Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4219-4223, September 1978
Biochemistry
Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits (dicyclohexylcarbodiimide-sensitive ATPase/H+ pathway/dicyclohexylcarbodiimide-binding subunit/soluble ATPase-binding subunit/reconstituted vesicles)
NOBUHITO SONE, MASASUKE YOSHIDA, HAJIME HIRATA, AND YASUO KAGAWA Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan 329-04
Communicated by Leon A. Heppel, June 19,1978
ABSTRACT The HW-translocating ATPase complex from the thermophilic bacterium PS3 (TFo.F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TFo). TFo was obtained by treating TFo0F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TFo contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TFo vesicles). The binding of TF1 to TFo present in vesicles restored energ-transducing activities, such as ) I exchange, dicyclohexylcarbodiimide-sensitive ATPase, and ATP-ependent enhancement of 8-anilinonaphthalene-l-sulfonate fluorescence. Treatments such as protease digestion and chemical modification with acetic anhydride, succinic anhydride, or diazobenzenesulfonic acid destroyed the TF1-binding activity, which was caused by band 6 protein. Band 8 protein was a proteolipid that reacted specifically with dicyclohexylcarbod iimide and seemed to play a central role in H+ conduction through the membrane. Accumulating evidence has indicated that biomembranes that synthesize ATP contain a reversible H+-translocating ATPase complex, named Fo-Fl, as an energy transducing coupling device (1-6). The N,N'-dicyclohexylcarbodiimide (DCCD)sensitive ATPase complex purified from the thermophilic bacterium PS3 (TFo-Fl), when reconstituted into liposomes, was actually able to translocate protons coupled to ATP hydrolysis (7, 8). The electrochemical proton gradient (A7gH+) formed (inside acidic and positive) by ATP hydrolysis was as much as 300 mV (7), and ATP synthesis took place when a L\IH+ of over 200 mV was imposed across the liposomal membranes (6, 9). It seems both interesting and important to determine the subunit structure of Fo-Fl, especially for clarifying the method by which this transducer works. The catalytic and peripheral moiety of the transducer is Fl-ATPase, which is composed of five subunits and has a molecular weight of about 3.8 X 105. Recently these five subunits (a, f3, y, 6, and c) were each purified in reconstitutively active forms from TF1 (thermophilic F1) and their roles in TFo-F1 were proposed (10, 11). The membrane integral part (Fo) of Fo-F1 was formerly defined as a factor(s) that renders F1-ATPase sensitive to energy transfer inhibitors, such as oligomycin and DCCD (12, 13). However, little is known about the constitution of Fo except that the smallest subunit of Fo, to which DCCD binds covalently, is extractable into organic solvents (14, 15). This component, DCCD-binding proteolipid, may be important as a H+ channel across membranes, since H+ conduction is known to be blocked by DCCD. Nelson et al. (16) claimed that a proteolipid fraction purified from lettuce chloroplasts mediated DCCD-sensitive H+ translocation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
TFo can be prepared from TF0-Fj by removal of TF1 (17, 18). Recently, we reported that this preparation contained three different polypeptides and mediated proton conduction that was blocked by addition of either TF1 or DCCD (19). This communication reports that a preparation of TFo consisting of only two kinds of subunits can mediate all the activities of Fo, such as H+ translocation, conferring of DCCD sensitivity, TF1 binding, and energy-transducing reactions with TF1. Evidence suggesting the individual roles of the two subunit proteins is also presented. MATERIALS AND METHODS Preparations. Thermostable ATPase (TFI) and the DCCD-sensitive ATPase complex (TFo0F1) were purified from the thermophilic bacterium PS3 (20) as described (19). TFo was prepared by treating TF0-Fj with 7 M urea as described (19), with slight modifications: a solution (20 ml) of purified TF0-Fj (40-100 mg of protein) in 7 M urea/0.2 M sucrose/5 mM EDTA/5 mM dithiothreitol/50 mM Tris-H2SO4, pH 8.0 was stirred for 2.5-3 hr at 50, diluted with distilled water (6 ml) to reduce the concentration of urea, and centrifuged at 200,000 X g for 40 min. This process was repeated once more and the resultant precipitate (TFo) was suspended in 10 mM N[tris(hydroxymethyl)methyl]glycine (Tricine)/NaOH (pH 8.0) containing 2 mM EDTA and stored at -80° until use. About one-fifth of TFo-F1 was recovered as TFo. The method for reconstitution of TFo or TF0-F1 into vesicles was as described (8, 19) except for the protein concentration of TFo used (0.15-0.3 mg/ml). TFo treated with CM-cellulose was prepared as follows: TFo (3 mg of protein) was incubated in 2 ml of 4 M urea containing 0.25% Triton X-100 and 10 mM phosphate buffer (pH 7.0) for 1 hr at 500 and then applied to a column of CMcellulose (1 X 6 cm) equilibrated with the same solution. The unabsorbed material was collected and precipitated with ammonium sulfate (45% saturation) in the presence of 2% cholate (18). Assay of Proton Permeability of Vesicles. An aliquot (0.2 ml) of reconstituted vesicles was incubated with 0.5 M KCI (2 ml), 0.1 M EDTA (0.1 ml), and 0.5 M dithiothreitol (0.02 ml) for 30 min at 55°. Then the mixture was cooled and centrifuged at 105,000 X g for 20 min, and the precipitate was suspended in 0.2 ml of 0.5 M sucrose containing 2.5 mM MgSO4. The proton-conducting activity of the K+-loaded vesicles obtained was followed by using 9-aminoacridine as an indicator of pH difference (7), in response to an artificial membrane potential (inside negative) imposed by K+ diffusion mediated by valinomycin as described (19). In some experiments the amount of proton uptake of these K+-loaded vesicles was also titrated with a pH electrode, as described (19). Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; H+-ATPase complex, proton-translocating ATPase complex (DCCD-sensitive ATPase, or Fo-Fj); Tricine, N-[tris(hydroxymethyl)methyl]glycine. 4219
4220
Biochemistry:
Sone et al.
Proc. Natl. Acad. Sci. USA 75 (1978) Valinomycin
A
T
0
20%
.0
Biochemistry
Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits (dicyclohexylcarbodiimide-sensitive ATPase/H+ pathway/dicyclohexylcarbodiimide-binding subunit/soluble ATPase-binding subunit/reconstituted vesicles)
NOBUHITO SONE, MASASUKE YOSHIDA, HAJIME HIRATA, AND YASUO KAGAWA Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan 329-04
Communicated by Leon A. Heppel, June 19,1978
ABSTRACT The HW-translocating ATPase complex from the thermophilic bacterium PS3 (TFo.F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TFo). TFo was obtained by treating TFo0F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TFo contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TFo vesicles). The binding of TF1 to TFo present in vesicles restored energ-transducing activities, such as ) I exchange, dicyclohexylcarbodiimide-sensitive ATPase, and ATP-ependent enhancement of 8-anilinonaphthalene-l-sulfonate fluorescence. Treatments such as protease digestion and chemical modification with acetic anhydride, succinic anhydride, or diazobenzenesulfonic acid destroyed the TF1-binding activity, which was caused by band 6 protein. Band 8 protein was a proteolipid that reacted specifically with dicyclohexylcarbod iimide and seemed to play a central role in H+ conduction through the membrane. Accumulating evidence has indicated that biomembranes that synthesize ATP contain a reversible H+-translocating ATPase complex, named Fo-Fl, as an energy transducing coupling device (1-6). The N,N'-dicyclohexylcarbodiimide (DCCD)sensitive ATPase complex purified from the thermophilic bacterium PS3 (TFo-Fl), when reconstituted into liposomes, was actually able to translocate protons coupled to ATP hydrolysis (7, 8). The electrochemical proton gradient (A7gH+) formed (inside acidic and positive) by ATP hydrolysis was as much as 300 mV (7), and ATP synthesis took place when a L\IH+ of over 200 mV was imposed across the liposomal membranes (6, 9). It seems both interesting and important to determine the subunit structure of Fo-Fl, especially for clarifying the method by which this transducer works. The catalytic and peripheral moiety of the transducer is Fl-ATPase, which is composed of five subunits and has a molecular weight of about 3.8 X 105. Recently these five subunits (a, f3, y, 6, and c) were each purified in reconstitutively active forms from TF1 (thermophilic F1) and their roles in TFo-F1 were proposed (10, 11). The membrane integral part (Fo) of Fo-F1 was formerly defined as a factor(s) that renders F1-ATPase sensitive to energy transfer inhibitors, such as oligomycin and DCCD (12, 13). However, little is known about the constitution of Fo except that the smallest subunit of Fo, to which DCCD binds covalently, is extractable into organic solvents (14, 15). This component, DCCD-binding proteolipid, may be important as a H+ channel across membranes, since H+ conduction is known to be blocked by DCCD. Nelson et al. (16) claimed that a proteolipid fraction purified from lettuce chloroplasts mediated DCCD-sensitive H+ translocation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
TFo can be prepared from TF0-Fj by removal of TF1 (17, 18). Recently, we reported that this preparation contained three different polypeptides and mediated proton conduction that was blocked by addition of either TF1 or DCCD (19). This communication reports that a preparation of TFo consisting of only two kinds of subunits can mediate all the activities of Fo, such as H+ translocation, conferring of DCCD sensitivity, TF1 binding, and energy-transducing reactions with TF1. Evidence suggesting the individual roles of the two subunit proteins is also presented. MATERIALS AND METHODS Preparations. Thermostable ATPase (TFI) and the DCCD-sensitive ATPase complex (TFo0F1) were purified from the thermophilic bacterium PS3 (20) as described (19). TFo was prepared by treating TF0-Fj with 7 M urea as described (19), with slight modifications: a solution (20 ml) of purified TF0-Fj (40-100 mg of protein) in 7 M urea/0.2 M sucrose/5 mM EDTA/5 mM dithiothreitol/50 mM Tris-H2SO4, pH 8.0 was stirred for 2.5-3 hr at 50, diluted with distilled water (6 ml) to reduce the concentration of urea, and centrifuged at 200,000 X g for 40 min. This process was repeated once more and the resultant precipitate (TFo) was suspended in 10 mM N[tris(hydroxymethyl)methyl]glycine (Tricine)/NaOH (pH 8.0) containing 2 mM EDTA and stored at -80° until use. About one-fifth of TFo-F1 was recovered as TFo. The method for reconstitution of TFo or TF0-F1 into vesicles was as described (8, 19) except for the protein concentration of TFo used (0.15-0.3 mg/ml). TFo treated with CM-cellulose was prepared as follows: TFo (3 mg of protein) was incubated in 2 ml of 4 M urea containing 0.25% Triton X-100 and 10 mM phosphate buffer (pH 7.0) for 1 hr at 500 and then applied to a column of CMcellulose (1 X 6 cm) equilibrated with the same solution. The unabsorbed material was collected and precipitated with ammonium sulfate (45% saturation) in the presence of 2% cholate (18). Assay of Proton Permeability of Vesicles. An aliquot (0.2 ml) of reconstituted vesicles was incubated with 0.5 M KCI (2 ml), 0.1 M EDTA (0.1 ml), and 0.5 M dithiothreitol (0.02 ml) for 30 min at 55°. Then the mixture was cooled and centrifuged at 105,000 X g for 20 min, and the precipitate was suspended in 0.2 ml of 0.5 M sucrose containing 2.5 mM MgSO4. The proton-conducting activity of the K+-loaded vesicles obtained was followed by using 9-aminoacridine as an indicator of pH difference (7), in response to an artificial membrane potential (inside negative) imposed by K+ diffusion mediated by valinomycin as described (19). In some experiments the amount of proton uptake of these K+-loaded vesicles was also titrated with a pH electrode, as described (19). Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; H+-ATPase complex, proton-translocating ATPase complex (DCCD-sensitive ATPase, or Fo-Fj); Tricine, N-[tris(hydroxymethyl)methyl]glycine. 4219
4220
Biochemistry:
Sone et al.
Proc. Natl. Acad. Sci. USA 75 (1978) Valinomycin
A
T
0
20%
.0
