ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 176, 127-135 (1976)

Energy-Linked Activities in Reconstituted Yeast Adenosine Triphosphatase Proteoliposome

Adenosine Triphosphate Formation Coupled with Electron Flow between Ascorbate and Ferricyanide

IVAN J. RYRIE AND PETER F. BLACKMORE’

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

Received February 3, 1976

(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine tri- phosphatase (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 migra- tion of K+ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-3ZP, ex- change. (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 light- dependent reaction using ATPase proteoliposomes reconstituted together with bacte- riorhodopsin.

As presently viewed, the oxidative phos- phorylation apparatus of the inner mito- chondrial membrane consists of two func- tionally separable segments: a highly or- dered, multicomponent electron transport chain and an ATPaseZ complex responsible

1 Present address: Department of Physiology, School of Medicine, Vanderbilt University, Nash- ville, Tenn. 37232.

’ Abbreviations used: ATPase, adenosine triphos- phatase; F,, mitochondrial coupling factor 1; F,, membrane factor required to confer oligomycin sen- sitivity on F,; PMS+-PMSH, oxidized and reduced forms of phenazine methosulfate; DCIP, 2,&dichlo- roindophenol; TPB-, tetraphenylboron anion; TPAs+; tetraphenylarsonium cation; CCCP, car- bony1 cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydra- zone; S-13, 5-chloro-3-tert-buty1-2’-chloro-4’-nitro- salicylanilide; Tricine, tris(hydroxymethyl)methyl- glycine.

for coupling ATP synthesis with the exer- gonic redox reactions. Despite extensive efforts no conclusive evidence has yet been found for a direct transfer of energy, cova- lent or otherwise, between the electron transport chain and the ATPase. On the other hand, considerable evidence has ac- cumulated 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 syn- thesis. Indeed recent speculation on the phosphorylation mechanism (2-5) is mostly in accord with the latter concepts, and interest seems centered on the mecha- nism by which the “proton motive force” is utilized.

An important corollary of the chemios- motic theory is that the purified ATPase

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

128 RYRIE AND BLACKMORE

complex, when inlaid into a proton-im- permeable phospholipid vesicle, might cat- alyze net ATP synthesis and the energy- linked exchange reactions. given an ade- quate transmembrane proton gradient. Mitochondrial ATP-32Pi exchange activity was first reconstituted from solubilized membrane components by Kagawa and Racker (6) who used phospholipids, a hy- drophobic membrane sector containing F,, the oligomycin-sensitivity-conferring pro- tein (OSCP), and F,. The membrane frac- tion, 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 es- tablished using bacteriorhodopsin and light (8) or electron flow through cyto- chrome c-cytochrome oxidase (9, 10).

Reconstitution of ATPb3”Pi exchange us- ing a purified ATPase was finally achieved in this laboratory using the oligomycin- sensitive ATPase from yeast (11, 12) and shortly thereafter by Kagawa’s group us- ing the dicyclohexyl carbodiimide-sensi- tive ATPase isolated from a thermophilic bacterium (13, 14). Neither preparation exhibited exchange activity prior to recon- stitution which suggests a requirement for a vesicular membrane structure contain- ing phospholipids. Interestingly, two bo- vine ATPase preparations have recently been isolated which contain intrinsic ATP-32Pi exchange activity (15, 16). HOW- ever, this may simply indicate the pres- ence of remaining vesicles. Indeed at least one of these preparations existed com- pletely as membrane fragments (15) while exchange activity in the other was mark- edly stimulated by addition of phospho- lipid vesicles (16).

It is the purpose of the present commu- nication to examine further the energy- transducing reactions of the reconstituted yeast ATPase complex. Conditions are de- scribed 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-

cyanide. Taken together, these observa- tions 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 puri- fication 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 di- alysis 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 Tricine- NaOH (pH 8.0). In a final volume of 1.0 ml, 1 mg of ATPase protein was combined with 10 mg of phos- pholipids 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. Gott- fried Wagner) was grown to stationary phase under illumination in 50-ml batches at 30°C. The growth medium and method for preparing the purple mem- branes were as described by Oesterhelt and Stoeck- enius (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 ferricyanide- containing 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 PROTEOLIPOSOMES 129

TABLE I

EFFECTS OF VALINOMYCIN + K+ ON ATP-32P, EXCHANGE ACTIVITY IN ATPA~E PROTEOLIPOSOMES~

Additions to dialysis me- Additions to assay ATP-32P, exchange dium

specific activ- Percent ityb

Experiment 1 None 833 100

50 mM KC1 768 92 1 rg/ml valinomycin 878 105 1 pg/ml valinomycin + 50 mM KC1 1566 188 25 TPB- /LM 1242 149 25 TPAs+ /.LM 789 95

Experiment 2 None 50 mM KC1 970 100

1 pg/ml valinomycin + 50 mM KC1 1510 156 1 pg/ml valinomycin + 50 mM KNO, 1415 146 1 pg/ml valinomycin + 25 mM KzSOa 1865 192

Experiment 3 None - 1712 100

1 pg/ml valinomycin + 50 mM KC1 2618 153 50 mM KC1 - 2415 100

1 Fg/ml valinomycin + 50 mM KC1 2905 120 50 mM KCH,COO - 1979 100

1 pg/ml valinomycin + 50 mM 2228 113 KCH,COO

25 mM K,SO, - 2800 100 1 pg/ml valinomycin + 25 mM K&SO., 2566 92

Experiment 4 25 mM K&SO, - 1428 100

1 pg/ml valinomycin 1618 113 50 mM K,SO, 1284 90 1 pg/ml valinomycin + 50 mM K,SO, 1513 106

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 val- inomycin (or monactin) in the presence of external K+. As shown in Table I, Experi- ment 1, valinomycin + K+ stimulated ex- change activity by 38% whereas neither reagent alone had much effect. The stimu- lation 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 nota- ble, first, that exchange activities were higher when K+ salts were included dur- ing reconstitution and, second, that now neither valinomycin + K+ nor TPB-,3 ef- fected very much increase in ATPe3’Pi ex- change. Where a small stimulation was observed it was much the same whether

’ I. J. Ryrie, unpublished observations.

130 RYRIE AND

the external K+ concentration was lower or higher than the internal one (Experi- ment 4).

Other conditions which affect the recon- stitution 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 sys- tem was not stimulated by inclusion of 5% (w/w) cardiolipin in the phospholipids.

Experience has shown that a critical fac- tor in achieving high ATPd3’Pi exchange activity is the removal of excess Triton X- 100 from the ATPase prior to reconstitu- tion. The final ATPase isolation step in- volves 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 concen- trate 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 pres- sure concentration of 50-ml aliquots of the ATPase using an Amicon XM-50 mem- brane.

ATP-Dependent Proton Uptake

As in natural (21, 22) and artificial (20) membranes where the catalytic F1 sector is

BLACKMORE

oriented outwards, addition of Mg2+-ATP to the reconstituted vesicles resulted in proton uptake which was significantly in- hibited by oligomycin and the uncoupler S- 13 (Fig. 2). Measurements were made at pH 6.25 where no net proton release occurs upon ATP hydrolysis (22). Like the vesi- cles reconstituted with mammalian mito- chondrial 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 activ- ity might be expected to catalyze net phos- phorylation 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 deter- mining the ApH values has been ques- tioned (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 consider- ably stimulated by inclusion of valinomy- tin. Oligomycin and the protonophorous uncouplers CCCP, FCCP, and S-13 mark- edly inhibited the reaction whereas the electron transport inhibitors rotenone, an- timycin A, and carbon monoxide did not. 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 ratios on the reconstitution of ATP-32P, exchange activity. Proteoliposomes were reconstituted by variations of the cholate dialysis procedure described in the text.

ENERGY-LINKED ACTIVITIES IN ATPase PROTEOLIPOSOMES 131

FIG. 2. ATP-dependent proton uptake in ATPase proteoliposomes. Reconstitution was car- ried 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), valinomy- tin (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 elec- trically 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). Phosphoryl- ation was largely dependent on the pres- ence of PMSH and was abolished when ascorbate was omitted or when the vesicles did not contain ferricyanide. [32PlATP for- mation was still observed when external ferricyanide was removed from the vesi- cles before assaying by predialysis against buffer containing 1 mM sodium ascorbate but not when ferricyanide was added to the vesicles (without sonication) immedi- ately before assaying. These latter find- ings are consistent with a requirement for internal ferricyanide. It should be men- tioned that the PMS-independent phos- phorylation (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 pro- ton, is uncertain.

The marked dependence on hexokinase indicates that the reaction product was y- labeled [32P]ATP; without a hexokinase- glucose trap the 13”P]ATP would be formed but degraded again by the ATPase.

Benzoquinone and DCIP, both of which produce protons upon oxidation, substi- tuted for PMS (Experiments 3 and 4). Phosphorylation was again sensitive to S- 13 but now was markedly inhibited by val- inomycin. Little or no [32P]ATP formation was observed with ferrocene however (Ex- periment 51, either in the presence or ab- sence of TPB- or valinomycin. Ferrocene is an electron but not proton carrier and is known to transfer electrons between exter- nal ascorbate and internal ferricyanide (especially in the presence of TPB-) with- out formation of a proton gradient (26).

Taken together the findings indicate that phosphorylation capacity in the vesi- cles can withstand brief sonication in me- dia containing 100 mM K3Fe(CN&. This was confirmed by measuring ATP-32P, ex- change: The vesicles used in Experiment

132 RYRIE AND BLACKMORE

TABLE II

ATP SYNTHESIS IN PROTEOLIPOSOMES LINKED WITH ELECTRON FLOW BETWEEN ASCORBATE AND FERRICYANIDE”

Assay [32PlATP formation

Specific activityb Percent

Experiment 1 Complete (50 PM PMS+ + 1 pg/ml vahnomycin) Minus valinomycin PhlS 100 ,uM CCCP

Plus 20 PM 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 TPB- Plus 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 A 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 TPB-

29.0 100 7.6 26 1.4 5

0 0 2.8 10 8.0 28

53.8 100 0 0

23.1 43 3.9 7

0 0 49.2 92 49.5 92 55.6 103

26.0 100 26.9 104 25.0 96 40.7 156 32.9 127

18.7 100 9.3 50 7.9 42

22.6 100 8.5 38 7.6 34

7.6 100 0 0

1.5 20 0 0 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 Vesi- cles Containing Bacteriorhodopsin

Independent evidence that the yeast ATPase complex is capable of net phospho-

rylation was obtained using bacteriorho- dopsin as a light-dependent proton translo- cator. Vesicles containing the ATPase to- gether 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 in- hibited by uncouplers and oligomycin (Ta- ble III). No phosphorylation was observed

ENERGY-LINKED ACTIVITIES IN ATPase PROTEOLIPOSOMES 133

TABLE III LIGHT-DEPENDENT PHOSPHORYLATION IN

PROTEOLIFQSOMES CONTAINING BACTERIORHODOP~IN~

Assay 13*PlATP formed (nmol/mg ATPase

protein)

Complete + 10 /&LM s-13 + 20 /.&M FCCP + 10 pg/ml oligomycin darkness vesicles without purple mem-

brane

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 dur- ing reconstitution.

DISCUSSION

Reconstitution of Energy-Linked Reac- tions in ATPase Proteoliposomes

ATPm3’Pi exchange activities of 2000- 3500 nmol of [32P]ATP formed per milli- gram of protein per 10 min are now rou- tinely 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 mitochon- dria and submitochondrial particles, re- spectively. Critical factors in the attain- ment of such high activities include the lowering of the Triton X-100 content in the purified ATPase prior to reconstitution, di- alysis at pH 8-9 in buffer containing both methanol (to prevent cold inactivation of the ATPase (12)) and salts, and use of opti- mal phospholipid/ATPase ratios. In gen-

eral terms it is worth noting the diversity of conditions where reconstitution of mem- brane-linked activities can be achieved (6, 20, 27-32); optimal conditions may vary widely depending on the protein compo- nents, and should be determined experi- mentally for each system.

The mechanism of the stimulation of ATPm3’Pi exchange activity by valinomy- tin + K+ remains uncertain but appar- ently is not identical to the valinomycin + K+-dependent enhancement of acid-base (33) and postillumination (34, 35) ATP synthesis in chloroplasts and chromato- phores. In the latter instances these re- agents act by creating a diffusion poten- tial, 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 ex- hibited little or no stimulation by valino- mycin + 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 mecha- nism by which the electrogenic proton movements during ATP hydrolysis and synthesis are favorably balanced by iono- phore-induced K+ countermovements. In future work, it is planned to examine these electrical effects more closely by us- ing probes such as the cyanine dyes (36) to monitor membrane potentials. The partial requirement for metal ions during recon- stitution is interesting and has been ob- served 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 transloca- tion in the reconstituted vesicles mimics that in mitochondria and submitochon- drial particles. In these native membranes such proton movement occurs in an appar- ently electrogenic way with a proton/ATP ratio approaching 2 (22,37) and with direc- tionality 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, how- ever, establish that the reconstituted ATP-

134 RYRIE AND BLACKMORE

ase molecules are all anisotropically ori- ented, only those with the F,-catalytic sec- tor facing outwards could function since substrates such as ATP are presumably impermeable and would not reach the ves- icle interior. This unidirectional approach of the substrate would itself impose the observed vectorial dimension to the reac- tion. 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.

brane movement of a hydrogen atom car- rier followed by reaction with an aniso- tropically located electron carrier.

ATP Synthesis Linked with Electron Flow between Ascorbate and Ferricyanide

The discovery that oligomycin- and un- coupler-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 phosphoryla- tion” has been demonstrated which is cou- pled with electron flow through an artifi- cial and nonprotein electron transport sys- tem. This observation is clearly in accord- ance with the chemiosmotic view (1) that phosphorylation is driven by an electro- chemical proton gradient (A,&+), other- wise termed a proton motive force, which is generated by proton translocation cou- pled with electron flow. From this stand- point, it is interesting that artificial transmembrane electron transport reac- tions such as the present one and the cytochrome c-cytochrome oxidase system (9, 10) are functional examples of the pro- ton translocating “loops” proposed by Mitchell (11, at least to the extent that the proton gradient is produced by transmem-

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 pre- vious 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 phosphoryl- ation was not a one-turnover reaction.

When PMSH was used as the permeant proton carrier, phosphorylation was markedly stimulated by inclusion of vali- nomycin + K+. To explain this, it must be noted that any outward movement of the PMS+ generated by internal PMSH oxi- dation would, in the short term, establish a membrane potential, negative inside, which would oppose phosphorylation ac- cording 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 catalyti- cally. 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 IN ATPase PROTEOLIPOSOMES

thy that where DCIP or benzoquinone was used as the proton carrier, phospho- rylation was not stimulated by valinomy- tin + K+. Unlike PMSH, neither DCIP or benzoquinone undergoes a charge change upon oxidation and would not, therefore, set up a membrane potential upon diffus- ing 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 thermo- philic bacterium PS3. In general terms, this obser- vation is consistent with the present findings.

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