Proc. NatL. Acad. Sci. USA
Vol. 74, No. 10, pp. 4185-4189, October 1977 Biochemistry
Location of protein(s) involved in oligomycin-induced inhibition of mitochondrial adenosinetriphosphatase near the outer surface of the inner membrane* (oxidative phosphorylation/trypsin/membrane topology)
HAMMOU MAiROUCH AND CATHERINE GODINOTt Laboratoire de Biologie et Technologie des Membranes du Centre National de la Recherche Scientifique, Universite Claude Bernard de Lyon, 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne, France
Communicated by Henry Lardy, July 11, 1977
ABSTRACT Mitoplasts, that is, mitochondria freed from their outer membranes, were prepared from pig heart. Sonication induced an inversion of these mitoplasts, giving inside-out vesicles. Added cytochrome c can be bound much better to mitoplasts than to sonicated vesicles; addition of trypsin increased adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) activity of sonicated vesicles without significantly affecting that of the mitoplasts. Since the site of fixation of cytochrome c was located on the outer side of the inner mitochondrial membrane and since the protein inhibitor of the mitochondrial ATPase is present on the inner face of the inner membrane and is very sensitive to trypsin, it can be concluded that mitoplasts are mainly oriented as normal mitochondria while sonicated vesicles are mainly inverted. Trypsin treatment can abolish the oligomycin sensitivity of ATPase activity of either mitoplasts or sonicated vesicles. However, trypsin induced the solubilization of the soluble F1ATPase of sonicated vesicles while the ATPase activity remained with the mitoplasts after t sin action. Therefore, trypsin destroyed the oligomycin effect rupturing the liaison between F1 and the membrane in sonicated vesicles. On the other hand, the effect of trypsin on mitoplasts must be attributed to the hydrolysis of a protein located near the outer surface of the inner membrane that is at least structurally involved in the oligomycin sensitivity of the ATPase complex. ATP synthesis and hydrolysis in mitochondria are catalyzed by enzymes intimately associated with the mitochondrial membrane structure (1-3). Extensive work reviewed recently (4-8) defines the complete adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) complex: It consists of (i) a headpiece called factor F1 made of five different polypeptides catalyzing ATPase activity and located on the matrix face of the inner mitochondrial membrane (9); (ii) a membrane sector which is believed to guide the flow of protons to F1 during oxidative phosphorylation (10-12); (Mii) an ATPase inhibitor peptide (13); and (iv) one or two other peptides, OSCP (oligomycinrsensitivity conferring protein) and F6 (14-16), involved in the binding of F1 to the membrane sector. ATP synthesis and hydrolysis are inhibited by oligomycin in mitochondria or in the complete ATPase complex (17), but not in isolated F1 (18). The oligomycin target is thought to be located in the membrane sector (19), and a correct binding of F1 to the membrane factor through OSCP and/or F6 (15, 16) is necessary to observe the oligomycin inhibition. The data presented in this paper indicate that a peptide susceptible to trypsin attack and involved in oligomycin sensitivity of ATPase activity is located on or near the surface of
the inner mitochondrial membrane which is exposed towards the cytoplasm. EXPERIMENTAL SECTION Membrane Preparations. Pig hearts obtained from the slaughterhouse and brought to the laboratory in ice were used within 30 min after the electrocution of the animals. Mitochondria were isolated by a procedure derived from that of Crane et al. (20), washed twice, and tested for respiratory control ratios, protein concentration, and ADP/O, as done previously (21). Purified mitoplasts were obtained as described by Maisterrena et al. (22): Washed mitochondria (400 mg of protein) were homogenized in 400 ml of 10 mM potassium phosphate at pH 7.4 and 0° and allowed to swell for 20 min. Then, the suspension was centrifuged at 105,000 X g for 60 min. The pellets were resuspended in 0.25 M sucrose/10 mM TrisHCI at pH 7.4 and centrifuged at 11,500g for 15 min in order to separate the outer membranes from the mitoplasts (inner membranes and matrix). To obtain sonicated vesicles, mitoplasts homogenized at 0° in 0.25 M sucrose/10 mM Tris-HCI at pH 7.4 at 20 mg of protein per ml were sonicated three times for 10 sec each time in 5-ml aliquots with a Branson sonifier B12. Care was taken to prevent the temperature from rising above 80 during sonication. Unbroken mitoplasts were centrifuged at 25,000 X g for 10 min. Sonicated vesicles were collected after centrifugation at 78,000 X g for 90 min and the pellets were suspended in 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 and 0°. The absence of outer membranes and the intactness of the mitoplasts were checked as before (22) by electron microscopy and by the lack of monoamine oxidase activity. ATPase. Oligomycin-insensitive F1-ATPase was prepared according to the procedure of Senior and Brooks (23) as modified earlier (24). A partially purified oligomycin-sensitive ATPase was obtained by washing the mitochondrial membranes (40 mg of protein) successively with 10 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4, containing 1 mg of Lubrol WX per ml and with the same medium containing 0.05% Triton X-100. After centrifugation (105,000 X g for 20 min), the pellets were extracted for 20 min with the same medium containing 0.2% Triton X-100 and the suspension was centrifuged at 105,000 X g for 20 min; the supernatant fluid contained the oligomycin-sensitive ATPase and was used as such without any further purification. Proteins were estimated by the Lowry procedure (25)
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. §1734 solely to indicate this fact.
work is part of the "These de Specialit6" in Biochemistry of H.M. t To whom correspondence should be addressed. * This
4185
4186
Biochemistry: Mairouch and Godinot
PProc. Natl. A Acad. Sci. UUSA 74 (1977)
Table 1. Comparative activities of enzymes involving cytochrome c in mitoplasts and sonicated vesicles Mmol substrate transformed/min per mg protein Enzyme activity Mitoplasts Sonicated vesicles NADH oxidase - Cytochrome c 0.20 0.86 + Cytochrome c 0.47 0.88 Succinate-cytochrome c reductase 0.12 0.05 Cytochrome oxidase - Lubrol 1.36 0.65 + Lubrol* 1.53 1.51 * 1 mg/mg of protein.
induced a high NADH oxidase activity, but no stimulation of NADH oxidase by cytochrome c could be detected. Succinate-cytochrome c reductase and cytochrome oxidase activities are low, and the latter is enhanced 2.3 times by the addition of Lubrol WX. All these observations are in agreement with those of Huang et al. (34), and indicate that these vesicles represent "inside-out" fragments that have a membrane orientation the opposite of that found in normal mitochondria. On the contrary, NADH oxidase activity of mitoplasts is low and increases in the presence of added cytochrome C; succinate-cytochrome oxidase and cytochrome oxidase activities are much higher than with sonicated vesicles; furthermore, the addition of Lubrol barely improves the cytochrome oxidase activity. All these experiments indicate a great accessibility of externally added cytochrome c to these mitoplasts and show that they have essentially retained the normal polarity of intact
modified by Wang and Smith (26) to eliminate Triton X-100 interference, when this detergent was present. Enzyme Assays. The rate of NADH oxidation was determined by oxypolarography in a Gilson Oxygraph equipped with a Clark electrode. Reaction mixture (1.8 ml) contained 0.1-0.4 mg of membrane proteins, and 66 mM potassium phosphate buffer (pH 7.4) with or without 0.1 mM cytochrome c. The reaction was initiated by the addition of 1 mM NADH. Temperature was 30°. Succinate-cytochrome c reductase was assayed spectrophotometrically and cytochrome oxidase (EC 1.9.1.3) polarographically as done by Sottocasa et al. (27). Monoamine oxidase (EC 1.4.3.4) was tested by the technique of Schnaitman and Greenawalt (28) after a modification of the spectrophotometric procedure of Tabor et al. (29). ATPase activity was determined by a spectrophotometric method using pyruvate kinase and lactate dehydrognease as auxiliary enzymes (24). Activity of trypsin (EC 3.4.21.4) was measured using N-ap-toluenesulfonyl-L-arginine methyl ester as substrate according to Rick (30). All reagents were of the purest available grade. Nucleotides and most enzymes were obtained from Boehringer. Trypsin (crystallized from bovine pancreas, Type III or Type XI), oligomycin, soybean trypsin inhibitor, and N-a-p-toluenesulfonyl-L-arginine methyl ester were from Sigma Chemical Co. All other reagents were obtained from Merck or Prolabo.
mitochondria. ATPase. Fig. 1 shows that trypsin treatment barely affects the ATPase activity of mitoplasts while it strongly increases the rate of ATP hydrolysis by sonicated vesicles. The stimulation of ATPase activity by trypsin depends on the trypsin concentration: 20 Atg of trypsin does not increase the ATPase activity of mitoplasts (1 mg of protein); a weak activation, 0.9-1.8 ,4mol of ATP hydrolyzed/min per mg of protein, is obtained in 1 hr with mitoplasts in the presence of 200 ,ug of trypsin. The same trypsin concentration increases by more than 10 times the
RESULTS AND DISCUSSION Orientation of mitoplasts and sonicated vesicles The differential orientation of the mitoplasts and of the sonicated vesicles was proved by measuring the activity of enzymes using cytochrome c and by looking at the effect of trypsin on the ATPase activity. Cytochrome c. Experiments from several laboratories (31, 32) have firmly established that the physiological location for cytochrome c is on the membrane surface normally oriented toward the cytoplasm. Moreover, cytochrome c can be easily removed from the membrane by hypotonic treatment (33). Our technique of mitoplast preparation includes a swelling in hypotonic phosphate buffer which should then remove, at least partly, the cytochrome c from its site. If the membrane is normally oriented, added cytochrome c can bind to its sites and the activity of enzymes involving cytochrome c must increase. On the contrary, if the membrane is inside out, added cytochrome c can no longer reach its binding sites and must have no effect on the activity of enzymes using cytochrome c. Table 1 shows a typical experiment reproduced with three different membrane preparations: Sonification of mitoplasts
ATPase activity of sonicated vesicles. To obtain reproducible results, it is important to check the specific activity of trypsin. Batches obtained from different manufacturers have greatly varying activities. The trypsin used here (from Sigma) could split 0.3-0.36 mmol of N-a-toluenesulfonyl-L-arginine methyl ester/min per mg in 10 mM CaCl2/40 mM Tris-HCI buffer at pH 8.1. Control experiments, in which mitoplasts or sonicated vesicles were incubated under similar conditions either in the absence of trypsin or in the presence of trypsin plus trypsin inhibitor, show no significant modification of the rate of ATP hydrolysis. Trypsin also increases the rate of ATP hydrolysis by oligomycin-sensitive ATPase solubilized from mitochondria in the presence of Triton X-100. Under the experimental conditions described here, trypsin (up to 200 ,ug/mg of Fl-ATPase) does not change the specific activity of the F1-ATPase prepared by a method (24) similar to that of Senior and Brooks (23), which provides an enzyme free of ATPase inhibitor (35). If the trypsin concentration reaches 500 ,g/mg of Fl-ATPase, the ATPase activity decreases. Racker et al. (32) demonstrated that F1-ATPase is oriented towards the matrix side of the membrane, and that exposure of sonicated particles to trypsin increased the rate of ATP hydrolysis 5- to 10-fold (36) because the ATPase inhibitor is easily attacked by trypsin (13, 37, 38) and that trypsin reaches this inhibitor if the membranes are "inside-out." The fact that trypsin barely increases the ATPase activity of the mitoplasts while it strongly stimulates the ATPase activity of the sonicated vesicles speaks again in favor of a membrane polarity of the mitoplasts similar to that of normal mitochondria and of an opposite orientation for the sonicated vesicles. Cytoplasmic orientation of a protein involved in oligomycin sensitivity of ATPase activity Fig. 2 shows that trypsin diminishes the percentage of inhibition of ATPase activity by oligomycin as well with the solubilized oligomycin-sensitive ATPase as with mitoplasts or sonicated vesicles. Controls made in the absence of trypsin or in the presence of trypsin inhibitor show a slight diminution of the
Biochemistiy:
Proc. Nati. Acad. Sci. USA 74 (1977)
Mairouch and Godinot
Mitoplasts Trypsin + inhibitor
3 2
F 1 ATPase
80 - Trypsin ± inhibitor 60
1
-i0 M itoplasts -
50
40 20 50 Oligomycin-sensitive ATPase Trypsin + inhibitor
._
01
E
1
E ,, 3 E
0
50 Sonicated vesicles Trypsin + inhibitor
5 4
11 50
'o
0 30 90 50 )Iigomycin-sensitive ATPase Trypsin
I-E 0 4 0
E
10
aw S a
a.5
0
1 1.25 1.25 1.15
0.06 0.06 0.09 0.12
0.09 0.6 1.05 0.8
0.03 0.06 0.09 0.12
0.16 4.4 5.5
0.13 1.2 3.6
Sonicated vesicles
2F I
N
Mitoplasts 0 30 60 90
3
._
I
Table 2. Effect of trypsin on oligomycin inhibition and solubilization of ATPase activity in mitoplasts and sonicated vesicles ATPase activity, umol ATP hydrolyzed/min No oligomycin Oligomycin Time of action Soluble Total Soluble Total of trypsin, min
Trypsin
4a
4187
0 50 Time of trypsin incubation, min
50
FIG. 1. Comparison of the effects of trypsin on ATPase activity of mitoplasts, sonicated vesicles, oligomycin-sensitive ATPase, and F1-ATPase. Mitoplasts, sonicated vesicles, and oligomycin-sensitive ATPase (1 mg of protein) were incubated in 1 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 at 300 in the presence of various concentrations of trypsin and in the presence or absence of trypsin inhibitor. At the indicated times 10-Al aliquots were removed and their ATPase activity measured spectrophotometrically at 300 in 1 ml of medium containing 40 mM Tris-SO4, 3 mM MgSO4, 4 mM phosphoenolpyruvate, 0.3 mM NADH, 2 mM Na2S, 2 mM ATP, 20 Mg of lactate dehydrogenase, and 30 g of pyruvate kinase at pH 7.5. Fj-ATPase (1 mg) was incubated in the presence of trypsin ± trypsin inhibitor in 0.2 M phosphate buffer/5 mM EDTA at pH 7.5. ATPase activity was determined on -Mgl aliquots. Trypsin concentration per ml: 0, 2 Mg; 200 yg; v, 500 ug. Open symbols, trypsin only; closed A, 20 ,g; symbols; trypsin + trypsin inhibitor (concentration five times higher than that of trypsin). Specific activity of trypsin was 320 gmol of N-a-toluenesulfonyl-L-arginine methyl ester/min per mg of protein (in 10 mM CaCl2/40 mM Tris.HCl at pH 8.1). 0,
sensitivity of ATPase activity during incubation at 300 of all enzyme preparations, but this diminution is in no way comparable to that obtained in the presence of trypsin. Table 2 demonstrates that a striking difference exists between mitoplasts and sonicated vesicles. With sonicated vesicles the ATPase activity becomes partly released to the supernatant fraction upon trypsin treatment while the ATPase activity of the mitoplasts remains associated with the membrane fraction even if the oligomycin sensitivity diminishes. Control experiments show no solubilization of ATPase activity of mitoplasts or sonicated vesicles either in the absence of trypsin or in the presence of trypsin plus trypsin inhibitor.
2.3 4.8 6.6
0.20 1.4 3.8
Mitoplasts and sonicated vesicles (1 mg of protein) were incubated at 300 in 1 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 in the presence of 0.2 mg of trypsin (78 Mmol of N-a-toluenesulfonyl-Larginine methyl ester split per min), as in Fig. 1. At the indicated times, aliquots were removed and trypsin inhibitor was added. ATPase activity in the presence or absence of oligomycin (1 ug) was measured either directly on these aliquots or in the supernatant fractions obtained after centrifugation in a Beckman Airfuge (airdriven ultracentrifuge) for 10 min at 100,000 X g. Similar results were obtained with two other experiments.
Trypsin diminishes the sensitivity of the ATPase to oligomycinlinthe mitoplastsaswellasiinthe sonicated vesicles. In the sonicated vesicles, the F1-ATPase is solubilized from the membrane. This solubilization of ATPase by trypsin was observed in other laboratories (37, 39) and can be explained by the great susceptibility to trypsin of OSCP (14) and of the F6 factor (15, 16), which have been reported to be the peptides involved in the binding of the factor F1 to the membrane factor Fo. Indeed, the integrity of these factors (OSCP and F6) and the correct attachment of the enzyme to the membrane are necessary to observe the inhibition of ATPase by oligomycin. Although the complete solubilization of the F1-ATPase appears more slowly than the oligomycin sensitivity disappears, it is likely that the functional properties of the peptides that bind the F1-ATPase to the membrane can be damaged before the total release of the FI-ATPase in the supernatant fraction. When mitoplasts were exposed to trypsin action, the sensitivity of ATPase to oligomycin progressively diminished. This indicates that a protein sensitive to trypsin and at least structurally involved in the oligomycin sensitivity is located near the outer surface of the mitochondrial inner membrane. The decrease in oligomycin sensitivity occurs without solubilization of the ATPase activity from the mitoplasts. This fact does not prove that the Fj-ATPase is still bound to the membrane since it could be solubilized within the matrix. However, the following experiment indicates that the FI-ATPase was not solubilized within the matrix. During the trypsin action, aliquots of mitoplasts were treated with 0.4 mg of Lubrol WX per mg of protein and centrifuged in a Beckman Airfuge; the super-
natant fraction was tested for ATPase activity. No increase in ATPase activity could be detected in this soluble fraction obtained after Lubrol treatment. At least 95% of the malate dehydrogenase activity was solubilized from the mitoplasts by this Lubrol treatment. In the absence of Lubrol, no change in the percentage of malate dehydrogenase activity could be detected in the supernatant fraction after 1 hr of trypsin treatment (200 Ag of trypsin per mg of mitoplasts). This indicates that extensive proteolysis, which would allow soluble proteins to pass through
4188
Biochemistry: Mairouch and Godinot
Proc. Natl. Acad. Sci. USA 74 (1977)
75
75
A-wlers
C .0
Oligomycin-sensitive ATPase EE 50 Trypsi n 0 + trypsin inhibitor 0I
50
D 25
25
75 Mitoplasts Trypsin + trypsin inhibitor
Sonicated vesicles Trypsin 550 + trypsin inhibitor I
25 F
.
4a 0.
50
.*.
0
A
50
75 I
c
0
:2 50 I C
25
50 Time of trypsin incubation, min
FIG. 2. Effects of trypsin on the percentage of inhibition by oligomycin of ATPase activity in mitoplasts, sonicated vesicles, and oligomycin-sensitive ATPase. Conditions are the same as in Fig. 1 except that the ATPase activity was measured at each indicated time in the presence and absence of 1 jg of oligomycin to calculate the percentage of oligomycin inhibition of ATPase activity.
the membrane, had not occurred during trypsin treatment. The fact that there is no significant increase in ATPase activity during treatment of mitoplasts by trypsin is another argument in favor of the relative intactness of the membrane. If the membrane was damaged, trypsin would have reached the ATPase inhibitor and the ATPase activity should have been
of the oligomycin sensitive-ATPase and destroy the oligomycin sensitivity of the complex.
increased. Juntti et al. (40), studying the oligomycin sensitivity of
nancial support was obtained from the Centre National de la Recherche Scientifique (ATP 2224) and from the Delegation Generale a la Recherche Scientifique et Technique (Contract 74-7-0183).
ATPase activity in submitochondrial particles of different orientation, could not detect any effect on this sensitivity using 20-40 Itg of trypsin per mg of protein in 10-min incubations. This might be related to the specific activity of the trypsin used, which may have been lower than the one used here. In conclusion, this study indicates that oligomycin inhibition is observed only, on one hand, when a peptide (or peptides) located somewhere near the outer surface of the inner membrane is intact and/or functional and, on the other hand, when the binding of F1 to the membrane is correct. These conclusions are in agreement with genetic studies (41, 42), which have shown that there are several genes coding for oligomycin sensitivity and therefore that there must be several peptides involved in this inhibition and a correct assembly of those peptides. Attempts to purify the oligomycin sensitive-ATPase complex from trypsinized mitoplasts have not been successful. Indeed, the peptide maps of mitoplasts are not very different after trypsin treatment than before it, but Triton X-100 extracts of mitoplasts contain many more peptides after trypsin treatment than before. Therefore, the oligomycin sensitive-ATPase cannot be purified from trypsinized mitoplasts in the same way as it may be from normal mitochondria. Further work is needed to purify this complex and decide which peptide(s) of the membrane have been digested by trypsin in order to test whether an Fo protein is really damaged or if the cleavage of another peptide in the membrane could affect the overall organization
We thank Prof. D. C. Gautheron for her valuable discussions and continuous interest in this research. We also thank Drs. J; Comte and D. Goldschmidt for their help and advice at the beginning of this study and F. Penin for his expert assistance in some of the experiments. Fi-
1. Fassenden-Raden, J. M. & Racker, E. (1971) in Structure and Function of Biological Membranes, ed. Rothfield, L. I. (Academic Press, New York), pp. 401-438. 2. Packer, L. (1972) Bioenergetics 3, 115-127. 3. Chance, B. (1972) FEBS Lett. 23,3-20. 4. Senior, A. E. (1973) Biochim. Biophys. Acta 801, 249-277. 5. Penefsky, H. S. (1974) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), Vol. 10, pp. 375-394. 6. Pedersen, P. L. (1975) Bioenergetics 6,243-275. 7. Kagawa,Y. (1974) in Methods in Membrane Biology, ed. Korn, E. D. (Plenum Press, New York), Vol. 1, pp. 201-269. 8. Gautheron, D. C. & Godinot, C. (1977) in Living Systems as Energy Convertors, eds. Buvet, R. & Massue, J. P. (North-Holland Publ. Co., Amsterdam), pp. 89-102. 9. Pullman, M. E., Penefsky, H. S., Datta, A. & Racker, E. (1960) J. Biol. Chem. 235,3322-3328. 10. Mitchell, P. (1973) FEBS Lett. 33,267-272. 11. Mitchell, P. (1974) FEBS Lett. 43, 189-193. 12. Hinkle, P. & Horstman L. L. (1971) J. Biol. Chem. 246,60246028. 13. Pullman, M. E. & Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769. 14. McLennan, D. H. & Tzagoloff, A. (1968) Biochemistry 7, 1603-1610. 15. Russel, L. K., Kinkley, S. A., Kleyman, T. R. & Chan, Sh. P. (1976) Biochem. Biophys. Res. Commun. 73,434-443. 16. Kanner, B. I., Serrano, R., Kandrach, A. & Racker, E. (1976) Biochem. Biophys. Res. Commun. 69,1050-1056.
Biochemistry: Mairouch and Godinot 17. Lardy, H. A., Johnson, D. & McMurray, W. C. (1958) Arch. Biochem. Biophys. 78,587-595. 18. Kagawa, Y. & Racker, E. (1966) J. Blol. Chem. 241, 24612467. 19. MacLennan, D. H. & Tzagoloff, A. (1968) Biochemistry 7, 1596-1603. 20. Crane, F. L., Glenn, J. & Green, D. E. (1956) Biochim. Blophys. Acta 135,599-613. 21. Godinot, C., Vial, C., Font, B. & Gautheron, D. (1969) Eur. J. Biochem. 8, 385-394. 22. Maisterrena, B., Comte, J. & Gautheron, D. (1974) Biochrm. Blophys. Acta 367, 115-126. 23. Senior, A. E. & Brooks, D. C. (1970) Arch. Biochem. Blophys. 140, 257-266. 24. Di Pietro, A., Godinot, C., Bouillant, M. L. & Gautheron, D. C. (1975) Biochimie 57, 959-967. 25. Lowry, 0. H., Rosebrough, N.J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 26. Wang, C. S. & Smith, R. L. (1975) Anal. Biochem. 63, 414417. 27. Sottocasa, G. L., Kuylenstierna, B., Ernster, L. & Bergstrand, A. (1967) J. Cell Blol. 32, 415-438. 28. Schnaitman, C. & Greenawalt, J. W. (1968) J. Cell Biol. 38, 158-175. 29. Tabor, C. W., Tabor, H. & Rosenthal, S. M. (1955) in Methods in Enzymology, eds. Kaplan, N. 0. & Colowick, S. P. (Academic Press, New York), Vol. 2, pp. 390-393. 30. Rick, W. (1974) in Methods of Enzymatic Analysis, ed. Bergmeyer, H. U. (Academic Press, New York), 2nd ed., Vol. 2, pp.
1021-1024.
Proc. Nati. Acad. Sc. USA 74 (1977)
4189
31. Harmon, H. J., Hall, J. D. & Crane, F. L. (1974) Blochim. BNophys. Acta 344,119-155. 32. Racker, E., Burstein, C., Loyter, A. & Christiansen, R. 0. (1970) in Electron Transport and Energy Conservation, eds. Tager, J. M., Papa, S., Quagliariello, E. & Slater, E. C. (Adriatica Editrice, Bari, Italy), pp. 235-241. 33. Jacobs, E. E. & Sanadi, D. R. (1960) J. Btol. Chem. 235, 531534. 34. Huang, C. H., Keyhani, E. & Lee, C. P. (1973) Biochim. Blophys. Acta 305, 455-473. 35. Brooks, J. C. & Senior, A. E. (1971) Arch. Blochem. Blophys. 147, 467-472. 36. Racker, E. (1963) Biochem. Blophys. Res. Commun. 10, 435439. 37. Horstman, L. L. & Racker, E. (1970) J. Biol. Chem. 245, 1336-1344. 38. Montecucco, C. & Azzi, A. (1975) J. Biol. Chem. 250, 50205025. 39. Knowles, A. F., Guillory, R. J. & Racker, E. (1971) J. Biol. Chem. 247,2351-2357. 40. Juntti, K., Torndall, V. B. & Ernster, L. (1970) in Electron Transport and Energy Conservation, eds. Tager, J. M., Papa, S., Quagliariello, E. & Slater, E. C. (Adriatica Editrice, Bari, Italy), pp. 257-270. 41. Avner, P. R., Coen, D., Dujon, B. & Slonimski, P. P. (1973) Mol.
Gen. Cenet. 125, 9-52. 42. Griffiths, D. E. (1975) in Genetics and Biogenesis of Mitochondria and Chloroplasts, eds. Birky, G. W., Pearlman, P. S. & Byers, J. J. (Ohio State University Press, Columbus, OH), pp. 117-135.
Vol. 74, No. 10, pp. 4185-4189, October 1977 Biochemistry
Location of protein(s) involved in oligomycin-induced inhibition of mitochondrial adenosinetriphosphatase near the outer surface of the inner membrane* (oxidative phosphorylation/trypsin/membrane topology)
HAMMOU MAiROUCH AND CATHERINE GODINOTt Laboratoire de Biologie et Technologie des Membranes du Centre National de la Recherche Scientifique, Universite Claude Bernard de Lyon, 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne, France
Communicated by Henry Lardy, July 11, 1977
ABSTRACT Mitoplasts, that is, mitochondria freed from their outer membranes, were prepared from pig heart. Sonication induced an inversion of these mitoplasts, giving inside-out vesicles. Added cytochrome c can be bound much better to mitoplasts than to sonicated vesicles; addition of trypsin increased adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) activity of sonicated vesicles without significantly affecting that of the mitoplasts. Since the site of fixation of cytochrome c was located on the outer side of the inner mitochondrial membrane and since the protein inhibitor of the mitochondrial ATPase is present on the inner face of the inner membrane and is very sensitive to trypsin, it can be concluded that mitoplasts are mainly oriented as normal mitochondria while sonicated vesicles are mainly inverted. Trypsin treatment can abolish the oligomycin sensitivity of ATPase activity of either mitoplasts or sonicated vesicles. However, trypsin induced the solubilization of the soluble F1ATPase of sonicated vesicles while the ATPase activity remained with the mitoplasts after t sin action. Therefore, trypsin destroyed the oligomycin effect rupturing the liaison between F1 and the membrane in sonicated vesicles. On the other hand, the effect of trypsin on mitoplasts must be attributed to the hydrolysis of a protein located near the outer surface of the inner membrane that is at least structurally involved in the oligomycin sensitivity of the ATPase complex. ATP synthesis and hydrolysis in mitochondria are catalyzed by enzymes intimately associated with the mitochondrial membrane structure (1-3). Extensive work reviewed recently (4-8) defines the complete adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) complex: It consists of (i) a headpiece called factor F1 made of five different polypeptides catalyzing ATPase activity and located on the matrix face of the inner mitochondrial membrane (9); (ii) a membrane sector which is believed to guide the flow of protons to F1 during oxidative phosphorylation (10-12); (Mii) an ATPase inhibitor peptide (13); and (iv) one or two other peptides, OSCP (oligomycinrsensitivity conferring protein) and F6 (14-16), involved in the binding of F1 to the membrane sector. ATP synthesis and hydrolysis are inhibited by oligomycin in mitochondria or in the complete ATPase complex (17), but not in isolated F1 (18). The oligomycin target is thought to be located in the membrane sector (19), and a correct binding of F1 to the membrane factor through OSCP and/or F6 (15, 16) is necessary to observe the oligomycin inhibition. The data presented in this paper indicate that a peptide susceptible to trypsin attack and involved in oligomycin sensitivity of ATPase activity is located on or near the surface of
the inner mitochondrial membrane which is exposed towards the cytoplasm. EXPERIMENTAL SECTION Membrane Preparations. Pig hearts obtained from the slaughterhouse and brought to the laboratory in ice were used within 30 min after the electrocution of the animals. Mitochondria were isolated by a procedure derived from that of Crane et al. (20), washed twice, and tested for respiratory control ratios, protein concentration, and ADP/O, as done previously (21). Purified mitoplasts were obtained as described by Maisterrena et al. (22): Washed mitochondria (400 mg of protein) were homogenized in 400 ml of 10 mM potassium phosphate at pH 7.4 and 0° and allowed to swell for 20 min. Then, the suspension was centrifuged at 105,000 X g for 60 min. The pellets were resuspended in 0.25 M sucrose/10 mM TrisHCI at pH 7.4 and centrifuged at 11,500g for 15 min in order to separate the outer membranes from the mitoplasts (inner membranes and matrix). To obtain sonicated vesicles, mitoplasts homogenized at 0° in 0.25 M sucrose/10 mM Tris-HCI at pH 7.4 at 20 mg of protein per ml were sonicated three times for 10 sec each time in 5-ml aliquots with a Branson sonifier B12. Care was taken to prevent the temperature from rising above 80 during sonication. Unbroken mitoplasts were centrifuged at 25,000 X g for 10 min. Sonicated vesicles were collected after centrifugation at 78,000 X g for 90 min and the pellets were suspended in 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 and 0°. The absence of outer membranes and the intactness of the mitoplasts were checked as before (22) by electron microscopy and by the lack of monoamine oxidase activity. ATPase. Oligomycin-insensitive F1-ATPase was prepared according to the procedure of Senior and Brooks (23) as modified earlier (24). A partially purified oligomycin-sensitive ATPase was obtained by washing the mitochondrial membranes (40 mg of protein) successively with 10 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4, containing 1 mg of Lubrol WX per ml and with the same medium containing 0.05% Triton X-100. After centrifugation (105,000 X g for 20 min), the pellets were extracted for 20 min with the same medium containing 0.2% Triton X-100 and the suspension was centrifuged at 105,000 X g for 20 min; the supernatant fluid contained the oligomycin-sensitive ATPase and was used as such without any further purification. Proteins were estimated by the Lowry procedure (25)
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. §1734 solely to indicate this fact.
work is part of the "These de Specialit6" in Biochemistry of H.M. t To whom correspondence should be addressed. * This
4185
4186
Biochemistry: Mairouch and Godinot
PProc. Natl. A Acad. Sci. UUSA 74 (1977)
Table 1. Comparative activities of enzymes involving cytochrome c in mitoplasts and sonicated vesicles Mmol substrate transformed/min per mg protein Enzyme activity Mitoplasts Sonicated vesicles NADH oxidase - Cytochrome c 0.20 0.86 + Cytochrome c 0.47 0.88 Succinate-cytochrome c reductase 0.12 0.05 Cytochrome oxidase - Lubrol 1.36 0.65 + Lubrol* 1.53 1.51 * 1 mg/mg of protein.
induced a high NADH oxidase activity, but no stimulation of NADH oxidase by cytochrome c could be detected. Succinate-cytochrome c reductase and cytochrome oxidase activities are low, and the latter is enhanced 2.3 times by the addition of Lubrol WX. All these observations are in agreement with those of Huang et al. (34), and indicate that these vesicles represent "inside-out" fragments that have a membrane orientation the opposite of that found in normal mitochondria. On the contrary, NADH oxidase activity of mitoplasts is low and increases in the presence of added cytochrome C; succinate-cytochrome oxidase and cytochrome oxidase activities are much higher than with sonicated vesicles; furthermore, the addition of Lubrol barely improves the cytochrome oxidase activity. All these experiments indicate a great accessibility of externally added cytochrome c to these mitoplasts and show that they have essentially retained the normal polarity of intact
modified by Wang and Smith (26) to eliminate Triton X-100 interference, when this detergent was present. Enzyme Assays. The rate of NADH oxidation was determined by oxypolarography in a Gilson Oxygraph equipped with a Clark electrode. Reaction mixture (1.8 ml) contained 0.1-0.4 mg of membrane proteins, and 66 mM potassium phosphate buffer (pH 7.4) with or without 0.1 mM cytochrome c. The reaction was initiated by the addition of 1 mM NADH. Temperature was 30°. Succinate-cytochrome c reductase was assayed spectrophotometrically and cytochrome oxidase (EC 1.9.1.3) polarographically as done by Sottocasa et al. (27). Monoamine oxidase (EC 1.4.3.4) was tested by the technique of Schnaitman and Greenawalt (28) after a modification of the spectrophotometric procedure of Tabor et al. (29). ATPase activity was determined by a spectrophotometric method using pyruvate kinase and lactate dehydrognease as auxiliary enzymes (24). Activity of trypsin (EC 3.4.21.4) was measured using N-ap-toluenesulfonyl-L-arginine methyl ester as substrate according to Rick (30). All reagents were of the purest available grade. Nucleotides and most enzymes were obtained from Boehringer. Trypsin (crystallized from bovine pancreas, Type III or Type XI), oligomycin, soybean trypsin inhibitor, and N-a-p-toluenesulfonyl-L-arginine methyl ester were from Sigma Chemical Co. All other reagents were obtained from Merck or Prolabo.
mitochondria. ATPase. Fig. 1 shows that trypsin treatment barely affects the ATPase activity of mitoplasts while it strongly increases the rate of ATP hydrolysis by sonicated vesicles. The stimulation of ATPase activity by trypsin depends on the trypsin concentration: 20 Atg of trypsin does not increase the ATPase activity of mitoplasts (1 mg of protein); a weak activation, 0.9-1.8 ,4mol of ATP hydrolyzed/min per mg of protein, is obtained in 1 hr with mitoplasts in the presence of 200 ,ug of trypsin. The same trypsin concentration increases by more than 10 times the
RESULTS AND DISCUSSION Orientation of mitoplasts and sonicated vesicles The differential orientation of the mitoplasts and of the sonicated vesicles was proved by measuring the activity of enzymes using cytochrome c and by looking at the effect of trypsin on the ATPase activity. Cytochrome c. Experiments from several laboratories (31, 32) have firmly established that the physiological location for cytochrome c is on the membrane surface normally oriented toward the cytoplasm. Moreover, cytochrome c can be easily removed from the membrane by hypotonic treatment (33). Our technique of mitoplast preparation includes a swelling in hypotonic phosphate buffer which should then remove, at least partly, the cytochrome c from its site. If the membrane is normally oriented, added cytochrome c can bind to its sites and the activity of enzymes involving cytochrome c must increase. On the contrary, if the membrane is inside out, added cytochrome c can no longer reach its binding sites and must have no effect on the activity of enzymes using cytochrome c. Table 1 shows a typical experiment reproduced with three different membrane preparations: Sonification of mitoplasts
ATPase activity of sonicated vesicles. To obtain reproducible results, it is important to check the specific activity of trypsin. Batches obtained from different manufacturers have greatly varying activities. The trypsin used here (from Sigma) could split 0.3-0.36 mmol of N-a-toluenesulfonyl-L-arginine methyl ester/min per mg in 10 mM CaCl2/40 mM Tris-HCI buffer at pH 8.1. Control experiments, in which mitoplasts or sonicated vesicles were incubated under similar conditions either in the absence of trypsin or in the presence of trypsin plus trypsin inhibitor, show no significant modification of the rate of ATP hydrolysis. Trypsin also increases the rate of ATP hydrolysis by oligomycin-sensitive ATPase solubilized from mitochondria in the presence of Triton X-100. Under the experimental conditions described here, trypsin (up to 200 ,ug/mg of Fl-ATPase) does not change the specific activity of the F1-ATPase prepared by a method (24) similar to that of Senior and Brooks (23), which provides an enzyme free of ATPase inhibitor (35). If the trypsin concentration reaches 500 ,g/mg of Fl-ATPase, the ATPase activity decreases. Racker et al. (32) demonstrated that F1-ATPase is oriented towards the matrix side of the membrane, and that exposure of sonicated particles to trypsin increased the rate of ATP hydrolysis 5- to 10-fold (36) because the ATPase inhibitor is easily attacked by trypsin (13, 37, 38) and that trypsin reaches this inhibitor if the membranes are "inside-out." The fact that trypsin barely increases the ATPase activity of the mitoplasts while it strongly stimulates the ATPase activity of the sonicated vesicles speaks again in favor of a membrane polarity of the mitoplasts similar to that of normal mitochondria and of an opposite orientation for the sonicated vesicles. Cytoplasmic orientation of a protein involved in oligomycin sensitivity of ATPase activity Fig. 2 shows that trypsin diminishes the percentage of inhibition of ATPase activity by oligomycin as well with the solubilized oligomycin-sensitive ATPase as with mitoplasts or sonicated vesicles. Controls made in the absence of trypsin or in the presence of trypsin inhibitor show a slight diminution of the
Biochemistiy:
Proc. Nati. Acad. Sci. USA 74 (1977)
Mairouch and Godinot
Mitoplasts Trypsin + inhibitor
3 2
F 1 ATPase
80 - Trypsin ± inhibitor 60
1
-i0 M itoplasts -
50
40 20 50 Oligomycin-sensitive ATPase Trypsin + inhibitor
._
01
E
1
E ,, 3 E
0
50 Sonicated vesicles Trypsin + inhibitor
5 4
11 50
'o
0 30 90 50 )Iigomycin-sensitive ATPase Trypsin
I-E 0 4 0
E
10
aw S a
a.5
0
1 1.25 1.25 1.15
0.06 0.06 0.09 0.12
0.09 0.6 1.05 0.8
0.03 0.06 0.09 0.12
0.16 4.4 5.5
0.13 1.2 3.6
Sonicated vesicles
2F I
N
Mitoplasts 0 30 60 90
3
._
I
Table 2. Effect of trypsin on oligomycin inhibition and solubilization of ATPase activity in mitoplasts and sonicated vesicles ATPase activity, umol ATP hydrolyzed/min No oligomycin Oligomycin Time of action Soluble Total Soluble Total of trypsin, min
Trypsin
4a
4187
0 50 Time of trypsin incubation, min
50
FIG. 1. Comparison of the effects of trypsin on ATPase activity of mitoplasts, sonicated vesicles, oligomycin-sensitive ATPase, and F1-ATPase. Mitoplasts, sonicated vesicles, and oligomycin-sensitive ATPase (1 mg of protein) were incubated in 1 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 at 300 in the presence of various concentrations of trypsin and in the presence or absence of trypsin inhibitor. At the indicated times 10-Al aliquots were removed and their ATPase activity measured spectrophotometrically at 300 in 1 ml of medium containing 40 mM Tris-SO4, 3 mM MgSO4, 4 mM phosphoenolpyruvate, 0.3 mM NADH, 2 mM Na2S, 2 mM ATP, 20 Mg of lactate dehydrogenase, and 30 g of pyruvate kinase at pH 7.5. Fj-ATPase (1 mg) was incubated in the presence of trypsin ± trypsin inhibitor in 0.2 M phosphate buffer/5 mM EDTA at pH 7.5. ATPase activity was determined on -Mgl aliquots. Trypsin concentration per ml: 0, 2 Mg; 200 yg; v, 500 ug. Open symbols, trypsin only; closed A, 20 ,g; symbols; trypsin + trypsin inhibitor (concentration five times higher than that of trypsin). Specific activity of trypsin was 320 gmol of N-a-toluenesulfonyl-L-arginine methyl ester/min per mg of protein (in 10 mM CaCl2/40 mM Tris.HCl at pH 8.1). 0,
sensitivity of ATPase activity during incubation at 300 of all enzyme preparations, but this diminution is in no way comparable to that obtained in the presence of trypsin. Table 2 demonstrates that a striking difference exists between mitoplasts and sonicated vesicles. With sonicated vesicles the ATPase activity becomes partly released to the supernatant fraction upon trypsin treatment while the ATPase activity of the mitoplasts remains associated with the membrane fraction even if the oligomycin sensitivity diminishes. Control experiments show no solubilization of ATPase activity of mitoplasts or sonicated vesicles either in the absence of trypsin or in the presence of trypsin plus trypsin inhibitor.
2.3 4.8 6.6
0.20 1.4 3.8
Mitoplasts and sonicated vesicles (1 mg of protein) were incubated at 300 in 1 ml of 0.25 M sucrose/10 mM Tris-HCl at pH 7.4 in the presence of 0.2 mg of trypsin (78 Mmol of N-a-toluenesulfonyl-Larginine methyl ester split per min), as in Fig. 1. At the indicated times, aliquots were removed and trypsin inhibitor was added. ATPase activity in the presence or absence of oligomycin (1 ug) was measured either directly on these aliquots or in the supernatant fractions obtained after centrifugation in a Beckman Airfuge (airdriven ultracentrifuge) for 10 min at 100,000 X g. Similar results were obtained with two other experiments.
Trypsin diminishes the sensitivity of the ATPase to oligomycinlinthe mitoplastsaswellasiinthe sonicated vesicles. In the sonicated vesicles, the F1-ATPase is solubilized from the membrane. This solubilization of ATPase by trypsin was observed in other laboratories (37, 39) and can be explained by the great susceptibility to trypsin of OSCP (14) and of the F6 factor (15, 16), which have been reported to be the peptides involved in the binding of the factor F1 to the membrane factor Fo. Indeed, the integrity of these factors (OSCP and F6) and the correct attachment of the enzyme to the membrane are necessary to observe the inhibition of ATPase by oligomycin. Although the complete solubilization of the F1-ATPase appears more slowly than the oligomycin sensitivity disappears, it is likely that the functional properties of the peptides that bind the F1-ATPase to the membrane can be damaged before the total release of the FI-ATPase in the supernatant fraction. When mitoplasts were exposed to trypsin action, the sensitivity of ATPase to oligomycin progressively diminished. This indicates that a protein sensitive to trypsin and at least structurally involved in the oligomycin sensitivity is located near the outer surface of the mitochondrial inner membrane. The decrease in oligomycin sensitivity occurs without solubilization of the ATPase activity from the mitoplasts. This fact does not prove that the Fj-ATPase is still bound to the membrane since it could be solubilized within the matrix. However, the following experiment indicates that the FI-ATPase was not solubilized within the matrix. During the trypsin action, aliquots of mitoplasts were treated with 0.4 mg of Lubrol WX per mg of protein and centrifuged in a Beckman Airfuge; the super-
natant fraction was tested for ATPase activity. No increase in ATPase activity could be detected in this soluble fraction obtained after Lubrol treatment. At least 95% of the malate dehydrogenase activity was solubilized from the mitoplasts by this Lubrol treatment. In the absence of Lubrol, no change in the percentage of malate dehydrogenase activity could be detected in the supernatant fraction after 1 hr of trypsin treatment (200 Ag of trypsin per mg of mitoplasts). This indicates that extensive proteolysis, which would allow soluble proteins to pass through
4188
Biochemistry: Mairouch and Godinot
Proc. Natl. Acad. Sci. USA 74 (1977)
75
75
A-wlers
C .0
Oligomycin-sensitive ATPase EE 50 Trypsi n 0 + trypsin inhibitor 0I
50
D 25
25
75 Mitoplasts Trypsin + trypsin inhibitor
Sonicated vesicles Trypsin 550 + trypsin inhibitor I
25 F
.
4a 0.
50
.*.
0
A
50
75 I
c
0
:2 50 I C
25
50 Time of trypsin incubation, min
FIG. 2. Effects of trypsin on the percentage of inhibition by oligomycin of ATPase activity in mitoplasts, sonicated vesicles, and oligomycin-sensitive ATPase. Conditions are the same as in Fig. 1 except that the ATPase activity was measured at each indicated time in the presence and absence of 1 jg of oligomycin to calculate the percentage of oligomycin inhibition of ATPase activity.
the membrane, had not occurred during trypsin treatment. The fact that there is no significant increase in ATPase activity during treatment of mitoplasts by trypsin is another argument in favor of the relative intactness of the membrane. If the membrane was damaged, trypsin would have reached the ATPase inhibitor and the ATPase activity should have been
of the oligomycin sensitive-ATPase and destroy the oligomycin sensitivity of the complex.
increased. Juntti et al. (40), studying the oligomycin sensitivity of
nancial support was obtained from the Centre National de la Recherche Scientifique (ATP 2224) and from the Delegation Generale a la Recherche Scientifique et Technique (Contract 74-7-0183).
ATPase activity in submitochondrial particles of different orientation, could not detect any effect on this sensitivity using 20-40 Itg of trypsin per mg of protein in 10-min incubations. This might be related to the specific activity of the trypsin used, which may have been lower than the one used here. In conclusion, this study indicates that oligomycin inhibition is observed only, on one hand, when a peptide (or peptides) located somewhere near the outer surface of the inner membrane is intact and/or functional and, on the other hand, when the binding of F1 to the membrane is correct. These conclusions are in agreement with genetic studies (41, 42), which have shown that there are several genes coding for oligomycin sensitivity and therefore that there must be several peptides involved in this inhibition and a correct assembly of those peptides. Attempts to purify the oligomycin sensitive-ATPase complex from trypsinized mitoplasts have not been successful. Indeed, the peptide maps of mitoplasts are not very different after trypsin treatment than before it, but Triton X-100 extracts of mitoplasts contain many more peptides after trypsin treatment than before. Therefore, the oligomycin sensitive-ATPase cannot be purified from trypsinized mitoplasts in the same way as it may be from normal mitochondria. Further work is needed to purify this complex and decide which peptide(s) of the membrane have been digested by trypsin in order to test whether an Fo protein is really damaged or if the cleavage of another peptide in the membrane could affect the overall organization
We thank Prof. D. C. Gautheron for her valuable discussions and continuous interest in this research. We also thank Drs. J; Comte and D. Goldschmidt for their help and advice at the beginning of this study and F. Penin for his expert assistance in some of the experiments. Fi-
1. Fassenden-Raden, J. M. & Racker, E. (1971) in Structure and Function of Biological Membranes, ed. Rothfield, L. I. (Academic Press, New York), pp. 401-438. 2. Packer, L. (1972) Bioenergetics 3, 115-127. 3. Chance, B. (1972) FEBS Lett. 23,3-20. 4. Senior, A. E. (1973) Biochim. Biophys. Acta 801, 249-277. 5. Penefsky, H. S. (1974) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), Vol. 10, pp. 375-394. 6. Pedersen, P. L. (1975) Bioenergetics 6,243-275. 7. Kagawa,Y. (1974) in Methods in Membrane Biology, ed. Korn, E. D. (Plenum Press, New York), Vol. 1, pp. 201-269. 8. Gautheron, D. C. & Godinot, C. (1977) in Living Systems as Energy Convertors, eds. Buvet, R. & Massue, J. P. (North-Holland Publ. Co., Amsterdam), pp. 89-102. 9. Pullman, M. E., Penefsky, H. S., Datta, A. & Racker, E. (1960) J. Biol. Chem. 235,3322-3328. 10. Mitchell, P. (1973) FEBS Lett. 33,267-272. 11. Mitchell, P. (1974) FEBS Lett. 43, 189-193. 12. Hinkle, P. & Horstman L. L. (1971) J. Biol. Chem. 246,60246028. 13. Pullman, M. E. & Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769. 14. McLennan, D. H. & Tzagoloff, A. (1968) Biochemistry 7, 1603-1610. 15. Russel, L. K., Kinkley, S. A., Kleyman, T. R. & Chan, Sh. P. (1976) Biochem. Biophys. Res. Commun. 73,434-443. 16. Kanner, B. I., Serrano, R., Kandrach, A. & Racker, E. (1976) Biochem. Biophys. Res. Commun. 69,1050-1056.
Biochemistry: Mairouch and Godinot 17. Lardy, H. A., Johnson, D. & McMurray, W. C. (1958) Arch. Biochem. Biophys. 78,587-595. 18. Kagawa, Y. & Racker, E. (1966) J. Blol. Chem. 241, 24612467. 19. MacLennan, D. H. & Tzagoloff, A. (1968) Biochemistry 7, 1596-1603. 20. Crane, F. L., Glenn, J. & Green, D. E. (1956) Biochim. Blophys. Acta 135,599-613. 21. Godinot, C., Vial, C., Font, B. & Gautheron, D. (1969) Eur. J. Biochem. 8, 385-394. 22. Maisterrena, B., Comte, J. & Gautheron, D. (1974) Biochrm. Blophys. Acta 367, 115-126. 23. Senior, A. E. & Brooks, D. C. (1970) Arch. Biochem. Blophys. 140, 257-266. 24. Di Pietro, A., Godinot, C., Bouillant, M. L. & Gautheron, D. C. (1975) Biochimie 57, 959-967. 25. Lowry, 0. H., Rosebrough, N.J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 26. Wang, C. S. & Smith, R. L. (1975) Anal. Biochem. 63, 414417. 27. Sottocasa, G. L., Kuylenstierna, B., Ernster, L. & Bergstrand, A. (1967) J. Cell Blol. 32, 415-438. 28. Schnaitman, C. & Greenawalt, J. W. (1968) J. Cell Biol. 38, 158-175. 29. Tabor, C. W., Tabor, H. & Rosenthal, S. M. (1955) in Methods in Enzymology, eds. Kaplan, N. 0. & Colowick, S. P. (Academic Press, New York), Vol. 2, pp. 390-393. 30. Rick, W. (1974) in Methods of Enzymatic Analysis, ed. Bergmeyer, H. U. (Academic Press, New York), 2nd ed., Vol. 2, pp.
1021-1024.
Proc. Nati. Acad. Sc. USA 74 (1977)
4189
31. Harmon, H. J., Hall, J. D. & Crane, F. L. (1974) Blochim. BNophys. Acta 344,119-155. 32. Racker, E., Burstein, C., Loyter, A. & Christiansen, R. 0. (1970) in Electron Transport and Energy Conservation, eds. Tager, J. M., Papa, S., Quagliariello, E. & Slater, E. C. (Adriatica Editrice, Bari, Italy), pp. 235-241. 33. Jacobs, E. E. & Sanadi, D. R. (1960) J. Btol. Chem. 235, 531534. 34. Huang, C. H., Keyhani, E. & Lee, C. P. (1973) Biochim. Blophys. Acta 305, 455-473. 35. Brooks, J. C. & Senior, A. E. (1971) Arch. Blochem. Blophys. 147, 467-472. 36. Racker, E. (1963) Biochem. Blophys. Res. Commun. 10, 435439. 37. Horstman, L. L. & Racker, E. (1970) J. Biol. Chem. 245, 1336-1344. 38. Montecucco, C. & Azzi, A. (1975) J. Biol. Chem. 250, 50205025. 39. Knowles, A. F., Guillory, R. J. & Racker, E. (1971) J. Biol. Chem. 247,2351-2357. 40. Juntti, K., Torndall, V. B. & Ernster, L. (1970) in Electron Transport and Energy Conservation, eds. Tager, J. M., Papa, S., Quagliariello, E. & Slater, E. C. (Adriatica Editrice, Bari, Italy), pp. 257-270. 41. Avner, P. R., Coen, D., Dujon, B. & Slonimski, P. P. (1973) Mol.
Gen. Cenet. 125, 9-52. 42. Griffiths, D. E. (1975) in Genetics and Biogenesis of Mitochondria and Chloroplasts, eds. Birky, G. W., Pearlman, P. S. & Byers, J. J. (Ohio State University Press, Columbus, OH), pp. 117-135.
