Eur. J . Biochem. 126, 149-153 (1982) FEBS 1982

ATP Synthetase (FIFO) of Escherichia cob K-12 High-Yield Preparation of Functional Fo by Hydrophobic Affinity Chromatography

Erwin SCHNEIDER and Karlheinz ALTENDORF

Fachrichtung Mikrobiologie, Fachbereich Biologie, Universitat Osnabruck

(Received March 17, 1982)

1. The purified ATP synthetase complex (FIFO) from Escherichia coli was adsorbed to immobilized poly- (L-1ysine)-deoxycholic acid. About 0.7 mg FIFO were bound per ml of settled gel. The hydrophilic F, part was dissociated from the complex by treatment with 7 M urea. F, was eluted in high yield either with deoxycholate (6 mM) or taurodeoxycholate (10 mM). About 14 of the total protein bound to the column was eluted as F,, which corresponds to 64% of the total F, in the FIFO complex.

2. The purified F, preparation obtained was composed of three different kinds of subunits with apparent molecular weights of 24000 (a), 19000 (b) and 8300 (c), respectively as determined by sodium dodecyl sulfate gel electrophoresis.

3. After incorporation into liposomes and the generation of a potassium diffusion potential by valinomycin, the F, preparation mediated H + translocation. This H + uptake is inhibited by either dicyclohexylcarbodiimide or purified F, ATPase.

4. Incubation of F,-containing liposomes with F, led to the reconstitution of an ATP-driven quenching of acridine-dye fluorescence. The quenching was abolished by uncoupler and prevented by dicyclohexylcarbodiimide.

ATP synthetase complexes, which play an important role in oxidative phosphorylation and in photophosphorylation, have been found in mitochondria, bacteria and chloroplasts (see [l - 31 for review). In Escherichia coli the complex (F, F,) is composed oftwo entities: the F1 part which is water soluble and carries the catalyticcenters of theenzyme and the F, part which is embedded in the cytoplasmic membrane and is involved in H + translocation. The properties of F, have been recently reviewed [4]. Procedures for the purification of the FIFO complex have been reported by Foster and Fillingame [5] as well as by Friedl and coworkers [6].

Several groups have recently reported on procedures for the isolation of theFopart startingfrom theFIFO complex[7-9].It seems now well established that F, is composed of three different kinds of subunits (a, b and c) [2]. However, DNA- sequencing studies may suggest that a fourth subunit exists [lo, 111 (see Discussion for detailed comments on the subunit composition).

As a prerequisite for further elucidation of the structure and function of F, and its individual subunits, purified F, has to be obtained in higher yields than have been achieved so far.

Recently, a new material has been introduced for hy- drophobic chromatography of membrane proteins by Cresswell [12]. I t contains deoxycholic acid covalently linked to poly(L-1ysine)-coupled agarose beads. This material, in con- trast to common hydrophobic gels, carrying alkyl or aryl groups, shows no interaction whatsoever with water-soluble proteins.

Erzzymr. ATPase (F,) or ATP synthetase (F,F,) (EC 3.6.1.3). Ahhuei~iations. EDTA (ethylenedinitrilo) tetraacetic acid; EGTA,

ethylene bis[oxoethylenenitrilo)]tetraacetic acid; Mes, 4-morpholineeth- anesulfonic acid; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]- glycine.

In the present communication we describe the successful use of this gel to isolate a functional F, in high yield starting from purified F,Fo.

MATERIALS AND METHODS

Chemicals were purchased from the following companies : soybean phospholipids, dicyclohexylcarbodiimide, deoxy- cholic acid, sodium salt, taurodeoxycholic acid, sodium salt, Mes and Tricine from Sigma (Miinchen); acrylamide (4 x recrystallized), A’,”-methylenebisacrylamide (1 x recrystal- lized), dithiothreitol and urea from Serva (Heidelbergj; cho- late, sodium salt and EGTA from Merck (Darmstadt); phe- nylmethylsulfonyl fluoride from Calbiochem (Giessen); p-aminobenzamidine from EGA (Steinheim); sodium dodecyl sulfate from BDH (Poole, UK j; immobilized poly(~-lysinej- deoxycholic acid from Pierce (Rockford, USA). 9-Amino-6- chloro-2-methoxyacridine and 4,5,6,7-tetrachIoro-2-tri- fluoromethylbenzimidazole were gifts from Dr. Friedl, Braunschweig. All other chemicals were of analytical grade.

Bacterial Growth

Escherichia coli K-12 Ym,, (i) was grown in the minimal medium of Davis and Mingioli [I31 with 0.2 glucose as a carbon and energy source. The cells were harvested in the late logarithmic phase.

Preparations

Soybean phospholipids were partially purified as described by Sone et al. [14]. Urea was recrystallized twice from ethanol. ATP synthetase complex was prepared as in [6] with the modification described in [8]. F ,-ATPase was isolated accord- ing to Vogel and Steinhart [lS].

150

Assays

Protein was determined by the method of Lowry et al. [I61 with the modification of Dulley and Grieve [17]. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was carried out according to Laemmli [18] on 13 % acrylamide gels at 2 mA/gel. The gels were stained as in [19]. Protein samples were denatured at room temperature 5 min prior to application to the gels.

The acridine dye fluorescence measurements were carried out with an Aminco fluorimeter SPF 500 using the wavelength 410 nm for excitation and 490 nm for emission. The assay medium in the cuvette (1 x 1 cm) was kept at 25 "C.

pH changes were monitored continously with a Radiometer electrode (no. G K 2321 C) connected to a Radiometer pH meter (model PHM 84) and an Abimed recorder (model Linear 800). The medium in the glass vessel was stirred and kept at 25 "C. Small amounts of standard concentrations of HCI were used for calibration.

Isolation of F,

Purified ATP synthetase (14.1 mg) in 0.5 ml 50 mM Tris/HCl, pH 8.0, 10 mM taurodeoxycholate, 1 mM MgCl,, 0.2 mM dithiothreitol, 0.2 mM EGTA, 0.1 mM phenylmethyl- sulfonyl fluoride, 6 mM p-aminobenzamidine, 20 % (v/v) methanol and 50 pg/ml soybean phospholipids was diluted and adjusted to 150 mM NaCl by the addition of an equal volume of 300mM NaCI. The solution was layered onto a column (1 x 20 cm) containing immobilized poly(L-1ysine)-deoxycholic acid, previously equilibrated with 10 mM TrislHCl, pH 8.0, 150 mM NaCl (buffer A). The loaded column was washed with the following buffers: (1) two bed volumes of buffer A ; (2) one bed volume of 10 mM Tris/HCl, pH 8.0,150 mM NaC1,5 mM EDTA, 5 mM dithiothreitol, 10% (v/v) glycerol, 7 M urea (buffer B); (3) two bed volumes of buffer A ; (4) one bed volume of 10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 10 mM tauro- deoxycholate (buffer C). Protein-containing fractions eluted by step [4] were pooled and concentrated by passage through an ultrafiltration membrane (Amicon XM-50). Subsequently the protein was frozen and stored under liquid nitrogen. All steps were carried out at 4 "C.

The column was regenerated by washing with several bed volumes of 10 mM Tris/HCl, pH 8.0,150 mM NaCl, 2 % (w/v) deoxycholic acid and subsequent reequilibrated with buffer A.

Reconstitution of H'-Translocating Activity

Liposomes were prepared and F, was incorporated as described by Okamoto et al. [20] with the following minor modifications. The weight ratio of phospholipid to protein was 80: 1, the dialysis buffer was changed once and the dialysis was carried out at 4°C for 18 h. F,-containing liposomes were loaded with K' as described by the method of Sone et al. [21].

Reconstitution of' A TP-Driven Acridine Dye Fluorescence Quenching

FIFO and F, were incorporated into liposomes by a modification of the freeze/thaw/sonication procedure of Kasahara and Hinkle [22]. Soybean phospholipids were sus- pended at 40 mg/ml in 20 mM Mes/NaOH, pH 6.3, 0.25 mM EDTA and sonicated (Branson B 14, microtip, output 50 W) in an ice-bath under a stream of nitrogen for 5 min. Appropriate amounts of protein were mixed with loop1 phospholipid

solution and MgCl, (2 mM) to a final volume of 150 pI and thoroughly vortexed. The mixture was then frozen in liquid nitrogen for at least 5 min, thawed at room temperature for about 15 min and sonicated in a bath type sonicator for 30 s at room temperature.

RESULTS

Isolation of F,

The purified ATP synthetase of E. coli K-12 was adsorbed to a column of immobilized poly(L-1ysine)-deoxycholic acid. Excess of enzyme which was not bound to the gel was removed by washing the column with equilibration buffer A. The binding capacity of the gel was about 0.7 mg F,F,/ml settled gel. Dissociation of the enzyme and elution of F, was achieved by washing the gel with buffer B, containing 7 M urea, 5 mM dithiothreitol, 5 mM EDTA, 10% (v/v) glycerol, 150 niM NaCl and 10 mM Tris/HCl, pH 8.0. Glycerol had to be present to obtain an intact Fo preparation. At 5 M instead of 7 M urea the y-subunit still remained bound. The eluted F, fraction, which always contained some F, F,, did not show any ATPase activity in the presence of urea, However, the enzymatic activity was partially restored by dialysis against 50 mM Tris/HCl, pH 8.0, 1 mM ATP, 1 mM MgCI,, 20% (v/v) methanol and 2.5 mM 2-mercaptoethanol (data not shown). We have tried several other dissociating agents to remove F, from the column. Washing the protein-loaded column with low-ionic strength buffer in the presence of EDTA resulted in a F, preparation which was still contaminated with rather large amounts of mainly c1 and p subunits. Sodium bromide (up to 3.5 M) did not dissociate any F, (data not shown).

After an intermediate washing step with buffer A, F, was eluted by buffer C, containing either 10 mM tauro- deoxycholate or 6 mM deoxycholate. Lower concentrations of the detergent were not effective. The elution profile of the column is shown in Fig. 1. About 14 o/, of the protein bound to the column was eluted in the F, fraction. The F,-containing

5 to 15 20 Fraction number

Fig. 1. Elution profile of hydrophohic chromatography. F,F, was applied onto the column of immobilized poly(L-1ysine)-deoxycholic acid and the washing steps were carried out as in Materials and Methods. Buffer A : 10 mM Tris/HCl, pH 8.0, 150 mM NaCl; buffer B: 10 mM Tris/HCI, pH 8.0,1 SO mM NaC1,S mM EDTA, S mM dithiothreitol, 10 % (v/v) glycerol, 7 M urea; buffer C: 10 mM Tris/HCl, pH 8.0, IS0 mM NaCl, 10 mM taurodeoxycholate. The flow rate was 1 .S ml/h and the fraction volume was S ml. The absorbance values at 280 nm (0) were corrected for fractions containing buffer B. The high absorbance of the first peak was in part caused by the buffer in which the FIFO protein was dissolved (see Materials and Methods). (0) Protein in mg

151

Table 1 . Summary of isolation steps of Fo Experimental details of the procedure for the isolation of Fo are described in Materials and Methods and in the legend to Fig. 1.

~

Step Protein Yield ~~ ~

mg x F,F, (bound to the column) 11.0 100 F, F, (applied to the column)

Buffer B eluate" 8.6 78 Buffer C eluate 1.5 14

14.1

a Containing urea Containing taurodeoxycholate

Table 2. Blocking of H + conduction in F,-containing liposomes by dicyclo- hexylcarhodiimide and F,-ATPase The experimental conditions are identical as in Fig. 3

Addition Initial velocity of H + translocation

nmol H+ x min- ' x mg-

None 2625 Dicyclohexylcarbodiimide

(20 PM) 38 F,-ATPase (40 pg) 71

A 0

C

0 d

Fig. 2. Densitometric traces of sodium dodecyl sulfarelpolyacrylamide gels of F,F, andF,. Gel electrophoresis, staining and destaining was carried out as described in Materials and Methods: lane A and B: 14 pg of F,F, and F,, respectively

fractions were pooled and concentrated by ultrafiltration through an Amicon XM-50 membrane. A summary of the isolation steps is given in Table 1.

On sodium dodecyl sulfate/polyacrylamide gels the F, preparation revealed three polypeptides with the apparent molecular weights of 24000, 19000 and 8300, respectively (Fig. 2). Minute amounts of the a and /3 subunits of F, and of a polypeptide with the apparent molecular weight of 14000 were still present (see Discussion).

Reconstitution of H + - Translocating Activity

The purified F, preparation was incorporated into lipo- somes prepared from partially purified soybean phospholipids by the cholate-deoxycholate-dialysis procedure [20]. The result- ing proteoliposomes were loaded with K + as previously described by Sone et al. [21]. Addition of valinomycin induced a K + diffusion potential across the membrane, internally neg- ative. This provided a driving force for H+ uptake, which in its turn was monitored by a pH-electrode. As shown in Table 2 the initial rate of H+ uptake was about 2600nmol H+ x min- ' x mg protein- Valinomycin-induced H+ uptake was completely blocked by the addition of either the specific inhibitor dicyclo-

A

0 C D

valino- mycin

Fig. 3. Proton conduction in F,-containing liposomes. (A) K+-loaded F,- containing liposomes (50 11, containing 10 pg of protein, prepared as in Materials and Methods) were suspended in a final volume of 2 ml in 0.2 mM Tricine/NaOH, 0.2 M Na2S04, 5 mM MgSO,. The initial pH was 7.16. After an equilibration period of about 10 min proton uptake was started by the addition of 20 ng of valinomycin in 2 p1 methanol. (B) F,- ATPase (40 pg) was added to 50 p1 ofF,-containing liposomes, incubated for 20 min at 25°C and subsequently suspended in assay buffer. (C) F,- containing liposomes were incubated in the assay buffer with dicyclohexyl- carbodiimide (20pM) for 20min at 25"C, prior to the addition of valinomycin. (D) Control liposomes, lacking Fo

hexylcarbodiimide or by purified F,-ATPase. Control lipo- somes, lacking F,, did not show any H+-translocating activity after the addition of valinomycin (Fig. 3; Table 2) .

Reconstitution of ATP-Driven Quenching of Acridine Dye Fluorescence

Incorporation of F, into liposomes by the freeze/ thaw/sonication procedure of Kasahara and Hinkle [22] and subsequent incubation with F, resulted in the reconstitution of an active FIFO complex. This was shown by the quenching of the fluorescence of the acridine dye 9-amino-6-chloro-2- methoxyacridine after the addition of ATP (Fig. 4). Maximal quenching was obtained with a F,/Fo weight ratio between two and three. Neither F,-containing liposomes alone nor F, together with pure phospholipid vesicles showed any quench- ing activity. The quenching was abolished by the uncoupler 4,5,6,7-tetrachioro-2-trifluoromethylbenzimidazole and com- pletely inhibited by preincubation of the proteoliposomes with dicyclohexylcarbodiimide (Fig. 4).

152

A

' f t

lmin - 1 t t t t t Y C L H H

t- k 6 6

ft t -a H

%

Fig. 4. Reconstitution ofATP-driven quenching of acridine-dyefluorescence. F,F,-containing and F,-containing liposomes were prepared as in Materials and Methods: 50 p1 of proteoliposomes were suspended in 20 mM Tricine/NaOH, pH 8.0, 10 mM MgCl,, 300 mM KCl to a final volume of 2 ml and incubated with valinomycin (1.6 pM) for 5 min at 37 "C. The fluorescence of the sample was set at zero. After the addition of 9-amino-6-chloro-2-methoxyacridine (2 pM) the fluorescence was set as 100 %. The reaction was started by the addition of ATP (2 mM). (A) FIFO- containing liposomes (27 pg); (B) Fo-containing liposomes (6.8 pg); (C) FI (20 pg) + pure liposomes; (D) F,-containing liposomes (6.8 pg) + F, (20 pg); (E) as D, but incubated with dicyclohexylcarbodiimide (80 pM) in test buffer for 20 min at 25 "C, prior to the addition of valinomycin. (I) 9-Amino-6-chloro-2-methoxyacridine; (11) 4,5,6,7-tetrachloro-2-tri- fluoromethylbenzimidazole (20 pM)

DISCUSSION

Preparations of the F, part of the ATP synthetase complex from E. coli have been published by three different groups [7 - 91. However, the yield in F, which can be obtained by these procedures is unsatisfactory. In the method employed by us so far [8] we obtained 2.5 mg of F, by treatment of a solution of 45 mg FIFO with urea. This corresponds to a protein yield of about 5.6 %. Negrinet al. [7] as well as Fried1 and Schairer [9] do not specify the yield of their methods.

As a prerequisite for further investigations on the H + translocation process it is necessary to obtain the purified Fo component in rather large amounts. Therefore we used the new approach to the isolation of F, described in this paper. The new method, involving the binding of purified FIFO to a column of immobilized poly(L-1ysine)-deoxycholic acid, increased the protein yield in F, up to 14 % which is more than twofold that obtained by our previous method [8].

Based on the assumptions that (a) the molecular weight of F,F, is 460000 [23, 241 and (b) the molecular weight of F, is 360000 [25], the molecular weight of F, can be calculated as

100000. A protein yield in F, of 14 % corresponds then to 64 % of the total F, present in the bound FIFo complex.

The success of the new procedure described here seems to be based on (a) the immobilization of the F , F, molecules on the deoxycholic-acid-coupled poly(~-1ysyl)-agarose prior to the attack by the dissociating agent and (b) the absolute lack of interaction of this gel with water-soluble proteins, e. g. the dissociated F, molecules. This is a fundamental advantage over other hydrophobic gels, such as octyl-Sepharose or phenyl- Sepharose. Therefore, this new hydrophobic material quite generally should be a useful tool for the purification of membrane proteins from water-soluble proteins.

The F, preparation described in this paper contained three different polypeptides with apparent molecular weights of 24000 (a), 19000 (b) and 8300 (c), respectively. This is in accordance with the results described by other groups [7,9]. On the basis of recent DNA sequencing studies these values have now to be corrected to 30267 for subunit a and 17212 for subunit b (10). The molecular weight of the smallest subunit c, which is identical to the dicyclohexylcarbodiimide-reactive protein, is deduced from its amino acid sequence as 8288

The results on the present F, preparation differ from our previous results [8, 271 in two respects. First, in the previous preparation we did not detect subunit a. However, it now appears that this was due to its extremely low affinity for the protein-staining dye, Coomassie blue. Using a modified proce- dure 1191 or the silver-staining method of Oakley et al. [28] we have demonstrated subunit a unequivocally. Second, in the previous preparation we found an additional polypeptide with the apparent molecular weight of 14000, which varies with different batches of cells, so that its role as a subunit of F, remains to be established (see also Fig. 2). However, there is now some evidence from DNA sequencing studies that a fourth subunit with approx, that molecular weight may exist [lo, 111. Experiments on the possible identity of this polypeptide with the gene product reported in [lo, 111 are in progress.

To be considered active a F, preparation has to fulfil the following criteria after incorporation into phospholipid ves- icles: the resulting proteoliposomes have to exhibit (a) H + translocation, utilizing a K + diffusion potential across the membrane as driving force and (b) binding of F, to reconstitute a functional FIFO complex; both activities must be sensitive to the specific inhibitor dicyclohexylcarbodiimide. Table 2 and Fig. 3 (see Results) clearly demonstrate that the F, preparation described here is active in H + translocation. The initial rate of H+ translocation corresponds to 4.4 H + x s-' x F, mole- cule- ', assuming that all F, molecules were active. This value is comparable with data which can be calculated from the results reported by other groups [7, 91.

The reconstitution of an active F IFO complex in liposomes from isolated F, and F, was demonstrated by the ATP-driven quenching of acridine-dye fluorescence (Fig. 4). The fact that this quenching was abolished by uncoupler and inhibited by preincubation of dicyclohexylcarbodiimide indicates that F, and F, were correctly assembled. Taken together, our Fo preparation fulfils the criteria stated above, thereby dispelling the notion that urea at 7 M would irreversibly damage the enzyme [9].

[261.

We thank Drs. Tilly Bakker-Grunwald, Evert Bakker and Roland Schmid for critical revision of the manuscript and useful discussions. This work was supported by the Deutsche Forschungsgemeirwhaft (A1 118/7-3), the Fonds der Chrmischen Industrie and the Ministeriurn f i r Cl/issrr?schafi und Kunst (Niedersachsen).

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E. Schneider and K. Altendorf, Fachrichtung Mikrobiologie, Fachbereich Biologie, Universitzt Osnabruck, Seminarstrane 20, D-4500 Osnabriick, Federal Republic of Germany