Plant Physiol. (1984) 74, 766-7720032-0889/84/74/0766/07/$0 1.00/0

Purifi'cation and Characterization of a Glycerol-Resistant CFo-CF1 and CF1-ATPase from the Halotolerant Alga Dunaliellabardawil'

Received for publication September 12, 1983

MOSHE FINEL, URI PICK*, SUSANNE SELMAN-REIMER2, AND BRUCE R. SELMAN2Biochemistry Department, The Weizmann Institute ofScience, Rehovot 76100, Israel

ABSTRACT

The isolation of the chloroplast ATP synthase complex (CF.-CF1) andof CF, from Dunaliella bardawil is described. The subunit structure ofthe D. bardawil ATPase differs from that of the spinach in that the D.bardawil a subunit migrates ahead of the , subunit and emigrates aheadof subunit II of CFO when separated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The CF, isolated from D. bardawil resemblesthe CF, isolated from Chladmydomonas reinhardi in that a reversible,Mg2+-dependent ATPase is induced by selected organic solvents. Glycerolstimulates cyclic photophosphorylation catalyzed by D. bardawil thyla-koid membranes but inhibits photophosphorylation catalyzed by spinachthylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-Pi exchange catalyzed by D. bardawil CFO-CF3 proteoliposomes butinhibits the activity with the spinach enzyme. The ethanol-activated,Mg2+-ATPase of the D. bardawil CF, is more resistant to glycerolinhibition than the octylgucoside-activated, Mg2'-ATPase of spinachCF, or the ethanol-activated, Mg2+-dependent ATPase ofthe C. reinhardiCF,. Both cyclic photophosphorylation and ATP-P; exchange catalyzedby D. bardawil CFo-CF, are more sensitive to high concentrations ofNaCI than is the spinach complex.

Dunaliella bardawil is a halotolerant, cell wall-less alga whichis capable of surviving in growth medium containing NaCIranging in concentration from 0.5 to 4 M. This is made possibleby the synthesis of an internal osmoticum which appears to bemainly glycerol (5, 7, 10). Thus, the internal concentration ofglycerol in the cell could range from 1 to 7.5 M (equivalent from7.5 to 55 volume per cent) at the physiological conditions inwhich the alga survives. Such unusually high glycerol concentra-tions undoubtedly alter the properties of the solution inside thecell and, therefore, one might expect to find special adaptationsof enzymic systems inside the alga that enable it to functionunder these unusual conditions. However, few comparisons be-tween D. bardawil and other higher plant enzymes with respect

'Supported in part by grants from the Weizmann Institute of Science(B. R. S., Meyerhoff Fellow), the college ofAgricultural and Life Sciences,University of Wisconsin (B. R. S.), the United States-Israel BinationalResearch Foundation (U. P. and B. R. S., BSF 2891/82), the UnitedStates-Israel Agriculture Research and Development Fund (U. P., BARDI-107-79); the United States Department of Agriculture, Science andEducation Administration, CRGO (B. R. S., 82-CRCR- 1-1 151) and theNational Institutes of Health (B. R. S., GM 31384-01).

2 Permanent address: Department of Biochemistry, University of Wis-consin, Madison, WI.

to their sensitivity to glycerol have been reported.In this work, we describe the isolation and characterization of

the chloroplast CFo-CFI3 ATP synthase and of the soluble CF,from D. bardawil. Although CF, has previously been isolatedfrom a large number of higher plants (20) as well as from a greenalga (27) and cyanobacteria (9), an active CFo-CF, complex hasonly been isolated from higher plants (23). We demonstrate herethat the CFo-CFI ATPase from D. bardawil differs from its higherplant counterpart in its subunit structure, resistance to glycerol,and sensitivity to NaCl, indicating that the structure of theprotein has been modified in order to enable it to function athigh glycerol concentrations.

MATERIAILS AND METHODS

Thylakoid Membranes Preparations. Dunaliella bardawil wasgrown outdoors in large (30 L) continuous cultures in a mediumcontaining 1.5 M NaCl and nitrate as previously described (8).D. bardawil thylakoid membranes were prepared by a modifi-cation of the procedure of M. Shefer (personal communication)as follows. Log-phase cultures were collected by centrifugationat 2,500g for 5 min, resuspended, and homogenized in buffercontaining 20 mM Na-Tricine (pH 8.0) and 15% (v/v) glycerol,and collected by centrifugation at 12,000g for 10 min at 5°C.The glycerol wash was repeated twice with extensive homogeni-zation in between each washing step. The cells were finallysuspended in a minimal volume (1-2 mg Chl/ml) of the glycerolwashing medium and stored on ice. Spinach thylakoids wereprepared as previously described (2). Chl was determined accord-ing to Arnon (1).ATPase Complexes. Extraction of the CFo-CF, ATPase com-

plex from D. bardawil was performed by a modification of theprocedure previously described for the extraction of the higherplant CFo-CF, complex (23). D. bardawil cells were collected asdescribed above and washed four times with 10 mm Na pyro-phosphate (pH 7.8) buffer. Cells were collected by centrifugationat 14,000g for 10 min at 5C. The solubilization medium con-tained 20 mm Na-Tricine (pH 8.0), 0.2 M sucrose, 50 mM DTT,3 mM MgCl2, 0.5% Na cholate, 30 mM octylglucoside, and D.bardawil thylakoid membranes (equivalent to 2 mg Chl/ml).(NH4)2S04 was omitted. In order to remove the residual carotenereleased during the solubilization, 1.0 ml of the Mg-Tricine-sucrose buffer solution was layered on top of the solubilizationmixture immediately prior to the high speed centrifugation

3Abbreviations: CFo-CF,, chloroplast ATP synthase complex; CF,,chloroplast coupling factor 1; PMS, phenazine methosulfate; OG, octyl-B-D-glucopyranoside (octylglucoside); FTS, the freezing-thawing-sonica-tion reconstitution technique; CD, the cholate-dilution reconstitutiontechnique; DCCD, dicyclohexylcarbodiimide.

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GLYCEROL-RESISTANT CFo-CF, AND CF, FROM DUNALIELLA BARDAWIL

(200,000g for 45 min at 3C in a Beckman type 65 rotor). Mostofthe orange-colored carotene was either extracted into the upperbuffer phase or appeared as a paste on the walls of the tube andwas carefully removed at the end of the centrifugation.The yellowish supernatant, which contained the ATPase, was

fractionated with (NH4)2SO4 between 30 and 50%, and theprecipitate was either stored frozen (crude CFo-CF,) or furtherpurified on a sucrose gradient containing 0.2% Triton X-100plus 0.1% soybean lipids. Other details regarding the purificationand the reconstitution of the CFo-CF, complex into proteo-liposomes were as previously described (23).

Coupling Factor 1 ATPases. Log-phase cultures of D. bardawil(6-10 L) were harvested by centrifugation (600g, 5 min) andwashed twice in 500 ml buffer containing 40 mM Na-Tricine(pH 7.4), 1.5 M NaCl, and 5 mM MgCl2. After the second wash,the cells were osmotically shocked by resuspension in 250 ml ofa hypotonic buffer containing 50 mm Na-Hepes (pH 7.6), 10 mmNaCl, and 1 mm EDTA, and incubated at 4°C for 5.0 min.Membrane fiagments were collected by centrifugation (3000g,10 min) and washed four times (250 ml/wash) with 10 mm Napyrophosphate (pH 7.8) buffer.CF. was extracted by chloroform and purified on a DEAE-

Sephadex column essentially according to Selman-Reimer et al.(27) with the exception that the dialysis prior to the DEAE-Sephadex purification was omitted.

Spinach (20) and Chlamydomonas reinhardi (27) couplingfactors were prepared as previously described.

Prior to use, aliquots of the (NH4)2SO4 precipitated proteinswere centrifuged (12,000g, 2.0 min), and the proteins wereredissolved in buffer containing 40 mM Na-Tricine (pH 8.0) and1.0 mM EDTA.Analytical Methods. Cyclic photophosphorylation was meas-

ured by the formation of [,y-32P]ATP from 32Pi and ADP afterillumination of thylakoid membranes (equivalent to 20 Mg Chl/ml) for 1.0 min at either 30°C (D. bardawil) or at 23°C (spinach)in a water bath (white light, 100,000 lux). The media contained20 mm Na-Tricine (pH 8.0), 3.0 mM ADP, 5.0 mm MgCl2, 4.0mM Pi (containing 0.1-0.2 ACi 32P/Mmol), and either 100 gMPMS plus 0.2 to 0.5 mm DTT (D. bardawil membranes) or 30Mm PMS (spinach thylakoid membranes) with or without 20%glycerol. The formation of[y-32P]ATP was determined accordingto Avron (2).ATP-Pi exchange was measured by the incorporation of 32P,

into ATP. Proteoliposomes containing the CFo-CF, complexes(40-80 Mg protein/ml) were incubated for 30 min at 37°C in amedium containing 20 mm Na-Tricine (pH 8.0), 5.0 mM MgC92,5.0 mM ATP, 1.0 mm K-phosphate (containing 0.5-1 MCi 32p,/Mmol), and 2.0 mm DTT with or without 20% glycerol.Ca-ATPase activation of CF, was performed by incubating the

enzyme (1-2 mg protein/ml) for 3 min at 62°C in a mediumcontaining 40 mM Na-Tricine (pH 8.0), 1.0 mm EDTA, 5.0 mMATP, and 5.0 mM DTT, followed by cooling to 23°. The Mg-ATPase activity was assayed in the presence of 25% ethanol (D.bardawil, or C. reinhardi CF,) or 30 mm octylglucoside (spinachCF,).

Unless otherwise stated, standard ATPase reaction mixturescontained in 0.1 ml total volume 0.5 Mg protein, 40 mm Na-Tricine (pH 8.0), 1.0 mm EDTA, 6.0 mM MgCl2 or CaCl2, and5.0 mM [y-32P]ATP (approximately 0.2 MCi 32P/Mmol). Reactionswere initiated by the addition of protein, incubated for 2 to 3min at 37°C, and terminated by the addition of2 ml ofa solutioncontaining 0.8 N HC104 and 1% (w/v) ammonium molybdate.Extraction of [32P]phosphate was performed as previously de-scribed (20).Other Methods. Ferricyanide reduction was measured in an

Aminco-Chance DW2 spectrophotometer by following the light-induced absorption changes at 420 minus 470 nm. 9-Aminoac-

ridine fluorescence quenching (for measurements of the trans-membrane ApH) was measured in an Eppendorf photometeradapted for fluorescence measurements as previously described(26). Protein was estimated either according to Lowry et al. (18)for the CF0-CF,-ATPase or according to Bradford (11) for CF,.The separation and staining of protein polypeptides were

performed by slab SDS-polyacrylamide gel electrophoresis(12.5%) essentially according to Laemmli (17). The polymeriza-tion solution contained 0.5% polyacrylamide (mol wt above 5 x106).Materials. Detergents and chemicals were obtained from

Sigma Chemical Co. y_-32P]ATP was synthesized by the illumi-nation of chloroplasts in the presence of 32Pi and ADP followedby the separation of [y_-32PJATP on polyethyleneiminocellulosecolumns according to Magnusson et al. (19).

RESULTS

Characterization of ATP Formation Catalyzed by D. bardawilThylakoid Membranes. Ben Amotz and Avron (6) have previ-ously reported that thylakoid membranes isolated from osmoti-cally shocked D. parva cells have the capacity to catalyze ATPformation in the light in the presence ofdifferent electron transfermediators; however, the rates that they measured were consid-erably lower than those obtained from higher plant thylakoidmembranes. In order to increase the phosphorylation activity ofDunaliella thylakoids, the effect of glycerol was tested.The presence of glycerol during the phosphorylation assay

stimulates the rate of PMS-dependent ATP formation with D.bardawil thylakoid membranes but inhibits the activity withspinach thylakoid membranes (Fig. 1). An optimal stimulationof about 2-fold is obtained by 20% (2.75 M) glycerol.

In order to determine if the glycerol stimulation of ATPformation was due to an increase in the electron transfer rate, toa lowered permeability of the thylakoid membrane to protons,or to a direct effect of glycerol on the ATP synthase, the effectof glycerol on the light-driven, PMS-dependent, transmembraneApH formation catalyzed by D. bardawil thylakoid membranes(measured by 9-aminoacridine fluorescence quenching [26]) was

250_

200c borow I0o / thylokoids0

50-

EO 000 /

a \ spinocho ~~~~~~~~thylakoids50_

10 20 30[glycerol] % (V/V)

FIG. 1. The differential effect of glycerol on PMS-dependent cyclicphotophosphorylation catalyzed by D. bardawil and spinach thylakoidmembranes. PMS-dependent photophosphorylation was measured in thepresence of the indicated glycerol concentrations. The control (100%)rates ofATP formation were 220 and 1700 Mmol ATP formed/mg Chl-h with D. bardawil and spinach thylakoid membranes, respectively.

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Plant Physiol. Vol. 74, 1984

determined. However, it was found that glycerol does not affectthe magnitude of the light-induced ApH formed across the D.bardawilthylakoid membranes (not shown). In addition, glycerolslightly inhibits the rates of ferricyanide reduction both in theabsence and in the presence of an uncoupler but does notsignificantly affect the ratio between the uncoupled to coupledrates. For example, without uncoupler, the rates of ferricyanidereduction in the absence and presence of 20% glycerol were 121and 104 geq/mg Chl h, respectively. In the presence of 1 Mmnigericin, the rates increased to 288 and 242 ueq/mg Chl . h,respectively. These results suggest, therefore, that the glycerolstimulation of the rate of ATP formation catalyzed by D. bar-dawil thylakoid membranes is not due to a lowering of themembrane permeability to protons or to a stimulation of theelectron transfer rate but rather is due to an effect of glycerol onthe CFo-CF, ATP synthase.

In comparison to spinach thylakoids, ATP formation catalyzedby D. bardawil thylakoid membranes is very sensitive to a highconcentration of NaCl (Fig. 2A). NaCl at 300 mm inhibits ATPformation catalyzed by D. bardawil thylakoid membranes byover 90% compared to only a 30% inhibition ofthe rate catalyzedby spinach thylakoid membranes. NaCl inhibition of photo-phosphorylation catalyzed by D. bardawil thylakoid membranesis not dependent on the presence or absence of glycerol (notshown). However, 300 mM NaCl does not affect the light-driven,PMS-dependent, ApH formation catalyzed by D. bardawil thy-lakoid membranes, indicating that salt does not alter the effi-ciency of the rate of electron transfer or the proton permeabilityof the membrane. Photophosphorylation catalyzed by D. bar-dawil thylakoid membranes was, therefore, routinely measuredin a medium containing 20% glycerol and low salt. The oppositeeffects of glycerol and of NaCl on photophosphorylation also

e A. thylakoids

0

,I100

E a

D bardawil0

0aB. CF0-GF1 protealiposomes

0~

H

i50 l;-D. bardowil

0Lo I=c ( 100 200 300

[NaCI], mM

FIG. 2. The differential sensitivity to NaCl of photophosphorylationand of the rates of ATP-Pi exchange catalyzed by preparation from D.hardawil and spinach. Cyclic photophosphorylation (A) was measuredin the presence (D. bardawil) or absence (spinach) of20% glycerol at theindicated NaCl concentrations as described under "Materials and Meth-ods." The control (100%) rates of ATP formation were 533 and 455,umol ATP/mg Chl-h with D. bardawil and spinach thylakoid mem-

branes, respectively. ATP-Pi exchange activity of CFo-CF, containingproteoliposomes prepared by the FTS reconstitution technique was meas-ured as described in the text. The control rates of ATP-Pi exchange were

140 and 20 nmol ATP formed/mg protein-min for spinach and D.hardawil complex containing liposomes, respectively.

strongly suggest that the glycerol-induced stimulation is not dueto an osmotic effect.

Inhibitors of the higher plant CFo-CF1 ATP synthase alsoinhibit PMS-dependent photophosphorylation catalyzed by D.bardawil thylakoid membranes (not shown). It is of interest tonote that tentoxin, a species-specific inhibitor ofphotophosphor-ylation catalyzed by higher plant thylakoid membranes, alsoinhibits photophosphorylation catalyzed by D. bardawil thyla-koid membranes, albeit at relatively high concentrations. TheK5o value for inhibition is 3 AM (compared to 0.2 Mm with spinachthylakoid membranes assayed under similar conditions), whereascomplete inhibition requires about 100lM tentoxin. In addition,antisera directed against the spinach CF, strongly inhibit photo-phosphorylation catalyzed by D. bardawil thylakoid membranes,suggesting an intimate structural similarity between the twoenzymes (not shown).

Characterization of the D. bardawil CF, ATPase. In contrastto the C. reinhardi ATPase, but similar to the spinach enzyme,the ATPase activity of the D. bardawil CF, is completely latent.Although the D. bardawil ATPase activity is not induced byproteolytic digestion of the enzyme, the latency is released byselective organic solvents or preincubation ofthe enzyme at 62Cfor 3.0 min in the presence of 5.0 mm DTT and 10 mm ATP.

Organic solvents induce a Mg2+-dependent ATPase activity.Among the most effective solvents tested were ethanol andmethanol. Optimal stimulation ofthe rates ofthe ATPase activitywas obtained at ethanol and methanol concentrations equivalentto 25 and 35% (v/v), respectively (Fig. 3A). These results aresimilar to those previously found for the C. reinhardi (27) andspinach CF, (25). In the presence of 25% ethanol, the Km forMgATP is 1.2 mm and Vmax is approximately 60 to 80 units/mgprotein. The detergent OG, which stimulates a Mg24-dependentATPase activity with the higher plant CF, (22; Fig. 3B), alsostimulates a Mg2+-dependent ATPase activity with the D. bar-dawil CF, (at similar concentrations, 30-40 mM). However, acomparison of Figure 3, A and B, demonstrates that ethanol ismore effective in stimulating the D. bardawil ATPase while OGis more effective in stimulating the Mg-ATPase of the higher

E0)E

-5

.21KE

-5~0E~Ht

0~

a,

[EtOH], % or [OG], mMFIG. 3. Ethanol and octylglucoside activation of the Mg2"-dependent

ATPase activity ofCF, isolated from D. bardawiland spinach. The Mg2+-dependent ATPase activity of the chloroform-extracted CFI from D.bardawil (A) and spinach (B) was assayed in the presence or absence ofthe indicated concentrations ofOG or ethanol as described in "Materialsand Methods."

768 FINEL ET AL.

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GLYCEROL-RESISTANT CFo-CF1 AND CF, FROM DUNALIELLA BARDAWIL

plant CF,.Heat treatment of the D. bardawil CF, induces a Ca2'-depend-

ent ATPase activity, in a manner similar to that of other higher(vascular) plant coupling factors (12). Whereas the solvent-in-duced, Mg2"-dependent ATPase activity is reversible upon theremoval of the solvent, the heat-induced, Ca2'-dependent acti-vation of the D. bardawil ATPase activity is irreversible. Thelatter absolutely requires the presence of both DTT and ATPduring the activation process. The addition of DTT after theheat treatment has no effect on the rate of the ATPase.A comparison of the glycerol sensitivity of the solvent-induced

algal and OG-induced spinach Mg-ATPase activities is shown inFigure 4A. Figure 4B shows a comparison of the glycerol sensi-tivity ofthe heat-activated, Ca-ATPase activity ofthe D. bardawiland spinach CF1. The ethanol-induced, Mg-ATPase activity ofthe D. bardawil CF, is the least sensitive to glycerol (in fact, asmall, 10 to 15%, but reproducible, stimulation is usually ob-served at about 5% [v/v] glycerol). The Mg-ATPase activities ofboth the spinach (OG-induced) and the C. reinhardi (ethanol-induced) coupling factors are more sensitive than the D. bardawilCF, to glycerol inhibition, the C. reinhardi enzyme being themost sensitive. On the other hand, the heat-activated Ca-ATPaseactivity of both the D. bardawil and spinach coupling factors arevery sensitive to glycerol inhibition. Under the conditions used,50% inhibition was obtained at about 5 to 8% (v/v) glycerol.

Isolation of the CFo-CF, ATP Synthase from D. bardawil.Isolation of the CFo-CF ATP synthase complex from D. barda-wil thylakoid membranes was achieved by a modification of theprocedure previously used for the isolation of the higher plantenzyme (23). The high carotene content (up to 30% of the totaldry weight of the alga [13]) interferes with the purification of theenzyme. For this reason, D. bardawil was cultured on a richgrowth medium which inhibits the biosynthesis of carotene (8).The solubilization procedure was also modified in order toremove the carotene globules released during the detergent sol-ubilization. This included layering buffer on top of the solubili-

0

F-.en0

0

a)

Q-

5C

100

5C

10 20

[glycerol] ,% (v/v)

30

FIG. 4. The differential effects of glycerol on the Mg2+- and Ca21-dependent ATPase activities of CF, preparations isolated from D. bar-dawil, C. reinhardi, and spinach. Mg2+-dependent ATPase activity (A)was measured in the presence of 25% (v/v) ethanol (D. bardawil and C.reinhardi CF,) or 30 mm octylglucoside (spinach). The Ca2"-dependentATPase (B) of heat-activated CF, from D. bardawil or spinach wasmeasured in the presence of the indicated glycerol concentrations asdescribed under "Materials and Methods."

zation mixture to extract the residual carotene as described under"Materials and Methods." Another modification introduced intothe isolation of the D. bardawil CFo-CF1 complex was to omitthe (NH4)2SO4 during solubilization. (The presence of(NH4)2SO4is essential for the isolation ofthe higher plant enzyme. Attemptsto solubilize the spinach CFo-CF1 complex in the absence of saltresulted in a massive solubilization of Chl and Chl-bindingproteins, both of which interfere with the purification of thecomplex [U. Pick, unpublished results].) Table I shows that, ifthe D. bardawil complex is solubilized in the presence of 10%(NH4)2SO4 ATP-Pi exchange activity catalyzed by CFo-CF, con-taining proteoliposomes is inhibited over 80%. The inhibitionby (NH4)2SO4 is not due to an interference in the solubilizationof the complex per se since the same recovery of solvent-acti-vated-ATPase activity is obtained in the extract regardless of(NH4)2SO4 (data not shown). It is possible, however, that thepresence of salt during the solubilization causes a specific inac-tivation of the CFo or a disruption in the coupling between CFoand CF,.The purified enzyme may be reconstituted into proteolipo-

somes with soybean phospholipids by either the freezing-thaw-ing-sonication technique (16) or the cholate-dilution technique(24). The FTS-reconstituted CFo-CF, proteoliposomes catalyzeATP-P1 exchange rates of about 50 nmol/mg protein min andthe activity is inhibited by protonophoric uncouplers, DCCDand tentoxin. The apparent Km values for ADP (0.8 mM) and Pi(0.75 mM) are similar to the Km values for the spinach enzyme.Reconstitution of the purified D. bardawil CFo-CF, complexwith bacteriorhodopsin and soybean phospholipids (1:2:80weight ratio) by the FTS procedure yields proteoliposomes whichcatalyze ATP synthesis in the light. The rates are linear for atleast 60 min and ranged from 10 to 20 nmol ATP/mg CFo-CFprotein min at 30°C.

Polypeptide Subunit Composition of the Purified D. bardawilCF, and CFo-CF3 complexes. A comparison of the polypeptidesubunit composition of the CFD-CF1 complex and of CF, fromspinach (lanes 1 and 2) and from D. bardawil (lanes 3 and 4) isshown in Figure 5. Four distinct bands can be seen in thechloroform-released preparations (lanes 1 and 4) while sevenmajor bands (as well as several minor contaminants) are presentin both CFo-CF, complexes (lanes 2 and 3). However, the relativemigration of several subunits of the D. bardawil complex differssubstantially from that of spinach. The most striking differenceis the location of the a subunit which migrates ahead of the #subunit in D. bardawil. The identification of the a and # subunitsof the D. bardawil CF, in comparison to spinach CF. was madeby immune cross-reactivity experiments using monospecific an-tisera and by chemical modification (28). Other differences be-tween the two enzymes are the slower migration of the D.bardawil 6 subunit and the faster migration of the D. bardawilsubunit with respect to the two larger polypeptide subunits of

Table I. Inhibitorv Effect ofAmmonium Sulfate When Present duringthe Solubilization on the Activity ofthe Reconstituted D. bardawil CFo-

CF, ATPase ComplexThe D. bardawil CFo-CF, ATPase was solubilized in the presence of

the indicated (NH4)2SO4 concentrations, and samples from the crudeenzyme fractions (30-50% saturation of (NH4)2SO4) were reconstitutedinto proteoliposomes by the FTS procedure and assayed for ATP-Piexchange.

[(NH4)2 S04] Rate of ATP-Pi Exchange% (w/v) nmol/mg protein* min

0 505 15

10 720 4

A. Mg-ATPase

)I D. bardowil

spinach

C. reinhardi

IIB. Co-ATPase

spinochD.bordawil

IX

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FINEL ET AL. Plant Physiol. Vol. 74, 1984

cplQC[ Ulurnahio e il....................

2

...I..* ..,

j -

4..t.* .....-......

FIG. 5. Comparison of the CF1 and CFo-CF1 polypeptides from D.hardawil and spinach by SDS-polyacrylamide gel electrophoresis. Thechloroform-extracted CF, from spinach (lane 1) and D. bardawil (lane4) and the purified CFo-CF, ATPases from spinach (lane 2) and D.bardawil (lane 3) were isolated as described under "Materials and Meth-ods" and separated on 1-2.5% SDS-polyacrylamide gel (17). Samplescontaining 30 ,g protein were applied to each slot. Greek letters designateCF, subunits and Roman letters designate CFo subunits.

the D. bardawil CFo (I and II). The DCCD-binding protein(subunit III) is not seen in this gel due to its poor staining. Theapparent mol wt of the D. bardawil CFo-CF1 subunits fi, a, 'y, 6,I, II, and e (calculated by comparison with the migration ofa setof polypeptide markers) are 52, 49, 35, 21, 17.8, 17, and 16.5kD, respectively.The Effects of Glycerol and Salt on the Activity of the D.

bardawil CFo-CF, Complex. Figure 6 shows that glycerol stimu-lates the rate of ATP-Pi exchange in proteoliposomes containingthe D. bardawil CFo-CF complex, but the magnitude of stimu-

[glycerol],% (V/V)

FIG. 6. Glycerol titration curve for the ATP-Pi exchange activitycatalyzed by D. bardawil and spinach CFo-CF1 containing proteolipo-somes. CFo-CF, proteoliposomes were reconstituted by CD or by FTSreconstitution techniques, and ATP-P1 exchange was measured in thepresence of tke indicated glycerol concentrations as described in "Mate-rials and Methods." The control (100%) rates of ATP-Pi exchange were

170, 20, and 45 nmol ATP/mg protein - min with spinach, D. bardawil(CD) and D. bardawil (FTS) complex containing proteoliposomes, re-spectively.

lation depends on the technique used for reconstitution. Glycerolat 10 to 20% only slightly stimulates (about 10%) the rate ofATP-Pi exchange with proteoliposomes prepared by the FTStechnique in which the lipid to protein ratio is high (50-100).However, glycerol at 20% greatly enhances the rate of ATP-Piexchange with CFo-CF, proteoliposomes prepared by the CDtechnique. These proteoliposomes have a lower lipid to proteinratio (8-10) and, in the absence ofglycerol, catalyze a lower rateof ATP-Pi exchange (between 10 and 50% of the maximal rate).At high glycerol concentrations, the activity of D. bardawil CFo-CF, complex containing proteoliposomes is only slightly in-hibited irrespective of the reconstitution procedure whereas theactivity of proteoliposomes containing the spinach enzyme isstrongly inhibited by glycerol (K50 = 15%).

In order to determine if the low glycerol stimulation and thehigh glycerol inhibition of the rate ofATP-Pi exchange catalyzedby D. bardawil CFo-CF, containing proteoliposomes are revers-ible, the reconstituted enzyme was preincubated with or withoutglycerol and subsequently assayed under different conditions.These results, summarized in Table II, demonstrate that prein-cubation ofCD-proteoliposomes with glycerol does not stimulatethe rate of ATP-Pi exchange. This stimulation requires the pres-ence of glycerol during the assay. Similarly, preincubation ofFTS-proteoliposomes with 50% glycerol does not inhibit the rateof ATP-Pi exchange to the same extent as does 50% glycerol inthe assay. These results indicate that both the low glycerolstimulation and the high glycerol inhibition of the rate of ATP-Pi exchange catalyzed by D. bardawil CFo-CF, containing pro-teoliposomes are reversible effects.The stimulation of ATP-Pi exchange in proteoliposomes con-

taining the D. bardawil CFo-CF, complex prepared by the CDmethod is not unique to glycerol and can also be obtained bycomparable concentrations of other polyols such as ethyleneglycol, sorbitol, or sucrose, as shown in Table III.The activity of D. bardawil CF0-CF, containing proteolipo-

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GLYCEROL-RESISTANT CFo-CF, AND CF, FROM DUNALIELLA BARDAWIL

Table II. The Reversibility ofthe Effects ofGlycerol on the Rate ofA TP-PA Exchange Catalyzed by Proteoliposomes Containing the D.

bardawil CFo-CF, ComplexD. bardawil CFo-CF, containing proteoliposomes were prepared either

by CD or by FTS techniques. The vesicles were incubated for 5 min at23°C in 1 ml containing 40 mm Na-Tricine (pH 8.0), 0.1 mM EDTA,and 0, 20, or 50% glycerol. The incubation was terminated by passingthe proteoliposomes through Sephadex columns by the centrifugationprocedure (21). The columns were pre-equilibrated with the same bufferat the indicated glycerol concentrations. Control rates (100%) of ATP-Piexchange were 52.4 and 47 nmol ATP formed/mg protein min in FTSand CD proteoliposomes, respectively.

Type Glycerol Concentration ATP-PiofExhne

Reconstitution Preincubation Sephadex Assay Exchange,% (v/v) % control

CD 0 20 10020 20 20 8620 0 0 7.30 0 0 2.5

FTS 20 20 10020 20 20 7550 20 20 6650 50 50 25

Table III. Polyol Specificityfor the Stimulation ofthe Rate ofA TP-PiExchange Catalyzed by D. bardawil CFo-CF1 Containing

ProteoliposomesD. bardawil CFo-CF, containing proteoliposomes were prepared by

the CD reconstitution technique, and ATP-Pi exchange was measured inthe absence or presence of 10 or 20% ethylene glycol (v/v), glycerol (v/v), sorbitol (w/v), or sucrose (w/v). The control (100%) rate of ATP-Pexchange was 9 nmol ATP formed/mg protein. min.

ATP-Pi Exchange with Polyol at

0 10% 20%% control

Ethylene glycol 100 138 138Glycerol 100 145 167Sorbitol 100 136 166Sucrose 100 115 150

somes is also more sensitive to high salt than is the activity ofproteoliposomes containing the spinach enzyme, as demon-strated in Figure 2B. NaCl inhibited the rate ofATP-Pi exchangeby 50% at 60 mm NaCl with D. bardawil CFo-CF complexcontaining proteoliposomes whereas 300 mm NaCl was requiredfor 50% inhibition with spinach CFo-CF1 complex containingproteoliposomes.The effects of salt and of glycerol on the purified D. bardawil

and spinach enzymes are, therefore, generally similar to theeffects of salt and glycerol on photophosphorylation and mayindicate that the differences in the sensitivity of D. bardawil andspinach thylakoid membranes to salt and glycerol are at least inpart due to the different properties ofthe CFo-CF. ATP synthases.

DISCUSSION

The results presented here demonstrate that D. bardawil thy-lakoid membranes, CFo-CF1 complex, and CF. exhibit a hightolerance to high glycerol concentrations and even, in some cases,a glycerol dependence when compared to similar systems foundin other higher plants and algae. These findings suggest that thephotosynthetic apparatus in D. bardawil is well adapted to func-

tion at the high glycerol concentrations which are accumulatedinternally in the cells under physiological growth conditions (7,10). Very few comparisons of the glycerol sensitivity of variousDunaliella enzymes with other high plant enzymes have beenperformed in the past. Ben Amotz (4) found, for example, thatwhereas the rate of electron transfer from water to Diuat cata-lyzed by lettuce thylakoid membranes is slightly inhibited by 3M glycerol, the rate of electron transfer catalyzed by D. parvathylakoid membranes was stimulated. The observation that 20%glycerol (about 2.7 M) stimulates ATP formation by D. bardawilthylakoid membranes is consistent with this observation. Theglycerol concentration (2.7 M) that optimally stimulates photo-phosphorylation with D. bardawil thylakoid membranes is closeto the optimal external osmolarity required for maximal photo-synthetic activity ofwhole Dunaliella cells (7). Tolerance to highglycerol concentrations is found not only for reactions catalyzedby D. bardawil thylakoid membranes but also for the reactionscatalyzed by the purified D. bardawil CFo-CF, complex whenreconstituted into proteoliposomes (Fig. 6) and for the Mg+-dependent ATPase activity of the D. bardawil CF1 (Fig. 4A).This suggests that the structure of the D. bardawil CFo-CFI ATPsynthase has been modified in comparison to the spinach or therelated alga C. reinhardi complexes. This is indeed indicated bythe altered migration of the D. bardawil CFo-CF, subunits onSDS-polyacrylamide gels reported here, by an altered amino acidcomposition, and by an increased sensitivity to various inhibitors(to be reported elsewhere).There are some intriguing observations regarding the effects of

glycerol on the D. bardawil ATPase. These include the differen-tial effect of glycerol on the rate of ATP-Pi exchange dependingupon the method chosen to reconstitute proteoliposomes (Fig.6), and the differential sensitivity ofthe induced D. bardawil CF,ATPase activity depending upon the method chosen for induc-tion (Fig. 4).The differential effect of glycerol on the stimulation of the rate

of ATP-Pi exchange in CD and FTS proteoliposomes catalyzedby the D. bardawil CFo-CF, complex may be related to thedifferent lipid/protein ratios used in the two reconstitution pro-cedures. The highest rates of ATP-Pi exchange catalyzed by thespinach CFo-CF, complex are obtained by the CD reconstitutiontechnique (U. Pick, unpublished observations). This procedure,however, is not very effective in the preparation of active proteo-liposomes containing the D. bardawil CFo-CF, complex. TheFTS procedure produces proteoliposomes with a much higheractivity. The ATP-Pi exchange activity ofthe D. bardawilenzymeapparently requires a higher lipid to protein ratio in the absenceof glycerol, but the activity may be improved by glycerol at alimiting lipid to protein ratio. This interpretation implies thatthe effect of glycerol is at least partly at the level of lipid-proteininteractions.A high tolerance of the D. bardawil CF, to glycerol is also

apparent with the soluble CF1. The Mg2"-dependent ATPaseactivity induced by solvents is virtually unaffected by glycerolconcentrations as high as 20 to 30% (v/v). However, the differ-ences between the glycerol sensitivity of the D. bardawil andspinach enzymes are smaller than those observed for reactionscatalyzed by thylakoid membranes and the CFo-CF, containingproteoliposomes. In contrast, the heat-induced Ca2"-dependentATPase activity of both enzymes is equally sensitive to glycerolinhibition (Fig. 4B). It may be noteworthy that the OG-activated,Ca-ATPase activity of spinach CF, is also more sensitive to highionic strength in comparison to the OG-dependent, Mg-ATPasectivity (22). A possible explanation for these differences is thatsoluble CF, (in the absence of organic solvents or OG) exists ina distorted conformation which alters its structural and catalyticproperties (allotopy). We have previously suggested that OG andorganic solvents may induce a structural change in solbule CF,

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772 FINEL ET AL.

converting it to a form that resembles its native conformationand then restores its catalytic properties (22). The differentialsensitivity of the D. bardawil CF, to glycerol in the presence orabsence of alcohols would be consistent with this view.Although a careful comparison between Dunaliella and other

higher plant enzymes with respect to their sensitivities to salt hasnot been performed, it has been demonstrated that the activitiesof Dunaliella membranes, as well as some soluble enzymes aresensitive to NaCl (6, 7, 10). The results shown in Figure 2 areconsistent with previous results and demonstrate that Dunaliellachloroplasts are even more sensitive to NaCl than other higherplant chloroplasts. This might suggest that the Dunaliella pho-tosynthetic system is adapted to a low ionic strength environ-ment.

Direct measurements of the internal NaCl content of Duna-liella cells and of the permeability of the Dunaliella outer mem-brane to NaCl are controversial. Indirect, earlier evidence (15)and more recent observations (14) suggest that the outer mem-brane of Dunaliella is quite permeable to NaCl and that theinternal salt concentration in the alga is high. In contrast morerecent direct measurements of the salt content of Dunaliella cellsgrown in high salt suggest that the internal NaCl concentrationin Dunaliella is low (M. Avron, personal communication).The results reported here demonstrate that the photosynthetic

system of D. bardawil is adapted to a high glycerol, low saltmedium and therefore is consistent with the idea that undernormal growth conditions (high NaCI), Dunaliella cells containhigh glycerol and low salt internally. The structural changes,reflected in the altered migration of D. bardawil CFo-CF, sub-units on SDS-PAGE may be related to this adaptation.

Acknowledgment-We are indebted to Prof. Mordhay Avron for his suggestionto purify CF, and CFo-CF, from Dunaliella which gave us the initiative for this

work.

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