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Factor B is essential for ATP synthesis by mitochondria
ABB Archives of Biochemistry and Biophysics 406 (2002) 271–274 www.academicpress.com
Factor B is essential for ATP synthesis by mitochondriaq Grigory I. Belogrudov* Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA Received 27 June 2002
Abstract Factor B is a water-soluble protein, which is required for the coupled activity of the mitochondrial ATP synthase complex. Specific removal of factor B from well-coupled bovine heart submitochondrial particles (SMP) results in uncoupling and the loss of ATP-driven membrane potential formation and reverse electron transfer from succinate to NAD. Addition of recombinant human factor B (molecular mass 20,341 Da) to factor B-depleted SMP (AE-SMP) restores these properties [G.I. Belogrudov, and Y. Hatefi, (2002) J. Biol. Chem. 277, 6097–6103]. This paper shows that extraction and purification of ATP synthase complex (complex V) from bovine heart mitochondria results in extensive loss of factor B. Addition of recombinant human factor B to AE-SMP completely restores the lost oxidative phosphorylation and ATP-32 Pi exchange activities of the particles and increases the ATP-32 Pi exchange activity of complex V by 2.5-fold. These results further indicate that factor B is an essential component of the mammalian ATP synthase complex. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Oxidative phosphorylation; Coupling; Mitochondria
Since its introduction into the mitochondrial oxidative phosphorylation literature in 1967 [1], the existence of factor B in mitochondria had remained controversial [2–4]. Recently, we cloned and purified human factor B, and showed that it reacts with uncoupled, factor B-depleted bovine heart submitochondrial particles (AESMP)1 and restores ATP-driven membrane potential formation and reverse electron transfer from succinate to NAD [5]. The mature human factor B is a watersoluble protein composed of 175 amino acids and has a molecular mass of 20,341 Da. It is inhibited by monothiol and especially dithiol-modifying reagents, which q Supported by United States Public Health Service Grant DK08126 to Y. Hatefi. This is Publication Number 15006-MEM from The Scripps Research Institute. * Present address: Department of Chemistry and Biochemistry, University of California at Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569, USA. Fax: 1-310-206-5213. E-mail address: [email protected] (G.I. Belogrudov). 1 Abbreviations used: SMP, bovine heart submitochondrial particles; AE-SMP, SMP depleted of factor B; OSCP, oligomycin sensitivityconferring protein; S1 -sup, deoxycholate extract of bovine heart mitochondria enriched in complex V (ATP synthase complex); Mr , relative molecular mass; DCCD, N,N 0 -dicyclohexylcarbodiimide.
likely react at its cysteine residues Cys-92 and Cys-94. Removal of factor B from SMP results in proton leakiness at the level of the ATP synthase membrane sector FO . The proton leak can be blocked by FO -specific inhibitors, such as oligomycin, DCCD, and organotin compounds. Recombinant human factor B also repairs the FO proton leak, but without inhibiting the ATP synthase complex [5]. The interaction with FO appears to be stoichiometric. As noted above, we have shown that human factor B restores ATP-driven reactions to depleted SMP. This paper shows that it also fully restores ATP synthesis and ATP-32 Pi exchange activity to uncoupled, factor B-depleted particles. In addition, it greatly increases the ATP-32 Pi exchange activity of preparations of bovine ATP synthase complex containing greatly diminished amounts of factor B.
Materials and methods Bovine heart ATP synthase complex (complex V), S1 sup, SMP, and AE-SMP were prepared as in the references cited [6–8]. The ATPase activities of complex V
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 4 3 1 - 9
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G.I. Belogrudov / Archives of Biochemistry and Biophysics 406 (2002) 271–274
and submitochondrial particles were measured spectrophotometrically by the coupled pyruvate kinase/lactate dehydrogenase method as before [6]. The complex V preparation used here was prepared according to [6], without additional purification by gel filtration [9], and is referred to as partially purified complex V throughout the manuscript. It exhibited an ATPase activity of 12 lmol of ATP hydrolyzed (min mg of protein)1 at 30 °C, pH 7.8, which was 97% inhibited by oligomycin. Recombinant human factor B was expressed and purified as described previously [5]. To prepare a polyclonal antiserum, white New Zealand rabbits were injected subcutaneously with pure recombinant human factor B in multiple sites on the back in 2-week intervals. After several immunizations, the serum tested positive against the recombinant protein, as determined by Western blot analysis, and was additionally affinitypurified as before [10]. SDS–polyacrylamide gel electrophoresis and immunoblotting were performed as described elsewhere [10]. The immune complexes were visualized using the West Pico chemiluminescence detection kit from Pierce. Protein concentration was determined using the BCA kit from Pierce. The ATP-32 Pi exchange assay was performed essentially as described in [6]. Complex V (28 lg), dissolved in a buffer containing 50 mM Tris–HCl, pH 8.0, 0.66 M sucrose, and 1 mM histidine was incubated alone or in the presence of variable amounts of recombinant human factor B with 5 mg/ml of sonicated phospholipids (20% L -a-lethicin, Avanti Polar-Lipids,) in a final volume of 50 ll for 30 min on ice. To this sample, about 0.5 ml of a reaction mixture containing 50 mM Tris–acetate, pH 7.5, 0.25 M sucrose, 15 mM MgCl2 , 20 mM potassium phosphate, pH 7.5, and 3 mg/ml bovine serum albumin was added, and the incubation was continued at 30 °C for 2–3 min. The ATP-32 Pi exchange reaction was started by adding neutralized [32 P]phosphate (1:5–2:0 106 cpm per assay) and 10 mM ATP in a final volume of 0.6 ml, and the incubation was continued at 30 °C for 10 min. The reaction was stopped by adding 50 ll of 35% HClO4 , the mixture was centrifuged to remove denatured proteins, and 0.5 ml of the supernatant was used for extraction with isobutanol-benzene/ammonium molybdate mixture [6]. Two milliliters of the aqueous phase was mixed with 5 ml of liquid scintillation fluid (BetaBlend, ICN Biomedicals,) and the radioactivity was counted in a Beckman liquid scintillation counter LS 1801. To determine the background of the exchange reaction, complex V in the above reaction mixture was supplemented with 50 ll of 35% HClO4 prior to the addition of [32 P]phosphate and ATP. The inhibitory effects of DCCD (final concentration 10 lM) and oligomycin (final concentration 3 lg/ml) on the ATP-32 Pi exchange activity were determined by preincubating the buffer containing complex V and phospholipid with each
inhibitor for 5 min at 30 °C prior to the addition of [32 P]phosphate and ATP. The ATP-32 Pi exchange activities of SMP, AE-SMP, and the particles reconstituted with recombinant factor B were assayed in a similar way as described above for complex V, except that the final concentration of SMP and AE-SMP in the reaction mixture were 0.083 and 0.141 mg/ml, respectively, and the reaction time was 5 min at 30 °C. The oxidative phosphorylation activities of SMP and AE-SMP alone or after reconstitution with recombinant human factor B were assayed as in [11]. Briefly, to 0.6 ml final volume of a buffer, containing 0.25 M sucrose, 50 mM Tris–acetate, pH 7.5, 0.5 mM EDTA, 50 mM glucose, 20 mM potassium phosphate, pH 7.5, 5 mM MgCl2 , and 0.07 mg/ml of hexokinase (type F-300, Sigma), was added 0.05 mg/ml SMP or 0.057 mg/ml AE-SMP and incubated for 5 min at 30 °C. Then after addition of 1:5–2:0 106 cpm [32 P]phosphate and 2 mM ADP, 0.5 mM NADH or 8 mM succinate was added as respiratory substrate and the mixture incubated for 4 min at 30 °C. After termination of the reactions with 50 ll of 35% HClO4 , the samples were processed as described above for the ATP-32 Pi exchange reaction to determine the amount of radioactivity in the [32 P]ATP formed. Results and discussion As was shown earlier, extraction of well-coupled bovine heart SMP with 0.6 mM EDTA in 0.25 M sucrose at pH 8.8 (adjusted with ammonium hydroxide) specifically removes factor B and renders the particles (AE-SMP) incapable of ATP hydrolysis-driven reverse electron transfer from succinate to NAD, and of membrane potential formation coupled to ATP hydrolysis or NADH oxidation [5]. The proton leak resulting from factor B removal could be repaired by addition of recombinant human factor B in amounts stoichiometric to the ATP synthases of the particles, resulting in restoration of the above ATP-driven reactions. Not investigated previously was whether recombinant human
Table 1 Effect of human factor B on the oxidative phosphorylation activitya of factor B-depleted bovine SMP Respiratory substrate
NADH
Succinate
Preparation SMP AE-SMP AE-SMP + factor B
1842 90 43 3 1865 110
915 40 39 4 1082 25
a Activity is expressed as nmol of ATP formed (min mg of particle protein)1 . The amount of human factor B added was 103 lg/mg AESMP. The activities shown are the means standard deviation of three separate experiments. For other details, see Materials and methods.
G.I. Belogrudov / Archives of Biochemistry and Biophysics 406 (2002) 271–274
factor B is also capable of restoring the ATP synthase activity of AE-SMP. As seen in Table 1, removal of factor B rendered the SMP nearly completely incapable of ATP synthesis driven by NADH or succinate oxidation, and addition of recombinant human factor B to AE-SMP completely restored their oxidative phosphorylation activity. Consistent with its recoupling effect, factor B addition also decreased the ATPase activity of AE-SMP from 4.9 lmol ATP hydrolyzed (min mg of protein)1 at 30 °C to 3.2 lmol (min mg of protein)1 . The full restoration by factor B of the oxidative phosphorylation activity of AE-SMP further indicates that the extraction of SMP with 0.6 mM EDTA at pH 8.8 is highly specific for the removal of factor B. The controversial status of factor B before our recent cloning, purification, and characterization of the active human protein was in part because a thorough analysis of the polypeptide composition of a highly purified preparation of bovine ATP synthase complex had failed to detect any factor B in that preparation [3,4]. Indeed, the concern about the existence of factor B as a component of the ATP synthase complex was fully justified, for the reasons detailed below. As indicated by the early work of Sanadi’s laboratory [2,12], bovine factor B (Mr 22 kDa) stains poorly with Coomassie brilliant blue. Fig. 1A shows SDS–polyacrylamide gels of bovine heart SMP (lane 1), bovine mitochondrial deoxycholate extract (S1 -sup) enriched in FO –F1 (lane 2), partially purified ATP synthase (lane 3), and purified recombinant human factor B (lane 4), all stained with Coomassie brilliant blue. It is seen that human factor B (Mr 22 kDa, same as the bovine protein) stains well with the dye used. Indeed we were able to clearly see on SDS gels as little as 0.3 lg of human factor B, but a protein band corresponding to the Mr 22 kDa of bovine factor B could not be seen in lanes 1, 2, and 3 of Fig. 1A. By contrast, Fig. 1B shows that although factor B was again undetectable on SDS gels of whole bovine heart mitochondria (lane 1), it was clearly detectable when the mitochondria were immunoblotted, using our antihuman factor B antiserum (Fig. 1B, lane 2). In the immunoblots of Fig. 1C, the conditions were so adjusted to give roughly comparable blot intensities for F1 a subunit and OSCP in SMP (lane 1), S1 -sup (lane 2), and the ATP synthase of Fig. 1A (lane 3), as shown in the second (F1 aÞ and the third (OSCP) rows. The top row of this figure shows the relative contents of factor B in SMP (lane 1), S1 -sup (lane 2), and partially purified ATP synthase (lane 3). It is seen that even at this stage of purification of the ATP synthase complex, the content of factor B was greatly diminished. This is not surprising, since a mild treatment of SMP with 0.6 mM EDTA at pH 8.8, a condition that does not harm the respiratory chain complexes or remove any other FO –F1 subunit, results in nearly complete extraction of factor B [5].
273
Fig. 1. (A) SDS–polyacrylamide gel electrophoresis patterns of SMP, 20 lg (lane 1); deoxycholate extract of bovine mitochondria (S1 -sup), 16 lg (lane 2); partially purified complex V, 14 lg (lane 3); and recombinant human factor B, 4 lg (lane 4). The proteins were separated by 15% SDS–polyacrylamide gel and stained with Coomassie brilliant blue. The electrophoretic positions of ATP synthase subunits a; b; c; d, b, OSCP, and d are indicated on the right. (B) SDS–polyacrylamide gel of bovine heart mitochondria (25 lg) stained with Coomassie brilliant blue (lane 1), and after transfer to nitrocellulose and immunoblotting with affinity purified human factor B antiserum (lane 2). In (A) and (B), molecular mass standards (in kDa) are indicated on the left. (C) SMP (lane 1), S1 -sup (lane 2), and complex V (lane 3) in the amounts indicated above were subjected to SDS–polyacrylamide gel electrophoresis, and immunoblotted with polyclonal antisera to human factor B (top row), bovine F1 a subunit (middle row), and bovine OSCP (bottom row).
As was shown previously [5] and in Table 1, the uncoupled state of factor B-depleted AE-SMP could be repaired by the addition of recombinant human factor B or by treating the particles with an FO inhibitor such as oligomycin, DCCD, or tributyltin chloride. As expected, the FO inhibitors blocked ATP hydrolysis and synthesis, but allowed respiration-driven membrane potential formation [5]. These results strongly suggested that factor B removal created a proton leak at the level of the membrane sector FO of the ATP synthase complex. However, since our partially purified ATP synthase preparation contained a greatly diminished amount of factor B, it was of interest to investigate its energy conserving activity in the absence and the pres-
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Table 2 Effect of human factor B on the ATP-32 Pi exchange activities of complex V and factor B-depleted bovine SMP Preparation
Factor B (lg/mg complex V or AE-SMP)
ATP-32 Pi exchange activitya
Complex Complex Complex Complex Complex
None 46 185 46 46
135 9 272 15 336 20 1:8 0:15 0.0
None 28 None 65 198
332 13 352 11 14 2 309 10 387 18
V V V V + DCCD V + oligomycin
SMP SMP AE-SMP AE-SMP AE-SMP
a ATP-32 Pi exchange activity is expressed as nmol (min mg of protein)1 . The values given represent means standard deviation for three separate experiments. Where indicated, complex V was treated with 10 lM DCCD or 3 lg of oligomycin/ml as described under Materials and methods.
ence of added human factor B. This was done by assay of ATP-32 Pi exchange activity, and the results are summarized in Table 2. It is seen that the oligomycinand DCCD-sensitive ATP-32 Pi exchange activity of our ATP synthase preparation was increased by 2.5-fold upon incubation with human factor B. For comparison, Table 2 also shows the ATP-32 Pi exchange activities of SMP and AE-SMP before and after treatment with human factor B. These and our previous results [5] make it clear, therefore, that factor B is essential for energy transduction by the mammalian ATP synthase complex. Acknowledgments The author thanks Dr. Youssef Hatefi for his support and Calixto Munoz for the preparation of mitochondria.
References [1] K.W. Lam, J.B. Warshaw, D.R. Sanadi, Arch. Biochem. Biophys. 119 (1967) 477–484. [2] D.R. Sanadi, Biochim. Biophys. Acta 423 (1982) 398–412. [3] J.E. Walker, R. Lutter, A. Dupuis, M.J. Runswick, Biochemistry 30 (1991) 5369–5378. [4] I.R. Collins, M.J. Runswick, S.K. Buchanan, I.M. Fearnley, J.M. Skehel, M.J. van Raaji, D.E. Griffiths, J.E. Walker, Biochemistry 33 (1994) 7971–7978. [5] G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097–6103. [6] D.L. Stiggall, Y.M. Galante, Y. Hatefi, Methods Enzymol. 55 (1979) 308–315. [7] Y. Hatefi, D. Stiggall, Methods Enzymol. 53 (1978) 5–10. [8] S. Joshi, D.R. Sanadi, Methods Enzymol. 55 (1979) 384–391. [9] Y.M. Galante, S.-Y. Wong, Y. Hatefi, J. Biol. Chem. 254 (1979) 12372–12378. [10] G. Belogrudov, Y. Hatefi, Biochemistry 33 (1994) 4571–4576. [11] A. Matsuno-Yagi, Y. Hatefi, J. Biol. Chem. 260 (1985) 14424– 14427. [12] L. Kantham, R. Raychowdry, K.K. Ogata, A. Javed, J. Rice, D.R. Sanadi, FEBS Lett. 277 (1990) 105–108.
Factor B is essential for ATP synthesis by mitochondriaq Grigory I. Belogrudov* Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA Received 27 June 2002
Abstract Factor B is a water-soluble protein, which is required for the coupled activity of the mitochondrial ATP synthase complex. Specific removal of factor B from well-coupled bovine heart submitochondrial particles (SMP) results in uncoupling and the loss of ATP-driven membrane potential formation and reverse electron transfer from succinate to NAD. Addition of recombinant human factor B (molecular mass 20,341 Da) to factor B-depleted SMP (AE-SMP) restores these properties [G.I. Belogrudov, and Y. Hatefi, (2002) J. Biol. Chem. 277, 6097–6103]. This paper shows that extraction and purification of ATP synthase complex (complex V) from bovine heart mitochondria results in extensive loss of factor B. Addition of recombinant human factor B to AE-SMP completely restores the lost oxidative phosphorylation and ATP-32 Pi exchange activities of the particles and increases the ATP-32 Pi exchange activity of complex V by 2.5-fold. These results further indicate that factor B is an essential component of the mammalian ATP synthase complex. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Oxidative phosphorylation; Coupling; Mitochondria
Since its introduction into the mitochondrial oxidative phosphorylation literature in 1967 [1], the existence of factor B in mitochondria had remained controversial [2–4]. Recently, we cloned and purified human factor B, and showed that it reacts with uncoupled, factor B-depleted bovine heart submitochondrial particles (AESMP)1 and restores ATP-driven membrane potential formation and reverse electron transfer from succinate to NAD [5]. The mature human factor B is a watersoluble protein composed of 175 amino acids and has a molecular mass of 20,341 Da. It is inhibited by monothiol and especially dithiol-modifying reagents, which q Supported by United States Public Health Service Grant DK08126 to Y. Hatefi. This is Publication Number 15006-MEM from The Scripps Research Institute. * Present address: Department of Chemistry and Biochemistry, University of California at Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569, USA. Fax: 1-310-206-5213. E-mail address: [email protected] (G.I. Belogrudov). 1 Abbreviations used: SMP, bovine heart submitochondrial particles; AE-SMP, SMP depleted of factor B; OSCP, oligomycin sensitivityconferring protein; S1 -sup, deoxycholate extract of bovine heart mitochondria enriched in complex V (ATP synthase complex); Mr , relative molecular mass; DCCD, N,N 0 -dicyclohexylcarbodiimide.
likely react at its cysteine residues Cys-92 and Cys-94. Removal of factor B from SMP results in proton leakiness at the level of the ATP synthase membrane sector FO . The proton leak can be blocked by FO -specific inhibitors, such as oligomycin, DCCD, and organotin compounds. Recombinant human factor B also repairs the FO proton leak, but without inhibiting the ATP synthase complex [5]. The interaction with FO appears to be stoichiometric. As noted above, we have shown that human factor B restores ATP-driven reactions to depleted SMP. This paper shows that it also fully restores ATP synthesis and ATP-32 Pi exchange activity to uncoupled, factor B-depleted particles. In addition, it greatly increases the ATP-32 Pi exchange activity of preparations of bovine ATP synthase complex containing greatly diminished amounts of factor B.
Materials and methods Bovine heart ATP synthase complex (complex V), S1 sup, SMP, and AE-SMP were prepared as in the references cited [6–8]. The ATPase activities of complex V
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 4 3 1 - 9
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G.I. Belogrudov / Archives of Biochemistry and Biophysics 406 (2002) 271–274
and submitochondrial particles were measured spectrophotometrically by the coupled pyruvate kinase/lactate dehydrogenase method as before [6]. The complex V preparation used here was prepared according to [6], without additional purification by gel filtration [9], and is referred to as partially purified complex V throughout the manuscript. It exhibited an ATPase activity of 12 lmol of ATP hydrolyzed (min mg of protein)1 at 30 °C, pH 7.8, which was 97% inhibited by oligomycin. Recombinant human factor B was expressed and purified as described previously [5]. To prepare a polyclonal antiserum, white New Zealand rabbits were injected subcutaneously with pure recombinant human factor B in multiple sites on the back in 2-week intervals. After several immunizations, the serum tested positive against the recombinant protein, as determined by Western blot analysis, and was additionally affinitypurified as before [10]. SDS–polyacrylamide gel electrophoresis and immunoblotting were performed as described elsewhere [10]. The immune complexes were visualized using the West Pico chemiluminescence detection kit from Pierce. Protein concentration was determined using the BCA kit from Pierce. The ATP-32 Pi exchange assay was performed essentially as described in [6]. Complex V (28 lg), dissolved in a buffer containing 50 mM Tris–HCl, pH 8.0, 0.66 M sucrose, and 1 mM histidine was incubated alone or in the presence of variable amounts of recombinant human factor B with 5 mg/ml of sonicated phospholipids (20% L -a-lethicin, Avanti Polar-Lipids,) in a final volume of 50 ll for 30 min on ice. To this sample, about 0.5 ml of a reaction mixture containing 50 mM Tris–acetate, pH 7.5, 0.25 M sucrose, 15 mM MgCl2 , 20 mM potassium phosphate, pH 7.5, and 3 mg/ml bovine serum albumin was added, and the incubation was continued at 30 °C for 2–3 min. The ATP-32 Pi exchange reaction was started by adding neutralized [32 P]phosphate (1:5–2:0 106 cpm per assay) and 10 mM ATP in a final volume of 0.6 ml, and the incubation was continued at 30 °C for 10 min. The reaction was stopped by adding 50 ll of 35% HClO4 , the mixture was centrifuged to remove denatured proteins, and 0.5 ml of the supernatant was used for extraction with isobutanol-benzene/ammonium molybdate mixture [6]. Two milliliters of the aqueous phase was mixed with 5 ml of liquid scintillation fluid (BetaBlend, ICN Biomedicals,) and the radioactivity was counted in a Beckman liquid scintillation counter LS 1801. To determine the background of the exchange reaction, complex V in the above reaction mixture was supplemented with 50 ll of 35% HClO4 prior to the addition of [32 P]phosphate and ATP. The inhibitory effects of DCCD (final concentration 10 lM) and oligomycin (final concentration 3 lg/ml) on the ATP-32 Pi exchange activity were determined by preincubating the buffer containing complex V and phospholipid with each
inhibitor for 5 min at 30 °C prior to the addition of [32 P]phosphate and ATP. The ATP-32 Pi exchange activities of SMP, AE-SMP, and the particles reconstituted with recombinant factor B were assayed in a similar way as described above for complex V, except that the final concentration of SMP and AE-SMP in the reaction mixture were 0.083 and 0.141 mg/ml, respectively, and the reaction time was 5 min at 30 °C. The oxidative phosphorylation activities of SMP and AE-SMP alone or after reconstitution with recombinant human factor B were assayed as in [11]. Briefly, to 0.6 ml final volume of a buffer, containing 0.25 M sucrose, 50 mM Tris–acetate, pH 7.5, 0.5 mM EDTA, 50 mM glucose, 20 mM potassium phosphate, pH 7.5, 5 mM MgCl2 , and 0.07 mg/ml of hexokinase (type F-300, Sigma), was added 0.05 mg/ml SMP or 0.057 mg/ml AE-SMP and incubated for 5 min at 30 °C. Then after addition of 1:5–2:0 106 cpm [32 P]phosphate and 2 mM ADP, 0.5 mM NADH or 8 mM succinate was added as respiratory substrate and the mixture incubated for 4 min at 30 °C. After termination of the reactions with 50 ll of 35% HClO4 , the samples were processed as described above for the ATP-32 Pi exchange reaction to determine the amount of radioactivity in the [32 P]ATP formed. Results and discussion As was shown earlier, extraction of well-coupled bovine heart SMP with 0.6 mM EDTA in 0.25 M sucrose at pH 8.8 (adjusted with ammonium hydroxide) specifically removes factor B and renders the particles (AE-SMP) incapable of ATP hydrolysis-driven reverse electron transfer from succinate to NAD, and of membrane potential formation coupled to ATP hydrolysis or NADH oxidation [5]. The proton leak resulting from factor B removal could be repaired by addition of recombinant human factor B in amounts stoichiometric to the ATP synthases of the particles, resulting in restoration of the above ATP-driven reactions. Not investigated previously was whether recombinant human
Table 1 Effect of human factor B on the oxidative phosphorylation activitya of factor B-depleted bovine SMP Respiratory substrate
NADH
Succinate
Preparation SMP AE-SMP AE-SMP + factor B
1842 90 43 3 1865 110
915 40 39 4 1082 25
a Activity is expressed as nmol of ATP formed (min mg of particle protein)1 . The amount of human factor B added was 103 lg/mg AESMP. The activities shown are the means standard deviation of three separate experiments. For other details, see Materials and methods.
G.I. Belogrudov / Archives of Biochemistry and Biophysics 406 (2002) 271–274
factor B is also capable of restoring the ATP synthase activity of AE-SMP. As seen in Table 1, removal of factor B rendered the SMP nearly completely incapable of ATP synthesis driven by NADH or succinate oxidation, and addition of recombinant human factor B to AE-SMP completely restored their oxidative phosphorylation activity. Consistent with its recoupling effect, factor B addition also decreased the ATPase activity of AE-SMP from 4.9 lmol ATP hydrolyzed (min mg of protein)1 at 30 °C to 3.2 lmol (min mg of protein)1 . The full restoration by factor B of the oxidative phosphorylation activity of AE-SMP further indicates that the extraction of SMP with 0.6 mM EDTA at pH 8.8 is highly specific for the removal of factor B. The controversial status of factor B before our recent cloning, purification, and characterization of the active human protein was in part because a thorough analysis of the polypeptide composition of a highly purified preparation of bovine ATP synthase complex had failed to detect any factor B in that preparation [3,4]. Indeed, the concern about the existence of factor B as a component of the ATP synthase complex was fully justified, for the reasons detailed below. As indicated by the early work of Sanadi’s laboratory [2,12], bovine factor B (Mr 22 kDa) stains poorly with Coomassie brilliant blue. Fig. 1A shows SDS–polyacrylamide gels of bovine heart SMP (lane 1), bovine mitochondrial deoxycholate extract (S1 -sup) enriched in FO –F1 (lane 2), partially purified ATP synthase (lane 3), and purified recombinant human factor B (lane 4), all stained with Coomassie brilliant blue. It is seen that human factor B (Mr 22 kDa, same as the bovine protein) stains well with the dye used. Indeed we were able to clearly see on SDS gels as little as 0.3 lg of human factor B, but a protein band corresponding to the Mr 22 kDa of bovine factor B could not be seen in lanes 1, 2, and 3 of Fig. 1A. By contrast, Fig. 1B shows that although factor B was again undetectable on SDS gels of whole bovine heart mitochondria (lane 1), it was clearly detectable when the mitochondria were immunoblotted, using our antihuman factor B antiserum (Fig. 1B, lane 2). In the immunoblots of Fig. 1C, the conditions were so adjusted to give roughly comparable blot intensities for F1 a subunit and OSCP in SMP (lane 1), S1 -sup (lane 2), and the ATP synthase of Fig. 1A (lane 3), as shown in the second (F1 aÞ and the third (OSCP) rows. The top row of this figure shows the relative contents of factor B in SMP (lane 1), S1 -sup (lane 2), and partially purified ATP synthase (lane 3). It is seen that even at this stage of purification of the ATP synthase complex, the content of factor B was greatly diminished. This is not surprising, since a mild treatment of SMP with 0.6 mM EDTA at pH 8.8, a condition that does not harm the respiratory chain complexes or remove any other FO –F1 subunit, results in nearly complete extraction of factor B [5].
273
Fig. 1. (A) SDS–polyacrylamide gel electrophoresis patterns of SMP, 20 lg (lane 1); deoxycholate extract of bovine mitochondria (S1 -sup), 16 lg (lane 2); partially purified complex V, 14 lg (lane 3); and recombinant human factor B, 4 lg (lane 4). The proteins were separated by 15% SDS–polyacrylamide gel and stained with Coomassie brilliant blue. The electrophoretic positions of ATP synthase subunits a; b; c; d, b, OSCP, and d are indicated on the right. (B) SDS–polyacrylamide gel of bovine heart mitochondria (25 lg) stained with Coomassie brilliant blue (lane 1), and after transfer to nitrocellulose and immunoblotting with affinity purified human factor B antiserum (lane 2). In (A) and (B), molecular mass standards (in kDa) are indicated on the left. (C) SMP (lane 1), S1 -sup (lane 2), and complex V (lane 3) in the amounts indicated above were subjected to SDS–polyacrylamide gel electrophoresis, and immunoblotted with polyclonal antisera to human factor B (top row), bovine F1 a subunit (middle row), and bovine OSCP (bottom row).
As was shown previously [5] and in Table 1, the uncoupled state of factor B-depleted AE-SMP could be repaired by the addition of recombinant human factor B or by treating the particles with an FO inhibitor such as oligomycin, DCCD, or tributyltin chloride. As expected, the FO inhibitors blocked ATP hydrolysis and synthesis, but allowed respiration-driven membrane potential formation [5]. These results strongly suggested that factor B removal created a proton leak at the level of the membrane sector FO of the ATP synthase complex. However, since our partially purified ATP synthase preparation contained a greatly diminished amount of factor B, it was of interest to investigate its energy conserving activity in the absence and the pres-
274
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Table 2 Effect of human factor B on the ATP-32 Pi exchange activities of complex V and factor B-depleted bovine SMP Preparation
Factor B (lg/mg complex V or AE-SMP)
ATP-32 Pi exchange activitya
Complex Complex Complex Complex Complex
None 46 185 46 46
135 9 272 15 336 20 1:8 0:15 0.0
None 28 None 65 198
332 13 352 11 14 2 309 10 387 18
V V V V + DCCD V + oligomycin
SMP SMP AE-SMP AE-SMP AE-SMP
a ATP-32 Pi exchange activity is expressed as nmol (min mg of protein)1 . The values given represent means standard deviation for three separate experiments. Where indicated, complex V was treated with 10 lM DCCD or 3 lg of oligomycin/ml as described under Materials and methods.
ence of added human factor B. This was done by assay of ATP-32 Pi exchange activity, and the results are summarized in Table 2. It is seen that the oligomycinand DCCD-sensitive ATP-32 Pi exchange activity of our ATP synthase preparation was increased by 2.5-fold upon incubation with human factor B. For comparison, Table 2 also shows the ATP-32 Pi exchange activities of SMP and AE-SMP before and after treatment with human factor B. These and our previous results [5] make it clear, therefore, that factor B is essential for energy transduction by the mammalian ATP synthase complex. Acknowledgments The author thanks Dr. Youssef Hatefi for his support and Calixto Munoz for the preparation of mitochondria.
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