with a peptide of about 68,000 M,. This peptide could be readily eliminated when ureatreated inner membrane vesicles were used
FIG. 1. SDS-polyacrylamide gel electrophoresis patterns of (A) rat liver mitochondria, and (B) phosphate transport fraction prepared from rat liver mitochondria using the Wohlrab procedure (8). In addition to a 30,000 and 35,000 M, component, there is also a 68,000 M, component in this preparation.
FIG. 2. SDS-polyacrylamide gel electrophoresis patterns of (A) urea particles (i.e., inner membrane vesicles treated with urea) and (B) phosphate transport fraction prepared as described under Methods and as outlined in Fig. 1. It will be noted that this purification scheme eliminates the 68,000 M, com-
ponent. The 30,000and 35,000M, components remain.
as starting material. Such vesicles from which the F,-ATPase has been removed are already markedly enriched in transport activities relative to whole mitochondria, but still contain a number of electrophoretically distinct polypeptides (Fig. 2A). Triton treatment of these vesicles followed by hydroxylapatite addition yields in the supernatant a protein fraction containing only two protein bands upon polyacrylamide gel electrophoresis in SDS, one of 30,000 M,. and one of 35,000 M, (Fig. 2B). Evidence that the Isolated Fraction Contains an NEM-Sensitive Phosphate Transport Protein Reconstitution of the isolated protein fraction into preformed phospholipid vesicles was accomplished by the freeze-thawsonication procedure (8). As shown in Fig. 3, chromatography of the reconstitution mixture on Sephadex G-100 indicates that 60-62s of the protein in various reconstitutions was actually incorporated into vesicles. Most of the reconstituted vesicles appear in the excluded volume although a small fraction is included. Pi transport mediated by the incorporated protein fraction was assayed by monitoring retention of Pi in the vesicles. As illustrated in Fig. 4, vesicles prepared without protein, or with protein isolated from N-ethylmaleimide-treated membranes retained a substantially higher
480
WEHRLE
AND
tor sensitivities very similar to the mitochondrial Pi/H+ symport system. In addition, they indicate that essential SH groups of the transporter are oriented outward in the reconstituted vesicles where t,hey are readily accessible to the impermeant inhibitors pCMB and DABS.
Void VOlUmC
10
b SPlOIC 1 (WI 6*
42-
-x-x’ I
I 10
J
yx-
-X
kA I 20 Fraction
PEDERSEN
I 30 Number
I 40
I
FIG. 3. Chromatography of liposomes reconstituted with the purified phosphate transport fraction on Sephadex G-200. Reconstitution was carried out exact,ly as described under Methods. Chromatography was then carried out on a 25-ml (bed volume) column of Sephadex G-100 (1 X 12 cm) using Buffer C.
amount of 32Pi after dilution than did vesicles prepared with active transport protein. It is clear from these results that the protein fraction isolated as described above contains an NEM-sensitive protein which promotes release of Pi from phospholipid vesicles.
Evidence that the 35,000 M, Band Contains the NEWSensitive Phosphate Transport Protein The impermeant membrane probe diazobenzene sulfonate is known to inhibit the Pi/H+ symporter via interaction at or very close to the critical SH group (19). Results presented in Fig. 5 show that labeling of the 35,000 M, species by DABS is prevented by prior treatment of inner membrane vesicles with NEM. No other inner membrane protein, including the 30,000 M, species, is protected from DABS labeling by NEM. These results, together with the inhibitor studies described above indicate that the 35,000 iIf, band contains
Evidence that the Isolated Fraction Contains the Pi/Hi Sympwt System When reconstituted into phospholipid vesicles, the phosphate transport fraction exhibits inhibitor sensitivities almost identical to those exhibited by the native Pi/H’ symport system. In intact mitochondria, the latter system is inhibited not only by N-ethylmaleimide (NEM) but by the impermeant mercurial agent p-chloromercuribenzoate (pCMB), and by the impermeant membrane probe diazobenzenesulfonate (DABS). As shown in Table I, Pi release from vesicles reconstituted with the isolated phosphate transport fraction is inhibited also by all three inhibitors. Inhibition by NEM and DABS is substantially slower than inhibition by mercurials due to the presence of Pi in the medium. In intact mitochondria, Pi is known to affect inhibition of the Pi/H+ symport system by NEM (6) and DABS (18). These studies show that the isolated fraction contains a reconstitutively active phosphate transport protein with inhibi-
I
I
EDpoLALp--.~ E E 1.0 a .-&
x
-X 1-.--..
x
st _______. (--------------, 0.5
1.0 t(min)
1.5
2.0
FIG. 4. Pi efhux from reconstituted liposomal vesicles catalyzed by the purified phosphate transport fraction. Transporter protein was reconstituted with liposomes and “Pi efflux assayed as described under Methods. The assay was initiated when the reconstituted vesicles containing 32Piwere placed in a dilute Pi medium. At the given time points, &MB (250 nmol) was added and the entire reaction mixture placed on a Dowex AG l-X8 column. 32Piwhich remains in the vesicles is eluted from the column whereas free Pi adheres to the anion exchange resin. l , Control, no vesicles (all 32Piadheres to the column, none is eluted); X, vesicles reconstituted with the purified phosphate transport fraction (-75% of the trapped 32Piis released in less than 0.5 min); A, vesicles without the Pi transporter fraction; 0, vesicles reconstituted with the Pi transport fraction isolated from inner membrane vesicles treated with NEM exactly as described in Fig. 5.
PHOSPHATE
the NEM-sensitive protein.
TRANSPORT
phosphate
PROTEIN
OF LIVER
TABLE
transport INHIBITION
Content of the 35,000 MT Protein Liver Mitochmdm’a
OF RECONSTITUTED
TRANSPORT
In experiments reported here, we summarize the first description of a substantially purified NEM-sensitive phosphate transport protein from rat liver mitochondria. We have demonstrated also that the fraction containing this protein when reconstituted into liposomal vesicles has inhibitor sensitivities identical to the Pi/ H+ symport system (Table I). In addition, the use of these inhibitors, two of which are impermeant (i.e., pCMB and DABS, (16,
600
Transport protein + pCMB (1 nmol/mg), 1’ + NEM (1 nmol/mg), 1’ + NEM (1 nmol/mg), 10 + DABS (1 nmol/mg), 15 NEM transport proteinb
J
100 0 94 6 0 0
a Reconstitution and transport assays were exactly as described under Methods. Reconstituted vesicles in a2Pi buffer C were incubated for the indicated times with inhibitors at 4°C prior to dilution and ion exchange chromatography. b Transport protein isolated from NEM-treated IMV.
20), indicate that essential SH groups of the transport system are oriented outward in the reconstituted vesicles. This phosphate transport protein of 35,000 M, has been purified almost 2000fold relative to intact rat liver mitochondria (Table II). The final fraction also contains a 30,000 M, species which comprises about 80% of the Coomassie dye staining material (Fig. 3), in analogy to the bovine heart preparation reported in a recent brief communication by Wohlrab (8). This 30,000 M, component has been shown in the bovine heart preparation to be (or to comprise) the ADP/ATP transport system.
TABLE BOTTOM
ACTIVITY”
Pi efflux (% control)
Treatment
DISCUSSION
t
I
in Rat
Data presented in Table II show that for every 300 mg of starting mitochondrial protein about 0.170 mg of 35,000 M, component is obtained. This constitutes less than 0.1% of the starting mitochondrial protein or about 17 pmol of 35,000 M, component/mg of rat liver mitochondria. This value representing a “yield” should also represent the minimal content of the phosphate transport protein in rat liver mitochondria.
TOP
481
MITOCHONDRIA
II
YIELD OF 35,000 M, COMPONENT FROM RAT LIVER MITOCHONDRIA
35K
FIN. 5. Inhibition of DABS labeling by iv-ethylmaleimide. IMV (25 mg/ml) in H medium were incubated with, n , or without, 0, N-ethylmaleimide (20 nmol/mg) for 10 min at O”C, washed with 1 mM DTT in H medium, centrifuged, and resuspended in H medium. IMV or NEM-IMV (25 mg/ml) were incubated with [“SIDABS (20 nmol/mg) for 10 min at 0°C washed with 1 mM DTT in H medium, centrifuged, and resuspended in Buffer A. Membranes were solubilized, electrophoresed, and radioactivity was assayed as described under Methods.
Fraction Mitochondria Inner membrane vesicles Urea particles Hydroxylapatite supernatant 35,000 M, band
Protein (mg) 300 60 43 0.85
0.17”
Percentage yield (100) 20 14 0.28 0.057
a Estimated from Coomassie blue staining to constitute 20% of the hydroxylapatite supernatant.
482
WEHRLE
AND
It is important to emphasize that in order to achieve a phosphate transport fraction from rat liver mitochondria, comparable to those obtained from bovine heart and flight muscle (8), it was essential to commence the preparation with Fi-depleted inner membrane vesicles (urea particles) rather than intact mitochondria. If intact mitochondria were used to initiate the purification an 68,000 M,. protein consistently appeared together with the 30,000 and 35,OOOM, components in the final preparation (Fig. 1). Since rat liver mitochondria are more metabolically complex than both bovine heart and flight muscle mitochondria, it seems likely that the 68,000 M, species may represent a subunit of an enzyme found only in liver mitochondria. It can be roughly estimated that the 35,000 M,. component catalyzes at 4°C a rate of Pi efflux from reconstituted liposomal vesicles of 8300 nmol/min/mg. This calculation is based on the following numbers: at least 1 nmol of Pi effluxes from the vesicles in 0.1 min (Fig. 4); 10 pg of Pi carrier fraction is mixed with the vesicles with an incorporation efficiency of 60%; 20% of the Pi carrier fraction is 35,000 M, component based on Coomassie blue staining (Fig. 2B). The efflux rate estimated is most likely a minimum figure because initial rates of efflux have yet to be followed carefully. Despite the fact that the modified Wohlrab procedure employed in this study provides a substantial purification of the Pi/ H+ transport system, it is clear that additional purification is in order before a homogeneous, Pi/H+ symport system can be achieved. The major question is how much additional purification will be required to achieve a homogeneous Pi/H+ symport system. An obvious first step would be to remove the 30,000 M, component. Recently, in a brief communication Kolbe et al. (12) claim to have done this with a porcine heart preparation using celite. However, their final preparation showing a single band in standard SDS gels showed four components upon gradient SDS-gel electrophoresis. In a more
PEDERSEN
recent brief communication, however, they indicate that these multiple bands may arise because of proteolytic cleavage of the native Pi/H+ carrier (21). Thus far, we have been unsuccessful in using celite to remove the 30,000 M, component. Previous estimates of the content of Pi carrier in rat liver mitochondria based on NEM labeling studies range from 30 to 60 pmol/mg (4, 5). The procedure described here yields about 0.17 mg of 35,000 M, component (~5 nmol) from 300 mg rat liver mitochondria (Table II), or about 17 pmol/ mg mitochondrial protein. Assuming that about 300 mg (protein) of mitochondria can be prepared from the liver of a single rat, it may be possible to prepare -1 mg of Pi/H+ carrier from no more than five to six animals. A two- to threefold purification of the preparation described in this report (i.e., by removal of the 30,000 M, component) may be all that is necessary to achieve a purified Pi/H+ carrier. Prior to carrying out any further studies on a partially purified preparation, which may provide ambiguous results, it will be our objective to first purify the rat liver Pi/H+ symport system to homogeneity. ACKNOWLEDGMENT We are grateful to Dr. Ronald S. Kaplan ically reading this manuscript.
for crit-
REFERENCES P. L., AND WEHRLE, J. P. (1982) ill Membranes and Transport (Martonosi, A., ed.), Vol. 1, pp. 645-663, Plenum, New York.
1. PEDERSEN,
2. FONYB, A., AND BESSMAN, S. P. (1966) Biochem. Biophys. Res. Cornmun. 24, 61-66. 3. CHAPPELL, J. B. (1968) Brit. Med. Bull 24, 150-
157. 4. COTY, W. A., AND PEDERSEN, P. L. (1975) J. Biol Chem. 250, 3515-3521. 5. HADVARY, P., AND KADENBACH, B. (1976) Eur. J B&hem. 67, 573-581. 6. BRIAND, Y., TOURAILLE, S., DEBISE, R., AND DURAND, R. (1976) FEB.3 Ldt. 65, 1-7. 7. COTY, W. A., AND PEDERSEN, P. L. (1974) J. BioL Chem. 249, 2593-2598. 8. WOHLRAB, H. (1980) J. Bid. Chem. 255,8170-8173. 9. BANERJEE, R. K., SHERTZER, H. G., KANNER, B. I., AND RACKER, E. (1977) B&hem. Biophys. Res. Commun. 75. 772-778.
PHOSPHATE
10. BANERJEE,
B&hem. 11. WOHLRAB,
TRANSPORT
PROTEIN
R. K., AND RACKER, 2, 203-205.
E. (1979) Membr.
H.,
N. (1982)
AND FLOWERS,
16. WEHRLE,
J. Biol.
12. KOLBE, H. V., BOTTRICH, J., GENCHI, G., PALMIERI, F., AND KADENBACH, B. (1981) FEBS Lett. 124, 265-269. 13. RICCIO, P., AWUILA, H., AND KLINGENBERG, M. (1975) FEBS Left. 56, 133-138. 14. PEDERSEN, P. L., AND HIJLLIHEN, J. (1978) J. Biol.
Ch,em. 253, 2176-2183. R. A., PETERS, G. A., AND SHAW, E. R. J.
(1972) Memtrr. Biol. 8, 163-180.
483
MITOCHONDRIA
J. B., CINTR~)N,
P. L. (1978)
Chem. 257, 28-31.
15. DILLEY,
OF LIVER
N. M., AND PEDERSEN,
J. Biol. Chem. 253, 8598-8603.
P. H. (1975) J. Biol. Chem. 250,4007-
17. O’FARRELL,
4021. Biochem. J. 144, 597-599.
18. DAWSON,
A. P. (1974)
19. WEHRLE,
J. P., AND PEDERSEN,
Bioenerg.
Biomembr.
P. L. (1981)
J.
13, 285-294.
20. BERG, H. C. (1969) Biochim.
Biophys.
Acta. 183,
65-78. 21. KADENBACH,
B., KOLBE,
H. V. J., AND MENDE,
(1982) i-n Second European ference 2, pp. 463-464.
Bioenergetics
P.
Con-
Isolation and Reconstitution of an A/-Ethylmaleimide-Sensitive Phosphate Transport Protein from Rat Liver Mitochondria’ JANNA
P. WEHRLE
AND
PETER L. PEDERSEN’
Laboratory for Molecular and Cellular Bioenergetics, Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received October 19, 1982, and in revised form February
1, 1983
An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of Fi-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent M,‘s of 30,000 and 35,000. Labeling of the 35,000 M, band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, pchloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H’ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F,-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 M, component.
the Pi/H+ symport system is inhibited by N-ethylmaleimide (2, 3). This differential inhibitor sensitivity was used earlier in this laboratory (4) and then in other laboratories (5, 6) to selectively label the Pi/ H+ transporter. The major protein labeled with radioactive NEM was shown to have a molecular weight slightly exceeding 30,000 M,. in both liver and heart mitochondria. (7, 8). More recently, a number of brief communications or preliminary reports have indicated that the Pi/H+ transport system can be solubilized and substantially purified from both bovine and porcine heart
Mitochondria contain two transport systems for inorganic phosphate, a Pi/H’ symport system and a Pi/dicarboxylate antiport system (For a recent review, see Ref. (1). Both systems are sensitive to mercurial SH reagents like pCMB3 but only ‘This work was supported by National Science Foundation Grant PCM 78-13249 to P.L.P. ‘To whom all correspondence should be addressed. 3 Abbreviations used: DABS, diazobenzenesulfonate; DTT, dithiothreitol; NEM, N-ethylmaleimide; pCMB, p-chloromercuribenzoate; IMV, inner membrane vesicles; SDS, sodium dodecyl sulfate; DATD, N,N’-diallyltartardiamide. 477
0003-9861/83 Copyright All rights
$3.00
IL 1983 by Academic Press, Inc. of reproduction in any form reserved
478
WEHRLE
AND
mitochondria (8-12) and from flight muscle mitochondria (8). The best preparations take advantage of the observation of Riccio et al. (13) that during purification of the ADP/ATP transporter some mitoafter solubilization chondrial proteins, with detergent, do not bind to hydroxylapatite powder. The ADP/ATP and Pi/H’ transport system appear to share this property (8, 12, 13). In this paper we describe a procedure developed for rat liver mitochondria for the isolation of a protein with both Pi transport activity and inhibitor sensitivities characteristic of the inner membrane Pi/H+ symport system. Unlike the procedures used for heart, and flight muscle (8), we found it necessary to commence the purification in the rat liver system with purified inner membrane vesicles (rather than intact mitochondria) and then to remove the Fi-ATPase with urea (14). Detergent treatment could then be employed to solubilize the Pi carrier and purification effected by using hydroxylapatite powder. The final preparation contains two major Coomassie blue-staining bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of the two bands exhibiting an M, of 35,000 has properties characteristic of Pi/H+ symport system. EXPERIMENTAL
PROCEDURES
Materials [%Y\r-ethylmaleimide and [35S]sulfanilic acid were purchased from New England Nuclear and used as provided. [‘*P]Orthophosphoric acid, also from New England Nuclear, was incubated for 3 h in 1 N HCl at lOO”C, diluted with KPI, and neutralized prior to use. Bio-Gel HTP hydroxylapatite powder was purchased from Bio-Rad Laboratories, as were all electrophoresis reagents except urea (ultrapure, Schwarz Mann) and ampholytes (LKB). Triton X-100 (scintillation grade) was purchased from Research Products International. Asolectin (soybean phosphatides) was purchased from Associated Concentrates. DABS was synthesized as described by Dilley et aL (15). All other chemicals were reagent grade and were used as provided.
Methods Preparation ofurea 1MV. Purified inner membrane vesicles (IMV) were prepared from fresh rat liver
PEDERSEN mitochondria exactly as described by Wehrle et al. (16). Urea IMV (depleted of F,-ATPase) were prepared as described by Pedersen and Hullihen (14). Urea IMV in volumes of 1 ml or less could be frozen in liquid Nz for several months or until needed. Isolation of phosphate transport protein. Urea IMV were washed twice by homogenization and centrifugation in 120 mM NaCl, 10 mM Nap,, 0.1 mM EDTA, pH 7.2, then resuspended in the same buffer to a protein concentration of 10 mg/ml. Sufficient 25% Triton X-100 (v/v) was added to give a final detergent concentration of 1.4%. The mixture was vortexed and allowed to stand for 5 min. Dry Bio-Gel HTP (0.5 g/ ml of solubilized membrane) was added, mixed thoroughly, and allowed to stand 5 min, then centrifuged at 27,OOOgfor 15 min. The supernatant was removed carefully and passed over a lo-ml (bed volume) column of G 25 Sephadex (1.5 X 10 cm) equilibrated with buffer C (10 mM NaPi, 10 mM Tris-Cl, 2 mM MgC&, pH 7.2), eluting with the same buffer. This preparation of phosphate transport protein could be stored for several days at 0-5’C. Freezing resulted in irreversible precipitation of protein. The entire isolation procedure was carried out at 0-5°C with the exception of the G 25 Sephadex step which was carried out at room temperature. Preparation of liposomes. Asolectin was purified before use and stored under Nz as the chloroform solution at 20°C. For preparations of liposomes, aliquots (7.5 pmol of lipid phosphorus) were dried under Nz in stainless-steel tubes, redissolved in ether, and dried under Nz followed by vacuum. Buffer C (2.0 ml) was added to one tube, the suspension sonicated at 0-5”C, 30 s/min for 15 min. This suspension was then transferred to a second tube of dry phospholipids and the additional phospholipid sonicated under the same conditions. The resulting suspension was saturated with N, and stored frozen for 1 week or less. Recmstitutim of P, transpmt. Phosphate transport protein in buffer C (lo-15 pg of protein) was mixed with preformed liposomes in buffer C and buffer C containing 32Pi. This mixture was quick frozen, partially thawed, then sonicated for 15 s with a sonicator microprobe. Assay of P, transpm-t. Pi efflux from phospholipid vesicles was assayed at 4°C essentially as described by Wohlrab (8). Vesicles reconstituted with buffer C containingZPi were diluted into nonradioactive buffer. At the times indicated, pCMB (250 nmol) was added and the entire reaction mixture applied to a lo-ml column (1 X 11 cm) of Dowex AG l-X8 (40- to 60mesh, Cl- form). Liposomes were eluted with 5% (v/ v) glycerol, 0.1 mM NaN,, and counted by liquid scintillation. Polyacllylamide gel electrophoresis. SDS slab gels (16% total acrylamide) were run using the procedure of O’Farrell (17), except that acrylamide was cross-
PHOSPHATE
TRANSPORT PROTEIN OF LIVER MITOCHONDRIA
479
linked with NJ’-diallyltartardiamide, DATD (1O:l) rather than methylene bisacrylamide. Gels were stained with Coomassie blue R-250. For determination of radioactivity unstained tracks were cut out, sliced in 2-mm pieces, incubated at 25°C for 30 min with 0.2 ml fresh 50% (w/v) NaI04, neutralized with HOAc, dark adapted, and counted by liquid scintillation in BudgetSolve. RESULTS
Isolation of a Phosphate Transport Fraction from Ra,t Liver Mitochondria Initial studies focussed on using the scheme devised by Wohlrab (8) for isolating a phosphate transport fraction from bovine heart and flight muscle mitochondria. In this scheme, whole mitochondria are solubilized with Triton X-100 and then treated with hydroxylapatite powder. The final scheme yields two protein bands upon polyacrylamide gel electrophoresis in SDS, one of 30,000 M,. which binds carboxyatractyloside and another of 34,000 M,. which appears to be (or to comprise) the Pi/H+ symport system (8) (Fig. 1). When applied to rat liver mitochondria, the Wohlrab procedure did in fact yield a substantially purified protein fraction containing peptides of molecular weights noted above (Compare Figs. 1A and 1B). However, the final fraction as shown in Fig. 1B was consistently contaminated with a peptide of about 68,000 M,. This peptide could be readily eliminated when ureatreated inner membrane vesicles were used
FIG. 1. SDS-polyacrylamide gel electrophoresis patterns of (A) rat liver mitochondria, and (B) phosphate transport fraction prepared from rat liver mitochondria using the Wohlrab procedure (8). In addition to a 30,000 and 35,000 M, component, there is also a 68,000 M, component in this preparation.
FIG. 2. SDS-polyacrylamide gel electrophoresis patterns of (A) urea particles (i.e., inner membrane vesicles treated with urea) and (B) phosphate transport fraction prepared as described under Methods and as outlined in Fig. 1. It will be noted that this purification scheme eliminates the 68,000 M, com-
ponent. The 30,000and 35,000M, components remain.
as starting material. Such vesicles from which the F,-ATPase has been removed are already markedly enriched in transport activities relative to whole mitochondria, but still contain a number of electrophoretically distinct polypeptides (Fig. 2A). Triton treatment of these vesicles followed by hydroxylapatite addition yields in the supernatant a protein fraction containing only two protein bands upon polyacrylamide gel electrophoresis in SDS, one of 30,000 M,. and one of 35,000 M, (Fig. 2B). Evidence that the Isolated Fraction Contains an NEM-Sensitive Phosphate Transport Protein Reconstitution of the isolated protein fraction into preformed phospholipid vesicles was accomplished by the freeze-thawsonication procedure (8). As shown in Fig. 3, chromatography of the reconstitution mixture on Sephadex G-100 indicates that 60-62s of the protein in various reconstitutions was actually incorporated into vesicles. Most of the reconstituted vesicles appear in the excluded volume although a small fraction is included. Pi transport mediated by the incorporated protein fraction was assayed by monitoring retention of Pi in the vesicles. As illustrated in Fig. 4, vesicles prepared without protein, or with protein isolated from N-ethylmaleimide-treated membranes retained a substantially higher
480
WEHRLE
AND
tor sensitivities very similar to the mitochondrial Pi/H+ symport system. In addition, they indicate that essential SH groups of the transporter are oriented outward in the reconstituted vesicles where t,hey are readily accessible to the impermeant inhibitors pCMB and DABS.
Void VOlUmC
10
b SPlOIC 1 (WI 6*
42-
-x-x’ I
I 10
J
yx-
-X
kA I 20 Fraction
PEDERSEN
I 30 Number
I 40
I
FIG. 3. Chromatography of liposomes reconstituted with the purified phosphate transport fraction on Sephadex G-200. Reconstitution was carried out exact,ly as described under Methods. Chromatography was then carried out on a 25-ml (bed volume) column of Sephadex G-100 (1 X 12 cm) using Buffer C.
amount of 32Pi after dilution than did vesicles prepared with active transport protein. It is clear from these results that the protein fraction isolated as described above contains an NEM-sensitive protein which promotes release of Pi from phospholipid vesicles.
Evidence that the 35,000 M, Band Contains the NEWSensitive Phosphate Transport Protein The impermeant membrane probe diazobenzene sulfonate is known to inhibit the Pi/H+ symporter via interaction at or very close to the critical SH group (19). Results presented in Fig. 5 show that labeling of the 35,000 M, species by DABS is prevented by prior treatment of inner membrane vesicles with NEM. No other inner membrane protein, including the 30,000 M, species, is protected from DABS labeling by NEM. These results, together with the inhibitor studies described above indicate that the 35,000 iIf, band contains
Evidence that the Isolated Fraction Contains the Pi/Hi Sympwt System When reconstituted into phospholipid vesicles, the phosphate transport fraction exhibits inhibitor sensitivities almost identical to those exhibited by the native Pi/H’ symport system. In intact mitochondria, the latter system is inhibited not only by N-ethylmaleimide (NEM) but by the impermeant mercurial agent p-chloromercuribenzoate (pCMB), and by the impermeant membrane probe diazobenzenesulfonate (DABS). As shown in Table I, Pi release from vesicles reconstituted with the isolated phosphate transport fraction is inhibited also by all three inhibitors. Inhibition by NEM and DABS is substantially slower than inhibition by mercurials due to the presence of Pi in the medium. In intact mitochondria, Pi is known to affect inhibition of the Pi/H+ symport system by NEM (6) and DABS (18). These studies show that the isolated fraction contains a reconstitutively active phosphate transport protein with inhibi-
I
I
EDpoLALp--.~ E E 1.0 a .-&
x
-X 1-.--..
x
st _______. (--------------, 0.5
1.0 t(min)
1.5
2.0
FIG. 4. Pi efhux from reconstituted liposomal vesicles catalyzed by the purified phosphate transport fraction. Transporter protein was reconstituted with liposomes and “Pi efflux assayed as described under Methods. The assay was initiated when the reconstituted vesicles containing 32Piwere placed in a dilute Pi medium. At the given time points, &MB (250 nmol) was added and the entire reaction mixture placed on a Dowex AG l-X8 column. 32Piwhich remains in the vesicles is eluted from the column whereas free Pi adheres to the anion exchange resin. l , Control, no vesicles (all 32Piadheres to the column, none is eluted); X, vesicles reconstituted with the purified phosphate transport fraction (-75% of the trapped 32Piis released in less than 0.5 min); A, vesicles without the Pi transporter fraction; 0, vesicles reconstituted with the Pi transport fraction isolated from inner membrane vesicles treated with NEM exactly as described in Fig. 5.
PHOSPHATE
the NEM-sensitive protein.
TRANSPORT
phosphate
PROTEIN
OF LIVER
TABLE
transport INHIBITION
Content of the 35,000 MT Protein Liver Mitochmdm’a
OF RECONSTITUTED
TRANSPORT
In experiments reported here, we summarize the first description of a substantially purified NEM-sensitive phosphate transport protein from rat liver mitochondria. We have demonstrated also that the fraction containing this protein when reconstituted into liposomal vesicles has inhibitor sensitivities identical to the Pi/ H+ symport system (Table I). In addition, the use of these inhibitors, two of which are impermeant (i.e., pCMB and DABS, (16,
600
Transport protein + pCMB (1 nmol/mg), 1’ + NEM (1 nmol/mg), 1’ + NEM (1 nmol/mg), 10 + DABS (1 nmol/mg), 15 NEM transport proteinb
J
100 0 94 6 0 0
a Reconstitution and transport assays were exactly as described under Methods. Reconstituted vesicles in a2Pi buffer C were incubated for the indicated times with inhibitors at 4°C prior to dilution and ion exchange chromatography. b Transport protein isolated from NEM-treated IMV.
20), indicate that essential SH groups of the transport system are oriented outward in the reconstituted vesicles. This phosphate transport protein of 35,000 M, has been purified almost 2000fold relative to intact rat liver mitochondria (Table II). The final fraction also contains a 30,000 M, species which comprises about 80% of the Coomassie dye staining material (Fig. 3), in analogy to the bovine heart preparation reported in a recent brief communication by Wohlrab (8). This 30,000 M, component has been shown in the bovine heart preparation to be (or to comprise) the ADP/ATP transport system.
TABLE BOTTOM
ACTIVITY”
Pi efflux (% control)
Treatment
DISCUSSION
t
I
in Rat
Data presented in Table II show that for every 300 mg of starting mitochondrial protein about 0.170 mg of 35,000 M, component is obtained. This constitutes less than 0.1% of the starting mitochondrial protein or about 17 pmol of 35,000 M, component/mg of rat liver mitochondria. This value representing a “yield” should also represent the minimal content of the phosphate transport protein in rat liver mitochondria.
TOP
481
MITOCHONDRIA
II
YIELD OF 35,000 M, COMPONENT FROM RAT LIVER MITOCHONDRIA
35K
FIN. 5. Inhibition of DABS labeling by iv-ethylmaleimide. IMV (25 mg/ml) in H medium were incubated with, n , or without, 0, N-ethylmaleimide (20 nmol/mg) for 10 min at O”C, washed with 1 mM DTT in H medium, centrifuged, and resuspended in H medium. IMV or NEM-IMV (25 mg/ml) were incubated with [“SIDABS (20 nmol/mg) for 10 min at 0°C washed with 1 mM DTT in H medium, centrifuged, and resuspended in Buffer A. Membranes were solubilized, electrophoresed, and radioactivity was assayed as described under Methods.
Fraction Mitochondria Inner membrane vesicles Urea particles Hydroxylapatite supernatant 35,000 M, band
Protein (mg) 300 60 43 0.85
0.17”
Percentage yield (100) 20 14 0.28 0.057
a Estimated from Coomassie blue staining to constitute 20% of the hydroxylapatite supernatant.
482
WEHRLE
AND
It is important to emphasize that in order to achieve a phosphate transport fraction from rat liver mitochondria, comparable to those obtained from bovine heart and flight muscle (8), it was essential to commence the preparation with Fi-depleted inner membrane vesicles (urea particles) rather than intact mitochondria. If intact mitochondria were used to initiate the purification an 68,000 M,. protein consistently appeared together with the 30,000 and 35,OOOM, components in the final preparation (Fig. 1). Since rat liver mitochondria are more metabolically complex than both bovine heart and flight muscle mitochondria, it seems likely that the 68,000 M, species may represent a subunit of an enzyme found only in liver mitochondria. It can be roughly estimated that the 35,000 M,. component catalyzes at 4°C a rate of Pi efflux from reconstituted liposomal vesicles of 8300 nmol/min/mg. This calculation is based on the following numbers: at least 1 nmol of Pi effluxes from the vesicles in 0.1 min (Fig. 4); 10 pg of Pi carrier fraction is mixed with the vesicles with an incorporation efficiency of 60%; 20% of the Pi carrier fraction is 35,000 M, component based on Coomassie blue staining (Fig. 2B). The efflux rate estimated is most likely a minimum figure because initial rates of efflux have yet to be followed carefully. Despite the fact that the modified Wohlrab procedure employed in this study provides a substantial purification of the Pi/ H+ transport system, it is clear that additional purification is in order before a homogeneous, Pi/H+ symport system can be achieved. The major question is how much additional purification will be required to achieve a homogeneous Pi/H+ symport system. An obvious first step would be to remove the 30,000 M, component. Recently, in a brief communication Kolbe et al. (12) claim to have done this with a porcine heart preparation using celite. However, their final preparation showing a single band in standard SDS gels showed four components upon gradient SDS-gel electrophoresis. In a more
PEDERSEN
recent brief communication, however, they indicate that these multiple bands may arise because of proteolytic cleavage of the native Pi/H+ carrier (21). Thus far, we have been unsuccessful in using celite to remove the 30,000 M, component. Previous estimates of the content of Pi carrier in rat liver mitochondria based on NEM labeling studies range from 30 to 60 pmol/mg (4, 5). The procedure described here yields about 0.17 mg of 35,000 M, component (~5 nmol) from 300 mg rat liver mitochondria (Table II), or about 17 pmol/ mg mitochondrial protein. Assuming that about 300 mg (protein) of mitochondria can be prepared from the liver of a single rat, it may be possible to prepare -1 mg of Pi/H+ carrier from no more than five to six animals. A two- to threefold purification of the preparation described in this report (i.e., by removal of the 30,000 M, component) may be all that is necessary to achieve a purified Pi/H+ carrier. Prior to carrying out any further studies on a partially purified preparation, which may provide ambiguous results, it will be our objective to first purify the rat liver Pi/H+ symport system to homogeneity. ACKNOWLEDGMENT We are grateful to Dr. Ronald S. Kaplan ically reading this manuscript.
for crit-
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1. PEDERSEN,
2. FONYB, A., AND BESSMAN, S. P. (1966) Biochem. Biophys. Res. Cornmun. 24, 61-66. 3. CHAPPELL, J. B. (1968) Brit. Med. Bull 24, 150-
157. 4. COTY, W. A., AND PEDERSEN, P. L. (1975) J. Biol Chem. 250, 3515-3521. 5. HADVARY, P., AND KADENBACH, B. (1976) Eur. J B&hem. 67, 573-581. 6. BRIAND, Y., TOURAILLE, S., DEBISE, R., AND DURAND, R. (1976) FEB.3 Ldt. 65, 1-7. 7. COTY, W. A., AND PEDERSEN, P. L. (1974) J. BioL Chem. 249, 2593-2598. 8. WOHLRAB, H. (1980) J. Bid. Chem. 255,8170-8173. 9. BANERJEE, R. K., SHERTZER, H. G., KANNER, B. I., AND RACKER, E. (1977) B&hem. Biophys. Res. Commun. 75. 772-778.
PHOSPHATE
10. BANERJEE,
B&hem. 11. WOHLRAB,
TRANSPORT
PROTEIN
R. K., AND RACKER, 2, 203-205.
E. (1979) Membr.
H.,
N. (1982)
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Ch,em. 253, 2176-2183. R. A., PETERS, G. A., AND SHAW, E. R. J.
(1972) Memtrr. Biol. 8, 163-180.
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Chem. 257, 28-31.
15. DILLEY,
OF LIVER
N. M., AND PEDERSEN,
J. Biol. Chem. 253, 8598-8603.
P. H. (1975) J. Biol. Chem. 250,4007-
17. O’FARRELL,
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18. DAWSON,
A. P. (1974)
19. WEHRLE,
J. P., AND PEDERSEN,
Bioenerg.
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P. L. (1981)
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13, 285-294.
20. BERG, H. C. (1969) Biochim.
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Bioenergetics
P.
Con-