Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome

ARCHIVES

OF

BIOCHEMISTRY

AND

Energy-Linked Adenosine

BIOPHYSICS

Activities in Reconstituted Yeast Adenosine Triphosphatase Proteoliposome

Triphosphate

Formation Coupled with Electron and Ferricyanide

IVAN J. RYRIE Department

176, 127-135 (1976)

of Developmental

AND

Flow between

Ascorbate

PETER F. BLACKMORE’

Biology, Research School of Biological Sciences, Australian University, CUnbeFFU, A.C.T. 2601, Australia

National

Received February 3, 1976 (1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-32Pi exchange. The vesicles so formed exhibit proton uptake upon addition of Mg2+-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-3*Pi exchange by valinomycin + K+ reported previously (Ryrie, I. J. (1975)Arch. Biochem. Biophys. 168,704-711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K+ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-3ZP, exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [32P]ATP formation from ADP and 32P,is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a lightdependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.

As presently viewed, the oxidative phosphorylation apparatus of the inner mitochondrial membrane consists of two functionally separable segments: a highly ordered, multicomponent electron transport chain and an ATPaseZ complex responsible 1 Present address: Department of Physiology, School of Medicine, Vanderbilt University, Nashville, Tenn. 37232. ’ Abbreviations used: ATPase, adenosine triphosphatase; F,, mitochondrial coupling factor 1; F,, membrane factor required to confer oligomycin sensitivity on F,; PMS+-PMSH, oxidized and reduced forms of phenazine methosulfate; DCIP, 2,&dichloroindophenol; TPB-, tetraphenylboron anion; TPAs+; tetraphenylarsonium cation; CCCP, carbony1 cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; S-13, 5-chloro-3-tert-buty1-2’-chloro-4’-nitrosalicylanilide; Tricine, tris(hydroxymethyl)methylglycine.

for coupling ATP synthesis with the exergonic redox reactions. Despite extensive efforts no conclusive evidence has yet been found for a direct transfer of energy, covalent or otherwise, between the electron transport chain and the ATPase. On the other hand, considerable evidence has accumulated supporting the concepts of Mitchell (1) that electron flow is coupled with proton translocation and that the electrochemical membrane potential so generated is utilized directly for ATP synthesis. Indeed recent speculation on the phosphorylation mechanism (2-5) is mostly in accord with the latter concepts, and interest seems centered on the mechanism by which the “proton motive force” is utilized. An important corollary of the chemiosmotic theory is that the purified ATPase 127

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

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complex, when inlaid into a proton-impermeable phospholipid vesicle, might catalyze net ATP synthesis and the energylinked exchange reactions. given an adequate transmembrane proton gradient. Mitochondrial ATP-32Pi exchange activity was first reconstituted from solubilized membrane components by Kagawa and Racker (6) who used phospholipids, a hydrophobic membrane sector containing F,, the oligomycin-sensitivity-conferring protein (OSCP), and F,. The membrane fraction, which constitutes approximately 80% of the reconstituted membrane protein (61, is rather ill defined, however, and contains numerous polypeptide components (7). Notwithstanding, the proteoliposomes were later shown to be capable of net ATP synthesis when a proton gradient was established using bacteriorhodopsin and light (8) or electron flow through cytochrome c-cytochrome oxidase (9, 10). Reconstitution of ATPb3”Pi exchange using a purified ATPase was finally achieved in this laboratory using the oligomycinsensitive ATPase from yeast (11, 12) and shortly thereafter by Kagawa’s group using the dicyclohexyl carbodiimide-sensitive ATPase isolated from a thermophilic bacterium (13, 14). Neither preparation exhibited exchange activity prior to reconstitution which suggests a requirement for a vesicular membrane structure containing phospholipids. Interestingly, two bovine ATPase preparations have recently been isolated which contain intrinsic ATP-32Pi exchange activity (15, 16). HOWever, this may simply indicate the presence of remaining vesicles. Indeed at least one of these preparations existed completely as membrane fragments (15) while exchange activity in the other was markedly stimulated by addition of phospholipid vesicles (16). It is the purpose of the present communication to examine further the energytransducing reactions of the reconstituted yeast ATPase complex. Conditions are described wherein ATPase proteoliposomes with extremely high ATP-32Pi exchange activity are formed and where net ATP synthesis in these vesicles can be driven by electron flow between ascorbate and ferri-

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cyanide. Taken together, these observations provide further support for the notion that electron flow is secondary and that phosphorylation is more directly linked with the proton motive force. They also confirm previous suggestions (11, 17) that the yeast ATPase complex contains the complete assembly of coupling proteins. EXPERIMENTAL PROCEDURES Purification of the ATPase and reconstitution of proteoliposomes. Growth of the yeast cells and purification of the ATPase were carried out as described previously (12) except that cells were grown under an air atmosphere in l-liter batches on a rotary shaker. Final purification of the ATPase through Sepharose 4B was often omitted since the purity is only slightly increased by this step (12). The ATPase proteoliposomes were formed by dialysis of an ATPase-phospholipid-cholate mixture. Partially purified (61 soybean phospholipids were used which were first sonicated (11) for 10 min under a nitrogen atmosphere in a solution containing 1 mM EDTA, 1% (w/v) sodium cholate, and 50 mM TricineNaOH (pH 8.0). In a final volume of 1.0 ml, 1 mg of ATPase protein was combined with 10 mg of phospholipids and 10 mg of sodium cholate then dialyzed for 20 h at 3°C against a medium containing 0.1 mM ATP, 0.2 mM EDTA, 1 mM dithiothreitol, 5% (w/v) methanol, 25 mM K,SO, and 10 mM Tricine-NaOH (pH 8.0). In some experiments (cf. Table I) K,SO, was either omitted or replaced by other salts. The medium was replaced three times during dialysis. Preparation of bacteriorhodopsin. Halobacterium hulobium (wild type, kindly provided by Dr. Gottfried Wagner) was grown to stationary phase under illumination in 50-ml batches at 30°C. The growth medium and method for preparing the purple membranes were as described by Oesterhelt and Stoeckenius (18). Methods. ATP-32P, exchange was measured as described previously (11). Inhibitors and ionophores added in methanol were taken to dryness before aqueous reagents were added. ATP synthesis linked with electron flow between ascorbate and ferricyanide was measured for 3 min at 37°C in reaction mixtures which contained, in 0.5 ml, 25 rnM K,SO,, 30 mM glucose, 5 mM MgS04, 1 mM ADP, 5 mM KP, (containing 10’ cpm of 32Pi), 30 units of yeast hexokinase (Sigma), 10 mM sodium ascorbate, 50 PM PMSH (or an alternative electron carrier), 1 mg of defatted bovine serum albumin, and 50 mM Tricine-NaOH (pH 7.5). Reactions were initiated by the addition of 50 ~1 of ferricyanidecontaining vesicles (40-45 gg of protein) which were prepared as follows: 1 M K,Fe(CN), (pH 8.0) was added to proteoliposomes to a final concentration of 100 mM, then the vesicles were sonicated for 3-5 s at

ENERGY-LINKED

ACTIVITIES

IN ATPase

TABLE EFFECTS OF VALINOMYCIN Additions

to dialysis dium

Experiment None

me-

Experiment None

I

+ K+ ON ATP-32P, EXCHANGE ACTIVITY IN ATPA~E PROTEOLIPOSOMES~ ATP-32P, exchange Additions to assay specific activityb

Percent

1 50 mM KC1 1 rg/ml valinomycin 1 pg/ml valinomycin 25 /LM TPB25 /.LM TPAs+

Experiment None

129

PROTEOLIPOSOMES

833 768 878 1566 1242 789

100 92 105 188 149 95

970 1510 1415 1865

100 156 146 192

+ 50 mM

1712 2618 2415 2905 1979 2228

100 153 100 120 100 113

+ 25 mM K&SO.,

2800 2566

100 92

+ 50 mM K,SO,

1428 1618 1284 1513

100 113 90 106

+ 50 mM KC1

2 50 mM KC1 1 pg/ml valinomycin 1 pg/ml valinomycin 1 pg/ml valinomycin

+ 50 mM KC1 + 50 mM KNO, + 25 mM KzSOa

3

50 mM KC1 50 mM KCH,COO 25 mM K,SO, Experiment 4 25 mM K&SO,

1 pg/ml valinomycin 1 Fg/ml valinomycin 1 pg/ml valinomycin KCH,COO 1 pg/ml valinomycin 1 pg/ml valinomycin 50 mM K,SO, 1 pg/ml valinomycin

+ 50 mM KC1 + 50 mM KC1

a Reconstitution of proteoliposomes was carried out by the cholate dialysis procedure. The dialysis medium contained either no K,SO, or the K+ salt additions shown in column 1. * Values are given in nanomoles of t3*PIATP formed per milligram of protein per 10 minutes. 0°C using a Branson Sonitier at a setting of 3 A. The preparation was then dialyzed for 30 min at lo-15°C against 25 mM K,SOI, 0.2 mM EDTA, and 10 mM Tricine-NaOH (pH 8.0) to lower the concentrations of methanol and K,Fe(CN),. The vesicles were used immediately. Separation of 32Pi before counting was carried out according to Avron (19) except that the ascorbate was first titrated with concentrated KI-I,. RESULTS

Reconstitution

of ATP-32Pi Exchange

Previous work (11) has shown that the ATPw3’Pi exchange activity reconstituted by the cholate dialysis procedure was markedly stimulated by the ionophore valinomycin (or monactin) in the presence of external K+. As shown in Table I, Experiment 1, valinomycin + K+ stimulated exchange activity by 38% whereas neither reagent alone had much effect. The stimulation was uniform over the lo-min assay

period.3 Interestingly, the TPB- anion also significantly stimulated whereas the TPAs+ cation did not. Exchange activity was enhanced where either KCl, KN03, or K&SO, was used as the K+ salt although K,SO, was the most effective (Experiment 2). The effect of valinomycin + K+ on the exchange activity in vesicles preloaded with internal K+ salts, i.e., vesicles formed by dialysis in the presence of K+ saIts, is shown in Experiment 3. It is notable, first, that exchange activities were higher when K+ salts were included during reconstitution and, second, that now neither valinomycin + K+ nor TPB-,3 effected very much increase in ATPe3’Pi exchange. Where a small stimulation was observed it was much the same whether ’ I. J. Ryrie,

unpublished

observations.

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the external K+ concentration was lower or higher than the internal one (Experiment 4). Other conditions which affect the reconstitution of ATP-32Pi exchange are shown in Fig. 1. Activities were highest where dialysis was carried out at more alkaline pH (Fig. 1A) in the presence of 10 mM MgSO, or 25 mM K,SO, (Fig. 1B). While reconstitution was normally carried out at a phospholipidlprotein ratio of 10/l (w/w) a ratio of 15-20/l was more optimal (Fig. 10. Unlike the vesicles reconstituted with mammalian mitochondrial components (201, exchange activity in the present system was not stimulated by inclusion of 5% (w/w) cardiolipin in the phospholipids. Experience has shown that a critical factor in achieving high ATPd3’Pi exchange activity is the removal of excess Triton X100 from the ATPase prior to reconstitution. The final ATPase isolation step involves concentration of an ATPase-0. 1% Triton X-100 solution by Amicon pressure filtration (12); since 0.1% Triton is largely micellar, pressure filtration will concentrate the detergent to such an extent that it cannot be adequately removed by the reconstitution dialysis. Vesicle formation, or subsequent assays, may therefore be disrupted by the endogenous detergent. The best method found for lowering the Triton concentration was repeated pressure concentration of 50-ml aliquots of the ATPase using an Amicon XM-50 membrane.

BLACKMORE

oriented outwards, addition of Mg2+-ATP to the reconstituted vesicles resulted in proton uptake which was significantly inhibited by oligomycin and the uncoupler S13 (Fig. 2). Measurements were made at pH 6.25 where no net proton release occurs upon ATP hydrolysis (22). Like the vesicles reconstituted with mammalian mitochondrial components (20), decay of the proton gradient was relatively slow with a halt-time of 60-80 s. Oxidative Phosphorylation Linked with Electron Flow between Ascorbate and Ferricyanide

Vesicles with ATPo3’Pi exchange activity might be expected to catalyze net phosphorylation given another source of proton pumping besides ATP hydrolysis. Deamer et al. (23) have suggested that a ApH of 4 units could be established in liposomes when a permeable carrier such as PMSH, which produces a proton on oxidation, was used as a redox mediator between external ascorbate and internal ferricyanide. While the exactness of their method for determining the ApH values has been questioned (24), it nonetheless seems certain that a proton gradient of considerable magnitude is established. As shown in Table II, Experiments l-3, phosphorylation could indeed be observed under these conditions and was considerably stimulated by inclusion of valinomytin. Oligomycin and the protonophorous uncouplers CCCP, FCCP, and S-13 markedly inhibited the reaction whereas the ATP-Dependent Proton Uptake electron transport inhibitors rotenone, anAs in natural (21, 22) and artificial (20) timycin A, and carbon monoxide did not. membranes where the catalytic F1 sector is Both TPB- and TPAs+ only slightly in-

PH

sa4t hQ.4,

mg P- lbd,ma AT Pari prate,n

FIG. 1. Effects of pH, metal ion concentration, and phospholipid/protein reconstitution of ATP-32P, exchange activity. Proteoliposomes were reconstituted of the cholate dialysis procedure described in the text.

ratios on the by variations

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FIG. 2. ATP-dependent proton uptake in ATPase proteoliposomes. Reconstitution was carried out by the cholate dialysis method. The vesicles were further dialyzed for 3 h at 15°C against buffer containing 25 mM K,SO, and 2 mM Tricine-NaOH (pH 7.0). Aliquots containing 0.32 mg of reconstituted ATPase protein in 0.4 ml were placed in a reaction chamber at 20°C and titrated to pH 6.25 with 5 mM HCl. Proton uptake was monitored using a Radiometer GK 2321 C combination electrode attached to a Radiometer pH meter 26 and a Rikadenki strip chart recorder. The following additions were made where indicated: HCl (10 nmol), valinomytin (0.3 pg), ATP-MgS04 (20 nmol), S-13 (20 nmol), oligomycin (20 pg).

hibited the reaction. It is interesting that in liposomes, the catalytic function of PMS+ is enhanced by formation of an electrically neutral complex with TPB- (25). No requirement for TPB- was observed in the present system, however, nor in the reconstituted proteoliposomes described by Backer and Kandrach (9, 10). Phosphorylation was largely dependent on the presence of PMSH and was abolished when ascorbate was omitted or when the vesicles did not contain ferricyanide. [32PlATP formation was still observed when external ferricyanide was removed from the vesicles before assaying by predialysis against buffer containing 1 mM sodium ascorbate but not when ferricyanide was added to the vesicles (without sonication) immediately before assaying. These latter findings are consistent with a requirement for internal ferricyanide. It should be mentioned that the PMS-independent phosphorylation (Experiment 2) is variable and has frequently not been observed in most recent experiments (e.g., Experiment 5). Whether it is due to some diffusion of ascorbate into the vesicles, followed by re-

action with ferricyanide to produce a proton, is uncertain. The marked dependence on hexokinase indicates that the reaction product was ylabeled [32P]ATP; without a hexokinaseglucose trap the 13”P]ATP would be formed but degraded again by the ATPase. Benzoquinone and DCIP, both of which produce protons upon oxidation, substituted for PMS (Experiments 3 and 4). Phosphorylation was again sensitive to S13 but now was markedly inhibited by valinomycin. Little or no [32P]ATP formation was observed with ferrocene however (Experiment 51, either in the presence or absence of TPB- or valinomycin. Ferrocene is an electron but not proton carrier and is known to transfer electrons between external ascorbate and internal ferricyanide (especially in the presence of TPB-) without formation of a proton gradient (26). Taken together the findings indicate that phosphorylation capacity in the vesicles can withstand brief sonication in media containing 100 mM K3Fe(CN&. This was confirmed by measuring ATP-32P, exchange: The vesicles used in Experiment

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TABLE

II

ATP SYNTHESIS IN PROTEOLIPOSOMESLINKED WITH ELECTRON FLOW BETWEEN ASCORBATE AND FERRICYANIDE” [32PlATP formation

Assay

Specific activityb Experiment 1 Complete (50 PM PMS+ + 1 pg/ml vahnomycin) Minus valinomycin PhlS

100

20

,uM

CCCP

FCCP Plus 10 PM s-13 Plus 10 pg/ml oligomycin Experiment 2 Complete (50 PM PMS+ + 1 pg/ml valinomycin) Minus ascorbate Minus PMS+ Minus hexokinase Vesicles without K,Fe(CN& Plus 25 ,uM TPBPlus 25 PM TPAs+ Plus 20 pM rotenone Experiment 3 50 FM PMS+ + 1 pg/ml vahnomycin 50 pM PMS+ + 1 pg/rnl vahnomycin + 0.5 pM antimycin 50 PM PMS+ + 1 pg/ml valinomycin + carbon monoxide 50 PM DCIP 50 PM DCIP + 1 Fg/ml valinomycin Experiment 4 50 PM DCIP 50 FM DCIP + 1 pg/ml valinomycin 50 PM DCIP + 10 PM S-13 50 pM benzoquinone 50 pM benzoquinone + 1 pg/ml vahnomycin 50 FM benzoquinone + 10 pM S-13 Experiment 5 50 FM benzoquinone Minus benzoquinone 50 pM ferrocene 50 pM ferrocene + 1 pg/rnl vahnomycin 50 FM ferrocene + 10 p,M TPBPlus

PM

A

Percent

29.0 7.6 1.4 0 2.8 8.0

100 26 5 0 10 28

53.8 0 23.1 3.9 0 49.2 49.5 55.6

100 0 43 7 0 92 92 103

26.0 26.9 25.0 40.7 32.9

100 104 96 156 127

18.7 9.3 7.9 22.6 8.5 7.6

100 50 42 100 38 34

7.6 0 1.5 0 0

100 0 20 0 0

a The proteoliposomes were formed by dialysis of an ATPase-phospholipid-cholate mixture. The vesicles were then preloaded with K,Fe(CN&, dialyzed briefly, then assayed. Details are given under Experimental Procedures. b Values are given in nanomoles of [32PlATP formed per milligram of ATPase protein.

2, Table I, retained activity of 994 nmol of [32P]ATP formed per milligram of protein per 10 min, compared with values of 1711 and 1609 in the parent vesicles and vesicles sonicated without ferricyanide. Light-Dependent ATP Synthesis in Vesicles Containing Bacteriorhodopsin Independent evidence that the yeast ATPase complex is capable of net phospho-

rylation was obtained using bacteriorhodopsin as a light-dependent proton translocator. Vesicles containing the ATPase together with purple membrane fragments were reconstituted by the cholate dialysis method, basically as described by Racker and Stoeckenius (8). Light-dependent ATP synthesis was observed which was inhibited by uncouplers and oligomycin (Table III). No phosphorylation was observed

ENERGY-LINKED

ACTIVITIES

TABLE III LIGHT-DEPENDENT PHOSPHORYLATION IN PROTEOLIFQSOMESCONTAINING BACTERIORHODOP~IN~

Assay

13*PlATP formed (nmol/mg ATPase protein)

Complete + 10 /&LMs-13 + 20 /.&MFCCP + 10 pg/ml oligomycin darkness vesicles without purple membrane

280 5 4 13 0 0

a Reconstitution was carried out essentially as described by Racker and Stoeckenius (8); purple membrane fragments containing 0.9 mg of protein were combined with 12 mg of sonicated phospholipid and 10 mg of sodium cholate (added as a 20% (w/v) solution, pH 8.0) in a final volume of 0.8 ml and sonicated at a 4 A setting for 5 s at 0°C. A 0.2-ml aliquot containing 1.1 mg of ATPase protein was then added and the preparation was dialyzed for 18 h at 3°C against a medium containing 25 mM K2SOs, 0.2 mM EDTA, 0.5 mM ATP, 5% (w/v) methanol, and 10 mM Tricine-NaOH @H 8.0). Assays were carried out for 10 min at 37°C in reaction mixtures which contained, in 0.5 ml, 30 mM glucose, 5 mM MgSO,, 1 mM ADP, 5 mM KP, (containing 10’ cpm of 3zPi), 2 units of yeast hexokinase, 1 mg of defatted bovine serum albumin, and 50 mM Tricine-NaOH (pH 7.5). The tubes were illuminated at high light intensity throughout.

where bacteriorhodopsin was omitted during reconstitution. DISCUSSION

Reconstitution of Energy-Linked tions in ATPase Proteoliposomes

Reac-

ATPm3’Pi exchange activities of 20003500 nmol of [32P]ATP formed per milligram of protein per 10 min are now routinely obtained using the cholate dialysis method described here; this compared with values of 650, 1010, and 1500 in the parent yeast membranes and in bovine mitochondria and submitochondrial particles, respectively. Critical factors in the attainment of such high activities include the lowering of the Triton X-100 content in the purified ATPase prior to reconstitution, dialysis at pH 8-9 in buffer containing both methanol (to prevent cold inactivation of the ATPase (12)) and salts, and use of optimal phospholipid/ATPase ratios. In gen-

IN ATPase PROTEOLIPOSOMES

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eral terms it is worth noting the diversity of conditions where reconstitution of membrane-linked activities can be achieved (6, 20, 27-32); optimal conditions may vary widely depending on the protein components, and should be determined experimentally for each system. The mechanism of the stimulation of ATPm3’Pi exchange activity by valinomytin + K+ remains uncertain but apparently is not identical to the valinomycin + K+-dependent enhancement of acid-base (33) and postillumination (34, 35) ATP synthesis in chloroplasts and chromatophores. In the latter instances these reagents act by creating a diffusion potential, positive inside. In the present system, however, high rates of ATPa3’Pi exchange were observed when vesicles were simply formed in the presence of K+ (or Mg2+) salts. These K+ preloaded vesicles now exhibited little or no stimulation by valinomycin + K+; when a small stimulation was observed, however, it occurred whether the external K+ concentration was lower or higher than the internal one. As presently viewed, these valinomycin effects may only reflect a subtle mechanism by which the electrogenic proton movements during ATP hydrolysis and synthesis are favorably balanced by ionophore-induced K+ countermovements. In future work, it is planned to examine these electrical effects more closely by using probes such as the cyanine dyes (36) to monitor membrane potentials. The partial requirement for metal ions during reconstitution is interesting and has been observed in other systems (28). Such ions probably lessen the electrostatic repulsion between polar phospholipid head-groups and thus assist vesicle formation. The ATP-dependent proton translocation in the reconstituted vesicles mimics that in mitochondria and submitochondrial particles. In these native membranes such proton movement occurs in an apparently electrogenic way with a proton/ATP ratio approaching 2 (22,37) and with directionality such that protons are moved to the side of the membranes opposite the F, sector. The vectorial proton translocation in the reconstituted vesicles does not, however, establish that the reconstituted ATP-

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ase molecules are all anisotropically oriented, only those with the F,-catalytic sector facing outwards could function since substrates such as ATP are presumably impermeable and would not reach the vesicle interior. This unidirectional approach of the substrate would itself impose the observed vectorial dimension to the reaction. The extremely high rates of ATP-32Pi exchange observed here suggest however that at least a major ;roportion of the ATPase molecules is oriented with the F, sector directed outwards. ATP Synthesis Linked with Electron Flow between Ascorbate and Ferricyanide

The discovery that oligomycin- and uncoupler-sensitive ATP synthesis can be driven by electorn flow between ascorbate and ferricyanide is notable from two points of view: First, these findings are the first instance where “oxidative phosphorylation” has been demonstrated which is coupled with electron flow through an artificial and nonprotein electron transport system. This observation is clearly in accordance with the chemiosmotic view (1) that phosphorylation is driven by an electrochemical proton gradient (A,&+), otherwise termed a proton motive force, which is generated by proton translocation coupled with electron flow. From this standpoint, it is interesting that artificial transmembrane electron transport reactions such as the present one and the cytochrome c-cytochrome oxidase system (9, 10) are functional examples of the pro“loops” proposed by ton translocating Mitchell (11, at least to the extent that the proton gradient is produced by transmem-

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brane movement of a hydrogen atom carrier followed by reaction with an anisotropically located electron carrier. Second, the present study is the first in which a purified mitochondrial ATPase complex has been shown to catalyse net ATP synthesis. This finding confirms previous suggestions (11, 17) that the yeast ATPase complex contains the complete assembly of coupling proteins required to transduce the proton gradient energy into net ATP synthesis. This latter conclusion was also confirmed by the demonstration of light-dependent phosphorylation in vesicles containing bacteriorhodopsin. In all cases where net ATP synthesis was measured, 5-100 mol of ATP were formed/ mol of ATPase, showing that phosphorylation was not a one-turnover reaction. When PMSH was used as the permeant phosphorylation was proton carrier, markedly stimulated by inclusion of valinomycin + K+. To explain this, it must be noted that any outward movement of the PMS+ generated by internal PMSH oxidation would, in the short term, establish a membrane potential, negative inside, which would oppose phosphorylation according to the chemiosmotic view and also prevent further egress of PMS+. Indeed the findings of Deamer et al. (23) suggest that PMS+ is only slowly released from liposomes and does not function catalytically. Whatever the extent of PMS+ efllux is in the present system, any membrane potential so produced would be abolished by a valinomycin-mediated K+ uptake. A summary of the redox reactions and of the proposed mechanism of the valinomycin effect is shown in Scheme 1. It is notewor-

ADP.P, -QTP -

SCHEME 1. The proposed mechanism of ATP formation linked with electron flow between external ascorbate and internal ferricyanide. The abbreviations used are: A-AH2-, BQ-BQH,, and PMS+-PMSH, oxidized and reduced forms of ascorbate, benzoquinone, and phenazine methosulfate respectively; Val, valinomycin.

ENERGY-LINKED

ACTIVITIES

thy that where DCIP or benzoquinone was used as the proton carrier, phosphorylation was not stimulated by valinomytin + K+. Unlike PMSH, neither DCIP or benzoquinone undergoes a charge change upon oxidation and would not, therefore, set up a membrane potential upon diffusing out again from the vesicle. Note added in proof. Following submission of this manuscript, a report (38) appeared demonstrating light-dependent ATP synthesis in proteoliposomes containing bacteriorhodopsin and the dicyclohexyl carbodiimide-sensitive ATPase from the thermophilic bacterium PS3. In general terms, this observation is consistent with the present findings.

1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

14.

15.

REFERENCES MITCHELL, P. (1966) Biol. Reu. 41, 445-502. MITCHELL, P. (1964) FEBS Lett. 43, 189-193. MITCHELL, P. (1975) FEBS Lett. 50, 95-97. BOYER, P. D. (1975) FEBS Lett. 58, 1-6. ROBERTSON, N. R., AND BOARDMAN, N. K. (1976) FEBS Lett. 60, 1-6. KAGAWA, Y., AND RACKER, E. (1971) J. Biol. Chem. 246, 5477-5487. CAPALDI, R. A., KOMAI, H., AND HUNTER, D. R. (1973) Biochem. Biophys. Res. Commun. 55, 655-659. RACKER, E., AND STOECKENIUS, W. (1974) J. Biol. Chem. 249, 662-663. RACKER, E., AND KANDRACH, A. (1971) J. Biol. Chem. 246, 7069-7071. RACKER, E., AND KANDRACH, A. (1973) J. Biol. Chem. 248, 5841-5847. RYRIE, I. J. (1975) Arch. B&hem. Biophys. 168, 704-711. RYRIE, I. J. (1975) Arch. B&hem. Biophys. 168, 712-719. YOSHIDA, M., SONE, N., HIRATA, H., AND KAGAWA, Y. (1975) J. Biol. Chem. 250, 79107916. SONE, N., YOSHIDA, M., HIRATA, H., AND KAGAWA, Y. (1975) J. Biol. Chem. 250, 79177923. SADLER, M. H., HUNTER, D. R., AND HAWORTH, R. A. (1974) Biochem. Biophys. Res. Commun. 59, 804-812.

IN ATPase

PROTEOLIPOSOMES

16. HATEFI, Y., STIGGALL, D. L., GALANTE, Y., AND HANSTEIN, W. G. (1974) B&hem. Biophys. Res. Commun. 61, 313-321. 17. RYRIE, I. J. (1975) J. Supramol. Strut. 3, 242247. 18. OESTERHELT, D., AND STOECKENIUS, W. (1974) Methods Enzymol. 31, 667-678. 19. AVIUJN, M. (1960) Biochim. Biophys. Actu 40, 257-272. 20. KAGAWA, Y., KANDRACH, A., AND RACKER, E. (1973) J. Biol. Chem. 248, 676-684. 21. MITCHELL, P., AND MOPE, J. (1965) Nature (London) 208, 1205-1206. 22. THAYER, W. S., AND HINKLE, P. C. (1973) J. Biol. Chem. 248, 5395-5402. 23. DEAMER, D. W., PRINCE, R. C., AND CROFTS, A. R. (1972) Biochim. Biophys. Actu 274,323-335. 24. FIOLET, J. W. T., BAKKER, E. D., AND VAN DAM, K. (1974) Biochim. Biophys. Actu 368, 432445. 25. HINKLE, P. C. (1973) Fed. Proc. 32,1988-1992. 26. HINKLE, P. (1970) Biochem. Biophys. Res. Commun. 41, 1375-1381. 27. KAGAWA, Y. (1972) Biochim. Biophys. Acta 265, 297-338. 28. RAZIN, S. (1972) Biochim. Biophys. Actu 265, 241-296. 29. RACKER, E. (1972) J. Membr. Biol. 10, 221-235. 30. RACKER, E. (1973) B&hem. Biophys. Res. Commun. 55, 224-230. 31. RACKER, E., CHIEN, T. F., AND KANDRACH, A. (1975) FEBS Lett. 57, 14-17. 32. EYTAN, G., MATHESON, M. J., AND RACKER, E. (1975) FEBS Lett. 57, 121-125. 33. SCHULDINER, S., ROTTENBERG, H., AND AVRON, M. (1972) FEBS Lett. 28, 173-176. 34. SCHULDINER, S., ROTTENBERG, H., AND AVRON, M. (1973) Eur. J. B&hem. 39, 455-462. 35. GROMET-ELHANAN, Z., AND LEISER, M. (1975) J. Biol. Chem. 250, 90-93. 36. SIMS, P. J., WAGGONER, A. S., WANG, C. H., AND HOFFMAN, J. F. (1974) Biochemistry 13, 3315-3329. 37. MITCHELL, P., AND MOYLE, J. (1968) Eur. J. Biochem. 4, 530-539. 38. YOSHIDA, M., SONE, N., HIRATA, H., KAGAWA, Y., TAKEUCHI, Y., AND OHNO, K. (1975) Biothem. Biophys. Res. Commun. 67, 1295-1300.