Characterization of the He-Pumping F1F0 ATPase ofVibrio alginolyticus
LEE R. KRUMHOLZ, URSULA ESSER, AND ROBERT D. SIMONI*
Department of Biological Sciences, Stanford University, Stanford, California 94305
Received 16 May 1990/Accepted 28 August 1990
The FIFO ATPase of Vibrio alginolyticus was cloned from a chromosomal A library. The unc operon, whichcontains the structural genes for the ATPase, was sequenced and shown to have a gene organization ofuncJBEFHAGDC. The sequence of each subunit was compared with those of other eubacterial ATPases. TheV. alginolyticus unc genes exhibited greater similarity to the Escherichia coli unc genes than to any of the otherbacterial unc genes for which the sequence is available. The ATPase was expressed in an E. coli unc deletionstrain, and the ATP hydrolytic activity was characterized. It has a pH optimum of 7.6 and is stimulated by theaddition of Triton X-100 or any of a variety of salts. The recombinant F1FO was purified 30.4-fold andreconstituted into proteoliposomes. This enzyme catalyzed the pumping of protons coupled to ATP hydrolysisas measured in fluorescence quenching experiments but would not pump Na' ions under similar conditions.
Vibrio alginolyticus is a facultatively anaerobic bacteriumthat exhibits optimal growth in medium containing relativelyhigh levels of NaCl (0.3 to 0.5 M). There has been recentinterest in its mechanism of regulating cation fluxes acrossthe cytoplasmic membrane. This interest stems from theinitial observation that between pH 7 and 8, a unique sodiumion pumping NADH:quinone oxidoreductase is active whichunder aerobic conditions can be used to generate a sodiumion gradient across the cytoplasmic membrane (26). Furtherresults indicated that V. alginolyticus could grow at pH 8.0in the presence of the protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP) and that under slightlyalkaline pH conditions, the AT was composed only of a Na+ion gradient with no ApH component (25). To produce ATPunder these conditions, this organism would require theATP-synthesizing machinery to be coupled to an Na+ gra-dient. The question of whether an Na'-translocatingATPase exists in V. alginolyticus was addressed by Dibrovet al. (7, 8). They showed that in intact cells ATP synthesiscould be induced by generating an NaCl concentrationgradient across the cytoplasmic membrane. The synthesis ofATP under these conditions was not abolished by theaddition of CCCP, suggesting that ATP synthesis was cou-pled directly to ApNa'.
In light of recent evidence that in the anaerobic marinebacterium Propionigenium modestum there exists an F1FO-type ATPase that is capable of pumping Na + coupled to ATPhydrolysis (20), it seemed possible that an Na+-dependentATPase was functioning under alkaline conditions in V.alginolyticus. The ATPase of P. modestum will pump pro-tons at low Na+ concentrations, but at NaCl levels of 1 mMor above Na+ is preferentially translocated over protons(21). This new finding that an F1FO-type ATPase can trans-locate both Na+ and H+ has had a significant influence onour current concept of the ion translocation mechanism.
It was the goal of this project to characterize the F1FOATPase of V. alginolyticus and in particular to determine itsion specificity.
* Corresponding author.
MATERIALS AND METHODS
Strains and culture conditions. V. alginolyticus 138-2 was agift of Hajime Tokuda. Escherichia coli 1100ABC, DK-8,and DK-6 all have chromosomal deletions of the eightstructural genes of the unc operon (16). In addition, E. coliDK-6 is a minicell-producing strain. All cultures used for thedetermination of membrane-bound ATPase activity weregrown aerobically in a modified LB medium unless other-wise indicated. This contained tryptone (10 g/liter), yeastextract (5 g/liter), NaCl (0.4 M), KCI (20 mM), MgSO4 (10mM), and glucose (2 g/liter). The pH was adjusted to 7.5 withNaOH.
Cloning and hybridization. V. alginolyticus chromosomalDNA was isolated as described previously (2). Southern blotanalysis was as previously described. For construction of XDNA libraries, chromosomal DNA was partially digestedwith EcoRI to yield fragments in the 10- to 20-kb size range.The entire partial digest was ligated into X Dash arms(Stratagene Cloning Systems, LaJolla, Calif.) previouslydigested with EcoRI. The ligated DNA was packaged with acommercial packaging preparation (Gigapack; Stratagene). Xclones containing unc DNA from V. alginolyticus wereselected by plaque hybridization (2) to a nick-translatedplasmid, pRPG44 (13), containing the E. coli genes uncEFHA. Fragments of unc DNA were initially subcloned intopBluescript SK and KS (Stratagene) for sequence analysis.The 9.0-kb Sacd fragment, which contains the structuralgenes uncBEFHAGDC (Fig. 1), was cloned into pUC19 tomake pLRK1. This plasmid was used for studies in which E.coli membrane vesicles were assayed. The same fragmentwas cloned into pCQV2 (23) to make pLRK2. The latterplasmid was used to express the F1F0 for purification. Thenucleotide sequence of this entire region has been published(17).
Preparation of membrane vesicles and quantitation ofATPase activity. Cells of V. alginolyticus, E. coli 1100, andE. coli 1100ABC(pLRK1) were grown to an optical density at600 nm of 1.0 and harvested by centrifugation. Membranevesicles were prepared as previously described (1). Fluores-cence quenching experiments were performed as previouslydescribed (1), except that the ATP concentration was 1.0mM. Assay of ATPase activity was done in a reactionmixture containing 50 mM Tris hydrochloride (pH 7.5), 5
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mM MgCl2, and 2.5 mM Na-ATP. Rates of ATP hydrolysiswere determined by quantitating phosphate released overtime (19).
Purification of F1FO. The purification procedure was amodification of that described by Friedl and Shairer (11). Allmanipulations were done with the samples on ice unlessotherwise indicated. The buffers used were identical, exceptthat buffer A contained 50 mM Tris (pH 7.5) and 100 mMKCl rather than morpholinepropanesulfonic acid. Buffer Dwas used at pH 7.5 rather than pH 8.0. E. coli DK-6(pLRK2)was grown at 30'C until it reached an optical density at 600nm of 0.5. The temperature was increased to 420C for 4 h toinduce expression of the ATPase genes from the heat-inducible X cI857 promoter. Cells were harvested and frozenin liquid N2. Samples of 120 g of cells were thawed andsuspended in 150 ml of buffer A. Cells were broken in aFrench press at 18,000 lb/in2, and cell debris was removed bycentrifugation at 16,000 x g for 20 min. The supernatantsolution was centrifuged at 180,000 x g for 2 h. Themembrane pellet was suspended in buffer A and centrifuged.The membranes were suspended, left on ice overnight, andcentrifuged. The membranes were again suspended in bufferA to 125 ml. Aminoxide WS-35 was added to this solution to11 mM and stirred on ice for 20 min. The mixture recentri-fuged for 2 h at 180,000 x g. The supernatant solution wasremoved and immediately made to 20% methanol and 25 mMaminoxide WS-35. This extract was run through a DEAE-Sepharose column (10 by 2.5 cm) previously equilibratedwith buffer D containing 80 mM KCI at 4TC. The protein waseluted with 150 ml of buffer D (110 mM KCl) and a 300-mlbuffer D gradient (110 to 300 mM KCl). Fractions of 10 mlwere monitored for ATPase activity, and those with ATPaseactivity were pooled to a total volume of about 70 ml.
Reconstitution. Pooled ATPase (1 to 5 ml) was made to 6mM MgCl2 and 12.5% polyethylene glycol (PEG) 8000. Thismixture was left on ice for 5 min and then centrifuged for 15min (17,000 x g) at 4°C. The supernatant solution wasremoved, and the pellet was centrifuged for 5 min. Theresidual supernatant solution was removed. The pellet wasthen used as the source of enzyme for reconstitution.Soybean phosphatidylcholine (Sigma type II-S) was ace-
tone precipitated and ether extracted. The ether was driedoff, and 80 mg of the residue was dissolved in 2 ml ofreconstitution buffer [Tricine-KOH (pH 7.5), 50 mM; meth-anol, 20%; p-aminobenzamidine, 6 mM; ethylene glycol-bis(,-aminoethyl ether)-N,N,N1,N1-tetraacetic acid, 0.2 mM;phenylmethylsulfonyl fluoride, 0.1 mM; dithiothreitol, 0.2mM; KCl, 100 mM]. This mixture was sonicated with amicroprobe tip (model W-220; Heat Systems-Ultrasonics,Inc.) for 5 to 10 min on ice until the solution becametranslucent. This solution was centrifuged for 10 min in amicrofuge at room temperature. The PEG-precipitated en-zyme was warmed to room temperature, and the lipidvesicle-containing solution was added to it at about 1 ml per2 mg of purified protein. The enzyme was dissolved byvortexing at room temperature. To this solution was added 1,l of 100 mM MgCl2 per 0.1 ml and vortexed thoroughly.This was maintained at room temperature for 5 min and thenon ice for 15 min. After the incubation, the solution hadchanged from translucent to opaque white. It was thendiluted with 5 to 10 volumes of purification buffer A madewith Tricine-KOH rather than Tris hydrochloride and cen-trifuged for 1 h at 260,000 x g. The pellet was suspended inthis same buffer A to a final concentration of 40 mg ofphospholipid per ml.Sodium ion transport experiments. The sodium ion trans-
unc GenesPI B EF H A G D C.. L I I I I
0 X0c
:m _
I
5.0 Kb 0n=)
p',0)0
4.0 Kb
m00:m
4.5 Kb
FIG. 1. Organization of the genes in the unc operon of V.alginolyticus. The individual genes are named in the same way asthe genes of the unc operon in E. coli.
port experiments were a modification of the procedure ofLaubinger and Dimroth (20). The incubation mixtures con-tained the following at pH 7.5 in 100 pl: 50 mM Tricine-KOH, 5 mM MgCl2, 3 U of pyruvate kinase, 6 mM phos-phoenolpyruvate, 2.5 VuM CCCP, 1 or 2 ptCi of 22NaCl (17.8Ci/mol), and 0.8 mg of proteoliposome lipids. NaCl wasadded at 1, 5, 20 and 100 mM with KCl added in each case tomake the final salt concentration 100 mM. K-ATP was addedto 1 mM to start the reaction. Controls were run with no ATPaddition. After a 10-min incubation the entire reaction mix-ture was run through a 0.5-ml Dowex 5OW-X8 K+ column.The vesicles were eluted with 9% (isoosmotic) sucrose andthen trapped by filtration through a nitrocellulose BA-85filter (Schleicher and Schuell). The filters were then countedin a Gamma-4000 gamma counter (Beckman Instruments,Inc.).
Vesicles could be preloaded with 22NaCl by adding 2 1xCito 0.5 ml of reconstitution buffer before sonication. Thesepreloaded vesicles were then used to test the efficiency of theDowex column and the filtration procedures.Other methods. Proteins were determined with the BCA
protein assay reagent (Pierce, Rockford, Ill.). Sodium dode-cyl sulfate (SDS)-gel electrophoresis was with the method ofLaemmli (18). Minicell experiments were performed aspreviously described (15) with the minicell strain E. coliDK-6. Amino acid similarities were determined with thealgorithm of Needleman-Wunch with the Genetics ComputerGroup sequence analysis software package v. 6.1a (6).
RESULTS
Organization of genes. Based on the analysis of openreading frames, the organization of the unc operon of V.alginolyticus was determined (Fig. 1). The organization isessentially the same as for the une operon of E. coli with theorder of genes uncIBEFHAGDC (13). Because of the inter-esting translational regulation that results in different levelsof each of the particular subunits (3), it was of interest tolook at the ribosome-binding sites and the intergenic regionsof the unc operons of E. coli and V. alginolyticus (Table 1).One interesting observation is the unique ribosome-bindingsite of uncH (GGGGG), which may be one of the factors thatinfluences the rate of synthesis of this subunit relative to thesubunits that surround it. Only one copy of the 8 subunit ismade per ATPase molecule. The other feature is the rela-tively short intergenic region between the uncD and uncCgenes of V. alginolyticus relative to that of E. coli. Otherthan these features, there is a considerable similarity be-tween the two operons.Amino acid similarity. The amino acid sequence similar-
ities of the individual ATPase subunits of E. coli and Bacillusmegaterium with V. alginolyticus have been determined
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TABLE 1. Comparison of the intergenic regions, start codons, and ribosome-binding sites of the urnc operons ofV. alginolyticus and E. coli
Ribosomal binding site Start codon Size of intergenic regionGene (subunit) Stoicheometry Region
E. coli V. alginolyticus E. coli V. alginolyticus E. coli V. alginolyticus
(Table 2). All of the E. coli subunits except i show verystrong similarity to the V. alginolyticus subunits; c was theleast conserved structural subunit. In contrast, the similaritywith the B. megateriurn subunits is very strong for et and Pand much weaker for the others; the least conserved werethe 8, b, and i subunits. The amino acid sequences of each ofthe bacterial a subunits that are available were aligned (Fig.2), and their similarities were determined (Table 3). Since thea subunit may be a candidate for the structural component ofthe proton pore (4), these results would give us an indicationof the relative structural relatedness of the ion pore. A clearresult is that the V. alginolyticus a subunit is structurally themost highly related to E. ccli when compared with any of theother ATPases that have been sequenced.Membrane-bound ATPase activities. Everted membrane
vesicles were prepared from E. coli 1100 and 1100ABC(pLRK1). The latter is an unc deletion strain with aplasmid containing all of the ATPase structural genes. Thesevesicles were used to determine fluorescence quenchingactivity. Fluorescence quenching is a technique used todetermine the level of ATP-dependent proton pumping ac-tivity carried out by membrane vesicles. Both the E. coliwild-type strain 1100 and 1100ABC(pLRK1) exhibited typi-cal ATP-dependent fluorescence quenching (Fig. 3). Whenthe deletion strain 1100ABC was examined, no ATP-depen-dent fluorescence quenching activity was detected. When V.alginolyticus membrane vesicles were prepared in the sameway as the E. coli vesicles, a small amount of fluorescencequenching activity was observed only when the MgCl2 levelwas reduced to 1 mM (data not shown).
TABLE 2. Amino acid similarities between the different subunitsof the F1FO ATPase of V. alginolyticus with E. coli and
Determined with the algorithm of Needleman and Wunch (6).
Levels of membrane-bound ATPase activity were deter-mined with the recombinant ATPase, the wild-type E. coliATPase, and the V. alginolyticus ATPase from V. alginelyti-cu's cells (Fig. 4). The activity of the V. alginolyticus ATPasewas stimulated by the addition of NaCl or Triton X-100. Thiseffect was additive; when both were added, the activityincreased beyond that of either one alone. An interestingfeature of the Triton X-100 stimulation was that it loweredthe pH optimum for activity. When crude membranes wereassayed in the presence of glutamate or chloride as theanion, a similar stimulation was observed with the additionof 100 mM of the sodium salt. This effect was not specific toNa+; similar activity was observed on addition of 100 mMpotassium or sodium glutamate. Both the recombinant en-zyme and the V. alginolyticus enzyme exhibited similar pHcurves and effects of Triton X-100 stimulation. The V.alginolyticus membranes contained a phosphatase activitythat was stimulated by NaCI and MgCl2. Evidence for thiscomes from experiments in which the specific FF0 ATPaseinhibitor N,Nl-dicyclohexylcarbodiimide (DCCD) was used.At 50 ,uM DCCD, V. alginclyticus membrane vesicleATPase activity was only inhibited by 9% with the reactionmixture at pH 8.3. When the vesicles were assayed at pH 6.8with 5 mM MgCI2 and 0.1% Triton X-100, conditions atwhich phosphatase activity is reduced relative to ATPaseactivity, 48% inhibition by DCCD was observed. When theactivity was assayed in the presence of 100 mM NaCl, thephosphatase was stimulated enough to create a very highbackground at the higher pH values (Fig. 4B). We also keptthe level of MgCl2 to 1 mM to reduce exogenous ATPaseactivities. Membranes prepared from E. coli 1100ABC hadless than 0.04 U of ATPase activity over the entire pH range.The recombinant ATPase exhibited a broad Mg2+ opti-
mum between 1 and 10 mM (Fig. 5), in contrast to the E. colienzyme, which had an Mg2+ optimum at 1 to 2 mM. Thisactivity was inhibited by 60 to 72% when the membraneswere incubated for 15 min with 50 ,uM DCCD at pH 6.8 or8.3 before the addition of ATP to start the reaction.
Purification of the recombinant ATPase. To further char-acterize the recombinant enzyme, this V. alginolyticusATPase was expressed from the heat-inducible promoter onpLRK2. To be sure that the full complement of unc geneswas expressed from this plasmid, pLRK2 was transformedinto a minicell strain of E. coli, and the proteins expressedfrom the plasmid were labeled and subjected to SDS-gelelectrophoresis. The autoradiogram (Fig. 6) shows the pres-ence of all of the subunits that are encoded by the plasmid.Each of the E. coli subunits was identified based on a
IADNVKDTFHGRN PLIAPLA IWC D 1IVEDFLPYPAEHWLGIPYLNGSVKDMYHGKS KLIAPLA DITWD Pt DLLPYIAEHVLGLPALRNLARTQIGEKEYRPWVPFI LTFF SGAIVI:KLIKLPSGELAAERDLAKNQIGEKEYRPWVPFV LF SGAI4V.FKLIHLPEGELTAJANMIRDNVG KEGMKYFPYI LG LP1YSFTFTSHIAVTAARGIINSTMDWQTGGRFLTLG MY LG LPFSVHVN GELWWKSGLVNSNMDWKTGGRFLTLG LG AIVI DHNLWWKS
FIG. 2. Amino acid sequence alignment of the a subunits of a variety of bacterial F1Fo ATPases. Highlighted amino acids are highlyconserved between the seven species. The eight transmembrane segments as determined by the topological analysis of Lewis et al. (22) areindicated by brackets. The species indicated are V. alginolyticus (VA), E. coli (EC), Synechococcus sp. strain 6301 (SY), Anabaena sp. strainPCC-7120 (AN), Rhodospirillum rubrum (RS), strain PS-3 (PS), and Bacillus megaterium (BM).
comparison of its pattern with published autoradiograms(16). The V. alginolyticus subunits were identified based ontheir molecular weights as calculated from their nucleotidesequences and from their levels of relatedness to the E. colisubunits.The ATPase was partially purified from washed mem-
branes of an E. coli unc deletion strain in two steps. Themembranes were originally extracted with 11 mM aminoxideWS-35, and the extract was purified with a DEAE-Sepharosecolumn. The ATPase activity eluted between 185 and 225mM KCl. These two steps resulted in a net purification of30-fold (Table 4) with a total purity of about 84% based on
peak area after densitometry of the gel (Fig. 7). The purifi-cation procedure that was used differed from the originalpurification procedure of Freidl and Schairer (11), mainly ina single step. This was the sodium cholate membrane extrac-tion, which was not used because it resulted in a large loss ofactivity of the V. alginolyticus ATPase preparation. Othertreatments that have been used with the ATPase of E. coli,such as treatment with 0.5% sodium deoxycholate or 0.2 mMEDTA, destroyed the activity of the V. alginolyticusATPase.The partially purified enzyme was activated by approxi-
mately 50% by the addition of 100 mM of either NaCl, KCl,
TABLE 3. Amino acid similarities of the a subunits of several eubacteriaa
% Similarity or identity with:Species
V. alginolyticus Synechococcus sp. E. coli B. megaterium Anabaena sp. PS-3 Rhodospirillum rubrum
a Determined with the algorithm of Needleman and Wunch. The percent similarity values are given in lightface type; the percent identityvalues are given in boldface type.
VA 65EC 68SY 73AN 60RS 52PS 46BM 45
TAGV
LI
VA 137EC 140SY 139AN 126RS 119PS 110BM 109
VA 213EC 216SY 204AN 191RS 180PS 174BM 175
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ATPase OF VIBRIO ALGINOLYTICUS 6813
LU ILL
0Cl)
0
-I~ ~ ~ ~~TM
LL
LU
LU
15%
1 mmn
TIME
FIG. 3. Fluorescence quenching profile of membrane vesiclesprepared from E. coli strains (I) 1100ABC(pLRK1), (II) 1100, and(III) 1100ABC. The reaction mixture contained 50 mM Tris hydro-chloride (pH 7.5), 10 mM MgCl2, and 1 ,uM ACMA. Additions at thearrows were of NADH, KCN, Na-ATP, and CCCP at 0.5 mM, 0.5mM, 0.75 mM, and 2.5 p.M, respectively.
or LiCl; all had similar effects. Na2SO4 had an effect similarto that of NaC1, indicating that the effect was not anionspecific. When Na' ions were completely replaced by K+ inthe reaction mixture, the activity was similar to that ob-served with NaCl alone. The activity was also increased withincreasing levels of NaCl up to 500 mM (Fig. 8). The specificactivity of the purified enzyme was determined over a broadpH range (Fig. 9). The activity had a pH optimum that was
the same as that of the crude membrane-bound form. Theeffect of the addition of 0.1% Triton X-100 was a shift downin pH optimum and a great stimulation of activity. Thepartially purified enzyme was completely inhibited by 50 ViMDCCD and 92% inhibited by as little as 10 ,uM DCCD. TheseDCCD results are consistent with the fact that the purifiedenzyme is an intact F1Fo ATPase. The addition of TritonX-100 to the reaction mixture will not reverse the effect ofDCCD of the V. alginolyticus ATPase.
Dmitriev et al. (10) have shown that the subunit homolo-gous to the P subunit in E. coli migrates at a higher molecularweight than that of the one homologous to the a subunitwhen separated by SDS-polyacrylamide gel electrophoresis.These results allowed us to locate these subunits on an SDSgel. We have decided to retain the E. coli nomenclature forthese as well as the other subunits. The molecular masses as
determined by SDS-gel electrophoresis are 56.4, 59.2, 39.8,24.3, 14.3, 26.1, 18.1, and 9.9 kDa for subunits a, ,, y, 8, E,
a, b, and c, respectively. The molecular masses as calculatedfrom the amino acid compositions determined from thenucleotide sequences of the unc genes, were 55.6, 50.7, 31.9,19.5, 15.3, 30.1, 17.3, and 8.6 kDA, respectively, for thesame subunits.
Characterization of the reconstituted ATPase. The recom-
binant ATPase was reconstituted directly from the PEG
0.6
0.5
0.4
0.3
0.2
0.1
C
0*-
co
E._C
a0E
C,
cL
0.
C.)
0
0.10
0.08
0.06
0.04
0.02
0
0.6-NaCaI0.6; ; >~Triton
0.4
0.2
0
6.0 7.0 8.0 9.0
pH
FIG. 4. Membrane-bound ATPase activities determined at dif-ferent pH values. Specific activities were determined for membranesprepared from (A) E. coli 1100ABC(pLRK1), (B) V. alginolyticus,and (C) E. coli 1100. The reactions were done in Tris-Imidazolebuffer (50 mM each) adjusted with HCl, 5 mM MgCl2 (1 mM for V.alginolyticus), and 2.5 mM ATP. Membranes were assayed with no
additions (NA), 0.1% Triton X-100, 100 mM NaCl, or both TritonX-100 and NaCl.
precipitate. In this way, we were able to avoid introducing a
large amount of aminoxide WS-35 into the proteoliposomepreparation.The percentage of ATPase molecules incorporated and the
relative percentage of those with the substrate-binding sitefacing outward were determined by using the protocol ofLaubinger and Dimroth (20). The PEG precipitation resultedin recovery of 74% of the activity. Recovery of 83% of thetotal activity in the PEG precipitate was found in theproteoliposomes when they were assayed in 0.1% TritonX-100. Control proteoliposomes had 52% of the activity seen
in the presence of 0.1% Triton X-100 after correction forstimulation by the detergent. This indicated that the orien-
a z XLz Ye 4I0.
IL
Triton ~*A NaCI
A 0
A"'' | NA
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6814 KRUMHOLZ ET AL.
1.5 .
E ci 110E. Coi 1100ABC
010
0 2 4 6 8 10
MgCI2 CONCENTRATION (mM)
FIG. 5. Membrane-bound ATPase activities determined at dif-
ferent MgCl2 levels. Membranes were prepared from each of the
indicated strains and assayed in 50 mM Tris hydrochloride (pH
7.5)-0.1% Triton X-100-2.5 mM ATP.
tation of the ATPase in the vesicles was almost completely
random.
Characterization of the reconstituted proteoliposomes was
done by looking at their capacity for ATP-coupled pumping
of Na+ or H+. We could not detect any ATP-coupled 22Na+uptake during any of our experiments. The recombinant
ATPase did have a strong capacity for proton pumping, as
determined through fluorescence quenching (Fig. 10). Nei-
ther potassium phosphate nor Tris hydrochloride buffer
allowed as much fluorescence quenching as that with Tricine
KOH. The addition of NaCl had no apparent effect either
inside or outside the vesicles as compared with KCl, except
that at least 1 mM KCl was required for optimal quenching.
This level of KCl was required for optimal activity of the K+ionophore valinomycin. When valinomycin was left out of
the reaction mixture, significantly less quenching was ob-
served, and the fluorescence would not rebound on the
addition of CCCP. In the presence of Tricine-KOH and
valinomycin, fluorescence quenching experiments were
done with the buffer at different pH values between 7.0 and
8.5. Under these conditions the internal pH should be the
same as the external pH before the start of the experiment.
No differences in fluorescence quenching activity were de-
tected. As with the purified enzyme, the ATPase activity of
the proteoliposomes as determined by fluorescence quench-
ing was completely inhibited by 50 ,uM DCCD.
DISCUSSION
Our primary goal in this study was to determine the ion
specificity of the F1F0 ATPase of V. alginolyticus. The best
way to determine these features is to purify the enzyme and
reconstitute it into artificial phospholipid vesicles. Vesicles
that were prepared with the reconstituted ATPase had a high
capacity for ATP-dependent proton pumping, yet under the
conditions tested they would not pump sodium ions. Further
evidence for the lack of ability to pump Na+ is derived from
experiments in which the concentration of Na+ exceeded
that of protons by 106-fold and yet no inhibition of proton
pumping was observed. In P. modestum, the ATPase will
EN
O CCU -J
CL CQ
Le)
0.C.
CAT-
4 b/
FIG. 6. Minicell experiment. Autoradiogram prepared from a12% polyacrylamide gel run with [35S]methionine-labeled minicells.The lane labels represent the plasmids used in each experiment witheach subunit or chloramphenicol acetyl transferase (CAT) labeled.E. coli ATPase subunits are produced from pRPG54, and V.alginolyticus subunits are produced from pLRK2.
pump protons at very low Na' levels, but with as little as 0.5mM Na' the Na'-pumping activity takes precedence overproton pumping (20). This level of Na' exceeds the level ofprotons by 5 x 103-fold. A feature of Na'-pumping enzymesis a stimulation of net activity on the addition of specificallyNa' to the reaction. A variety of Na' transport decarbox-ylases require Na' for catalytic activity (9). The ATPaseactivity of the P. modestum ATPase is greatly stimulated bythe presence ofNa' (20). This feature was not observed withthe enzyme cloned from V. alginolyticus. Activity wasstimulated by NaCl, but the effect was not specific to Na'and was observed with all of the other salts tested. Anonspecific stimulation of ATPase activity by the addition ofsalt is also observed with E. coli membranes (Fig. 4).
TABLE 4. Purification of the V. alginolyticus FIFO ATPasea
Total Sp act FoldStep Vol activity Protein (~Lmol/ purifi-(ml) (,umolI (mg/ml) mi/mg) cation
a Assays were conducted in the presence of 50 mM Tris hydrochloride (pH7.5)-S5 mM MgCl2-0.1% Triton X-100-2.5 mM Na-ATP.
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1.2
1.0 _-
uJ
z4m
m0CD)
0.8 _-
0.6
0.4
0.2
180 160 140 120 100 80 60 40 20 0
DISTANCE FROM ORIGIN (mm)
FIG. 7. Densitometric scan of a 12% SDS-polyacrylamide gel ofthe purified V. alginolyticus ATPase. The gel was loaded with 45 ,ugof protein and stained with Coomassie blue. kD, Kilodaltons.
This leaves us in the difficult situation of explaining resultsindicating that an Na'-dependent ATPase exists in V. algi-nolyticus (8). There are several difficulties that one alwaysencounters when attempting to prove that an enzyme doesnot possess a particular activity. One possibility is that theconditions that were used to measure the Na'-translocatingactivity may not have been ideal. What seems to us like a
more likely explanation is that there is another ATP synthasethat is Na' coupled and that is not of the F1F0 type. An F1F0ATPase mutant of Vibrio parahaemolyticus has been char-acterized and shown to possess an ATP synthase activitythat is dependent on an Na' gradient and is independent ofthe F1F0 He-dependent ATP synthase activity (24). InStreptococcus faecalis, there is a vanadate-sensitive ATPasethat is capable of pumping potassium ions (12).The addition of Triton X-100 to the reaction mixture
stimulated activity of the V. alginolyticus ATPase and low-ered its pH optimum. Triton X-100 stimulated the P. mod-estum ATPase (20) but did not significantly stimulate the E.coli enzyme. Octyl glucoside stimulated the activity of theBacillus firmus ATPase and increased the pH optimum (14).
0.8.
0.7-
L 0.6
E
E ,/
an3.0.41
A"A
0.3 1 IJ
0 100 200 300 400
NaCI CONCENTRATION (mM)500
FIG. 8. ATPase activities of the purified V. alginolyticus ATPaseat increasing levels of NaCl in the reaction mixture. The solubleATPase was assayed in 50 mM Tris hydrochloride (pH 7.5)-5 mMMgCl2-2.5 mM Na-ATP.
1.5
a.i. .'
5 0c( m
s EC) CE-g
en EI-,
1.0
0.5
6.0 7.0 8.0 9.0
pH
FIG. 9. ATPase activities of the purified V. alginolyticus ATPasedetermined at different pH values. Conditions were the same as
those for the assays shown in Fig. 4.
When Triton X-100 was added to E. coli membranes previ-ously incubated with 50 FiM DCCD, inhibition was relieved.This indicated that the Triton X-100 caused the F0 todissociate from the F1. This effect was not observed with theV. alginolyticus ATPase.The recombinant ATPase was similar to the ATPase found
on membranes isolated from V. alginolyticus cells in everyaspect tested. These included pH optimum, the effect ofTriton X-100, and DCCD sensitivity. In addition, the a and, subunits have been isolated from V. alginolyticus mem-
A B
z
0
LU>
~~~~~15%LU
TIME
FIG. 10. Fluorescence quenching profiles with the recombinantATPase reconstituted into proteoliposomes. The proteoliposomeswere prepared in the presence of (I and II) Tricine NaOH and NaClor (III) Tricine KOH and KCI as described in Materials andMethods. The reaction mixtures contained the following: (I) 50 mMTricine NaOH (pH 7.5), 100 mM NaCl, 5 mM MgC12, and 1 p.MACMA; (II) I plus 1 mM KCl; (III) 50 mM Tricine KOH (pH 7.5),100 mM KC1, 5 mM MgCl2, and 1 V.M ACMA. Reactions were
carried out with (A) or without (B) 5 p.M valinomycin. Also addedwere 1 mM Na-ATP (I and II) or 1 mM K-ATP (III) and 2.5 p.MCCCP.
14.4 kD 21.5 kD
7
b
C
VOL. 172, 1990
a
97.4 kD
p31.0 kD 42.7 kD
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6816 KRUMHOLZ ET AL.
branes, and the amino acid sequence of the N terminus hasbeen determined (10). This sequence was the same as thesequence of the a and ,B subunits determined from thenucleotide sequence of the cloned V. alginolyticus uncoperon. The above factors indicate that the -activity of thecloned ATPase is similar to if not the same as that of thenative enzyme.A chromosomal EcoRI digest of V. alginolyticus DNA
was prepared, and a Southern blot was done. The blot washybridized with a labeled pRPG54, a plasmid containing allof the structural genes of the E. coli unc operon. Only asingle hybridizing band was observed with a relatively largesize (>15 kb). The X clone from which the V. alginolyticusunc genes were obtained contained an EcoRI fragment of15.5 kb. Since the E. coli unc gene-containing probe hasbeen shown to be specific for a variety of F1F0 ATPases, itseems likely that the enzyme that we have cloned is the onlyF1F0-type ATPase present in V. alginolyticus. Therefore, ifthere is a sodium-dependent ATPase in V. alginolyticus, it isnot likely to be an F1F0-type ATPase.The V. alginolyticus unc genes are arranged in a single
operon with the same order as that observed in several othercomplete bacterial unc operons (Fig. 1). This same arrange-ment has been observed in three other procaryotes, B.megaterium, E. coli, and strain PS-3. The unc operons of thethree photosynthetic procaryotes that have been sequenced(Fig. 2) are split up into two different loci on the chromo-some. The organization of the une genes in the seveneubacteria that have been characterized show a relationshipbetween the type of operon and the ability or lack thereof tophotosynthesize, rather than a relationship between phylo-genetic divergence and unc operon structure. This indicatesthat the splitting of the operons in the photosynthetic micro-organisms is a feature that confers particular phenotypiccharacteristics to the ATPase. It may not necessarily be theresult of conservative evolution but rather a mutation,required for an ideal photosynthetic lifestyle, that may haveoccurred many times.The a subunit has been studied in detail because it is
believed to be a key component of the proton channel in theF0 part of the ATPase (4, 5). In the past, a consensussequence for the a subunit was obtained by comparing the E.coli a sequence with that of eucaryotic a subunits (4). Acomparison of the a subunits of several eubacteria mayreveal important sequences that may no longer be present intheir eucaryotic counterparts. Beginning at position 63 of theV. alginolyticus subunit a sequence, there is a string ofconserved amino acids occurring every fourth to sixthposition (Fig. 2). In particular, the P, Q, and E occurring atpositions 69, 73, and 77, respectively, likely generate animportant surface of the first cytoplasmic loop. Other re-gions of the a subunit that show a high degree of similaritybetween species are found starting at positions 104, 180, 203,and 251 within membrane spans 2, 4, 5, and 8, respectively.This indicates that conservation of structure within certaintransmembrane-spanning regions is important.The overall similarities between the a subunits of the
different eubacteria have been summarized in Table 3. Theseclearly show a much closer overall relationship between thea subunits found in three pairs of eubacteria. In bothsimilarity and identity, E. coli and V. alginolyticus, Synecho-coccus sp. and Anabaena sp., and PS-3 and B. megateriumare very closely related. When we examine the similarities ofeach of the individual subunits of V. alginolyticus with E.coli and B. megaterium, we observe some interesting trends.The a and P subunits are highly conserved, yet the i subunit
is poorly conserved, between the two pairs. Among theother subunits, a comparison with E. coli reveals a relativelylow percent identity between the c subunits; still, the percentsimilarity is quite high. Since the c subunit is largely hydro-phobic, this likely represents a mutation of hydrophobicresidues to other related residues. The relationship with B.megaterium reveals less similarity among all the subunits ascompared with E. coli; the 8 and b subunits have signifi-cantly less similarity than the others. This type of compari-son tells us that in the latter two subunits structural conser-vation is critical for only a small part of their structure.
ACKNOWLEDGMENTS
We thank Richard Leder for assistance with the a subunit align-ment.
This work was supported by Public Health Service awardsGM-13572 (to L.R.K.) and GM-18539 (to R.D.S.) from the NationalInstitutes of Health.
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