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Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic activities of the Ca2+ and Mg2+-activated adenosine triphosphatase of Escherichia coli
.4RCHIVES
OF
Effect
HIOCHEMISTKY
ANI)
of Removal
BIOPRYSIVS
169,
66&670
or Modification
Coupling
Factor
and
Mg*+-Activated
and
of Subunit
Hydrolytic
P. D. BRAGG, of Biochemistry,
Received
of the
Triphosphatase
on the Ca2+ of
coli
P. L. DilVIES,
University
Polypeptides
Activities
Adenosine Escherichia
Deparlme,lt
(1973)
of Brifish August
AND
Columbia,
c.
HOU
Vancouver
8, B. C., Ca?larla
23, 1973
The effect of removal or modificat.ion of t.he polypeptide subunits (a, p, y, 6, and 6) of the Ca*+ and Mg2+-activated ATPase of Escherichia coli was investigated. Removal of the d-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of t,he r-polypeptide, as found in the ATPase of an energy transfer coupling mutant, (k-15), resulted in diminut.ion of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.
Calcium or magnesium ion-act,ivaDed adenosine triphosphatase (ATPase) has been implicat’ed in the coupling of electron transport to the formation or use of ATP in mitochondria (I), chloroplasts (l), and bacteria (2, 3). The molecular weight of the membrane-bound, Ca2+ or LIg2+-activat’ed ATPase solubilized from different organisms is remarkably similar. Thus, the molecular weight of the enzyme from rat liver and beef heart mitochondria is 360,000 (4), from yeast mitochondria, 340,000 (5), from spinach chloroplasts, 325,000 (6), from Escherichia coli, 365,000-390,000 (7), from Bacillus ~negateriunz, 379,000~410,000 (8), and from Streptococcus &ecu&, 385,000 (9). It is likely that the enzyme from d/icrococcus lysodeikticus has a molecular weight in the same range since it has a sedimentation const’ant of 1415 s (10). With the exception of the enzymr from 5’. faecalis (9) which is mado up of t,no nonidentical polypeptidos of RIW 33,000, the other enzymes show a similar polypeptide composit’ion. The mit,ochondrial ATPase from beef heart contains polyCopyright ;\I1 rights
@ 1973 by Academic Press, of reproduction in my form
Inc. reserved.
pept,idcs of NW 53,000, 50,000, 25,000, 12,500, and 7,500 (4), while that from chloroplasts has subunits of MW 59,000, 56,000, 37,000, 17,500, and 13,000 (11). The enzyme from E. coli also contains five polypcpt,ldes (av MW: 56,800; 51,800; 30,500; 21,000; 11,500) (12). Fewer polypeptides have been found in the ATPases of X. lysodeikticus (13) and B. megaterium (8). This is probably due to insufficient, protein having been applied to the gels to detect the smallrlr polypeptides since it is t,he two subunits of highest molecular weight which stain most st’rongly with coomassie blue. However, the major polypeptides of the enzyme from R. nzeguterium had molerular weights of 68,000 and 6.5,OOO (8), while those of the M. lysodeikticus ATPase were 62,000, 60,000, and 35,000 (13). Recently Nt>lson et al. (11, 14) haw rclport.ed on the function of the polypcptides of the chloroplast enzymes, but much still remains unknown. The similarity in fun<:tion and polypeptide composit’ion of the ATPase from the various sources raises the
E’. coli
Ca*+
AND
Mg2’
problem of whether t’he polypeptides also have an analogous funct’ion in the different enzymes. The availability of a number of mutants of E. coli in which the ATPase activity has been altered (2,15,16) prompted us to study the functions of the polypeptide subunits of the ATPasc from this organism. The initial result’s arc given in this paper. For convenience of identification of the five subunit polypeptides of the E. coli ATPasc we follow t,hr proccdurc of Nelson et al. (11) and rcfrr t’o them as the (Y,p, y, 6, and Epolypcptides in order of decreasing molecular wights. METHODS Escherichia coli strains NRC-482, ML308-225 (wild type) and it’s electron t,ransfer coupling mutant etc-15 (17), were grown with vigorous aeration on a minimal salts-glucose (O.l(yfi) medium containing 12 ).LM ferric citrate. The cells were harvest.ed in the 1at.e exponent.ial phase and rtrnverted to washed respiratory particles as described previously (18). ATPase was stripped from these particles by dialysis against 0.5 mM EDTA at low ionic strength, and purified to homogeneit,y by a combination of chromatography on DEAE-cellulose and Sepharose 6B, followed by sucrose density gradient centrifugation. The procedure followed the method used before (12). The st.ripped part.icles were suspended in 50 mM Tris-H&O4 buf?‘er, pH 7.8, containing 10 rn>f MgClz , and lyc bovine serum albumin, at a concentration of lo-20 mg protein/ml. Protein, Caz+-activated ATPase, aerobicand ATP-driven energy-dependent transhydrogenase activities were measured exactly as described previously (18) except that the final concentration of sucrose in the transhydrogenase assays was 0.133 M. ATPase aet,ivity was determined in the presence of 5 mM CaCll The activities of the energy-dependent t,ranshydrogenases were corrected for that of the energy-independent transhydrogenase. Enzyme activities were measured at 37°C. For reconstitution of transhydrogenase activity the stripped particles (50 ~1) in a cuvette with 1 ml Tris-HzSO, buffer, pH 7.8, containing 10 rnM bovine serum albumin, 0.1 rn~ MgCIz , O.lt>i dit,hiothreitol, and 0.16 hi sucrose, were preincuhat.ed for 5 min at, 37°C with various amounts of ATPase. Following preincubation, the transhydrogenase assay was carried out directly on the cont,ents of the cuvette (18). The final volllme of the complete assay mixture was 1.2 ml. Analytical disc gel electrophnresis was performed either on 5”; polyacrylamidc gels prepared
ACTIVATED
ATP~sE:
665
and run in 0.05 M Tris-glycine buffer, pH 8.7, or on 7.5y0 polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS). For the latter system the protein sample was depolymerized at 37°C for 1 hr, and then ate 100°C for 3 min, in 4 M urea-l% SDS-l(r, mercaptoethannl, and run in system 1 as described previously (19). Protein bands were stained with Coomassie blue. When the ATPase was purified on polyacrylamide gels a slab gel vertical clectrophoresis apparatus (E-C Apparatus Corp.) was used. Partially purified ATPase of E. coli NRC-4X2 (1.2 mg protein) obtained following chromatography on Sepharose 6B (12) was applied t,o a 4 X 100 mm slot in a 59> polyacrylamide gel made in 0.05 M Tris-glycine buffer, pH 8.7, which had been prerun to remove charged compounds. The buffer cornpartment,s contained the same buffer. I.;lectrophoresis was carried out at 300 V for 90 min at which time the bromophenol blue marker dye had migrated CR. 13 cm through the gel. During electrophoresis the gel was cooled by circldating water at. 10°C. Guide strips were cut from the gel slab aft,er electrophoresis and were stained for ATPase ac%ivity using the assay reagents normally used to quantitate the enzyme. The area containing t,he ATPase was cut out from the remainder of the gel and extracted with 0.05 M Tris-HCl buffer, pH 7.5, cont’aining0.5 mM EDTA, 0.1 m&f dithiothreitol, and 107, glycerol. The enzyme solution was concentrated immediately to about 0.7 mg protein/ml by ultrafiltration through a PM 10 membrane (Amico Corp.). The recovery of ATPase activit)y from the gel was about 357). RESULTS
&feet
of gel electrophoresis
on ATPase
of
E. coli LVCR-482. In experiment’s designed to purify ATPase the almost homogcnt>ous enzyme was separated on polyacrylamide geIs at pH 8.7. Although t.hc enzyme could be rcext’racted from the gels thercb \vas a considerable loss of clnzymc units. Furthcrmore, t)here was some decline in the specifk activity of the enzyme (Tablr I) even though the enzyme appeared to migrate as a single protein band on the gel. When the extracted enzyme was disaggregatcd into its const,ituent polypeptides by heating ivith 1 y0 SDS in the presence of 4 XI uwa and 1% mercaptoethanol and examined by c+ctrophoresix in SDS-containing gels, the 6polypeptide of the ATPaw was found to be missing (Fig. 1). Sinw thc>scgels n-erc ovcrloaded with sampleto show the minor bands, the a- and ,kpolypcq)tidcs haw not rwolwd
666
BRAGG, TABLE
HOMOGI.:NXOUS NTIV.411:D
PREPARATIONS
Final
OF
cb~moles,‘min/mg
purification
Sucrose gradient Gel electrophoresis Sucrose gradient Sucrose gradient protein.
HOU
CA*+COLI
.___-NRC-482 NRC-482 MT,308-225 elc-15
AND
I
ATPASl':IRoLATEDFRoME.
Strain
IlAVIES,
step
Specific activity& ..38.9 26.9 31.3 18.6
172
3
132
3
from one another on these gels. In other gels 4 (not, shown) where less sample was applied t,o the gel the (Y- and P-polypeptides were found to bc identical in the two prepara5 5 tions. In the gel shown in Fig. 1 there is a trace of another polypeptidc band migrating more slowly than the y-subunit of the ATPase obt’ained from t’he gel. It is not clear if t’his represents an impurity or whether this is dclrived from the missing polypeptide. Although the increased molecular size of this band over the &polypeptide could be FIG. 1. Ele&rophoresis of purified preparations explained by the formation of disulfide of the ATPase of E. culi NRC-482 purified in the bridges between two molecules of the 6- final step either by sucrose densit,y gradient, rerltrifugation (left gel) or by gel electrophoresis at polypept,idc, it seems unlikely that these gel). The samples were depolymbridges I\-ould not have been cleaved by the pH 8.7 (right as described in Met,hods and elertrodisaggregation technique used to prepare t,he erized phoresis was carried out in the presence of ().I”;, sample for analysis on the SDS-containing SDS. Bands l-5 indicate polypeptides ol-e. gels. Removal of ATPase from membrane pardependent transhydrogcnasc: activities than t’icles of E. coli results in loss of both aerobict.hc parent strain, 1lL308-225, lvhil(a the and ATP-driven transhydrogenase activities. nonenergy dependent transhydrogenasc, as These activit’ics can be restored to the st,ripped particles by incubating them in the measured by the reduction of Z-acetylpyridine-NAD+ by NADPH, was much less presence of the purified ATPase and magaffected. Moreover the specific act’ivit) nesium ions or salts (12). Figure 2 shows the of the membrane-bound Ca2+-activatcld restoration of these activities to stripped ATPase of the mut’ant’ was about half that of particles by an clectrophoretically homothe parent (Table II). In mitochondria th(a geneous ATPase purified on a sucrose gradienergy-dependent transhydrogenase enzyme cnt,. In contrast to this ATPase, the ATPase extracted from the polyacrylamide gel had appears t,o bc the same clnzyme t’hat functions in the rloncncrgg-dcpcndent reaction no ability to r&ore either of t,hc encrgy(20). Thus, the lower activity of the clncrgydependent transhydrogenase activities even dependent reactions suggested that, th(l aftc,r preincubation with mercaptoethanol supply of ctnergy to the transhydrogenast: or dithiothreit’ol. enzyme was impaired. This would agree wit,h A TPase of electron transfer coupling mutant. Preliminary experiments with E. coli the results of Hong and T
E. co7i CaZ+
AND
Mg2+
45
0
ACTIVATED
90
0
ATPass
667
45
Factor-p
FIG. 2. Effect of purified ATPase (“factor”) on the aerobic-driven (A) and ATP-driven (B) transhydrogenase activities of stripped particles of E. coli NRC-482. The final step in the purification of the ATPase of E. coli NRC-482 was either sucrose density gradient centrifugation (solid points) or gel electrophoresis (open points). Activity is expressed as nmoles NADPH formedjmin. Stripped particles, 0.94 mg protein. TABLE SPECIFIC
.\NU ATP-DRIVEN ICNERGY-NONDEPENDENT* OF WASHED
ML308-225 Particles
BND AND
II OF
ACTIVITIES~
ATPAsE,
AEROBIC-
TRINSHYDROGENMX, TRANSHYDKOGEN.ME STRIPPED
PARTICLES
OF
.IND
E.
COLZ
ETC-15
ATPase
AerobicTH
Washed ML308-225 100 etc.15 4x Stripped ML308-225 etc.15 0 nmoles/min/mg protein. h Assayed as in Ref. 23 as pyridine-NAD+ by NADPH.
ATPTH
NondependentTH
74 24
38 14
424 345
35 14
0 0
922 646
reduction
of 3.aretyl-
when compared to the wild type. In order to see if the energy defect n-as related to the lower specific activity of t’he ATPase lve purified this enzyme to homogcncity. The homogeneous enzyme had a lower specific activity than that from JIL308-225 (Table I) although the two enzymes comigrated on polyacrylamide gels at pH 8.7 (Fig. 3; left gel). Examination of the subunit st’ructure of the two enzymes on polyacrylamide gels in the prescncc of SDS revealed a distinct difference in the rate of migration of the ypolypeptidr of the ATPasc (Fig. 3; middle and right g&). The incwawd rate of migra-
tion of this polypeptide, although small, has been verified numwous times and appears to be a real difference between the two cnzymcs. No difference in Dherate of migration of the a, 0, 6, and E-polypeptides bctwen the mutant and t,he parent could be detected. The increased rate of migration of t)hc ysubunit’ in the enzyme from the mutant would bc consistent \vith the dclction of a small part of thr polypoptide chain. The ability of the clcctrophorctically homogeneous ATPascs from JIL308-‘225 and etc.15 to restore aerobic- and ATEdependent transhydrogmation t’o stripped particles of both skains is shown in Fig. 4. On a weight’ basis the mutant, ATPaw appoarcd to bc Iws &cient’ in restoring cncrgy-de pcndcnt transhydrogenation than the (‘nzyme from the parent, strain. Howwr, the final activity achieved with both c~nzgmw was similar. These results suggest that the mutant ATPasc may bind lesscfhcic,ntly to the stripped particles such that largw amounts are required for saturation of the binding sites. It is also clear from Fig. 4 that an atlditional defect t’o that present in th(, =\TPase must occur in thr mut,ant strain. Evcm at saturating levels of the ATPasr th(b cntrrgydependent t,ranshydrogcnasc act,ivitics \\-cre lowr than in the parent’ strain. It is probably this defect, and not that, in the ATPaw, n-hich :twounts for tlw drastic Io\vwirlg
668
Blli\GG,
l)AVIES,
AND
HOIJ
4. Effect of purified ATPases (“factor”) of coli ML308-225 (solid points) or etc.15 (open points) on t,he aerobic-drive (A, C) and ATPdriven (B, 11) transhydrogennse activities of stripped particles from ML3OH-225 (C, I)) or efc-16 (A, B). Activit,y is expressed as nmoles NADPH formed!min. The amounts of stripped particles of ML308-225 and eic-16 were 0.73 and 0.89 mg protein, respectively. FIG.
E.
FIG. 3. Electrophoresis of purified preparations of the ATPases of E. roli ML308-225 and e(c-15. The gels were run using the split gel technique (24) with the enzyme from strain ML308-225 applied to t,he left half of the top of each gel and with that. of e/c-15 to the right half. The middle and right-hand gels were run in the presence of O.ll,‘;, SDS following depolymerization of the enzymes as described in Methods. Smaller amounts of the two enzymes were applied to the right-hand gel to obtain resolution of the w and 8-polypeptides. The durations of electrophoresis for the middle and right-hand gels were 2 and 3.5 h, respectively. Bands l-5 indicate polypeptides a--t. The enzymes on the left-hand gel were not depolynerized an d were rlul in Tris-glycine bufl’er, PIT x.7.
of c,nc~rg~-dependent t’ranshydrogenasc and transport activitic,s which initially prompted examination of th(> ATPasc of etc-15. JIuch remains t,o bc discovered about the function of the individual subunit polypept,ides of the coupling factor ATI’ascs of mitochondria, chloroplasts, and the bacterial cytoplasmic membrane. Th(b most extcnsiw studies have been carried out with thca ATPaw from chloroplasts by Nelson et al. (11, 14). These aorkers observed that a pwparation having LY,p, and y polypeptides
was still active as a coupling factor for photophosphorylat,ion, and retained its ATPase activity (14). The pwcisc function of the cy, fi, and y subunits \vas not determined. However, antibodies against both cr and y polypeptidcs \I-cre required to inhibit the ATI’asc activit,y of the compl& coupling factor. Antibodies against /3 or 6 polypept,idcs \vcrc ineffectiw. This suggestc>d that tho LYand y subunits might have the active site for ATP hydrolysis (I 1). The E polypcptide appcsawd to have a regulatory function since: it \vould inhibit the AkT!‘as(b activity of the complete coupling factor (I 4). It seems to be analogous in function t)o the ATPasc inhibitor of mammalian mitochow dria which is b&vcd to control the back flow of csncrgy from ATE’ to t’hc mitochondrial electron- and ion-transport systerns (21). No function for th(b 6 subunit XV:W d&crmined. The onI>- previous information regarding th(l role of the subunits of ATPase in bact,crial systems has bwn provided by Salton and Schor (13). Thcsc workers solubilized the ATPasc from the mombrancs of MiwoCOCCUSlysodeikticus by but,anol trwtmcwt.
IS. coli
C;t2+
AN11
Mg *+ ACTIVATED
It, differed from another form of ATPase which was released by a washing procedure in that it had only the a and p polypeptides, t,hat it would not rebind to ATPase-depletod membranw, and that, it \\-as not stimulated by trypsin. Thus, for ATPasc act,ivity t)hr 01 and 0 subunits had to be present, whercas one (or more) of the other subunits was required for binding of the enzyme to tho mc>mbrancl and for the masking of the ATPasc activity. It is possib10 that one of bhrse other polypeptides might correspond to ncctin. Scctin is a protein (MW 37,000) which is not found as a subunit of the ATPaw of &~eptococcus fczecalis, as isolated, but is rcquircd for the binding of the enzyme to t,hc cytoplasmic membrane (22). The ATPases of Jif. Zysodeilcticus (13) and of E. coli (12) prepared by washing procedures do not’ require a dissociable binding protein like m&in for attachment to the mcmbranc, but a protein with this funct,ion is presumably a subunit of thcw ATPascs as isolated. The results described in the prcscnt paper extend the information given above particularly with regard to the coupling factor activity of the ATPasc in bacterial systems. Modification of the y-polypcptide as occurs in the ATPase of etc.15 is associated with a somewhat lower ATPase act,ivit’y. This agrrcs with the results for chloroplast ATPasc where the y-subunit has some involwment in ATPase activity. The coupling fact,or act’ivity is part’ly affected in that more of the enzyme from the mutant than from t!he parent is required to obtain full stimulation of the energy-dependent transhydrogenase. This might be due to impairment of binding of t’he ATPasc to the membrane but wc have no clear evidcncc for this yet’. Removal, or possibly modification, of the &subunit of thr ATPase by alkaline gel rlwtrophoresis has a much greater effect on coupling factor activity than on ATPase activity. Thus, although this result agrees with those of Salton and Schor (13) and of N&on et al. (14) which indicate t,his subunit is not directly involved in ATP hydrolysis, it diffws from the findings of the latter workws n-ith th(l chloroplast ATPase in that it. appears t,o be essential for energy coupling in I<. coli. If this diffcrcncc is substant,iated,
ATP.\sfi:
(X9
it, will show that, regardless of th(l apparently similar polypc’ptide subunit atruct’ure of ATI’ases from various sources, the function of the individual polypeptidw in thrw cxnzymclsmay not be the same. Thtl wason \vhy gel clcctrophorcsis appears to remove t’hc d-subunit from the ATPaxc of B. coli has not been complctcly de&mined. If the prowss simply involved dissociation of the subunit at the alkalino pH of t’hc gel then t’he 1iberatc.d subunit should be detectable on alkaline gels heavily loaded with the enzyme. However, MYhave not been able to detect t>he released subunit under these conditions. The reason for this is unclear, but this result may indicat,cl that the &subunit’ has undergone some modification on the gels. It is possible that the other subunits of the ATPase may have been modified also, but we have no evidence for this. Att’empts to duplicate the results of gel elcctrophoresis by taking the enzyme to pH 9 during the purificat)ion step on the sucrose gradient have at present been only partiall? successful in causing removal of the 6subunit. ACKNOWLEDGMENTS We are pleased to acknowledge the generosity of Dr. H. R. Kaback in providing us with cultures of E. coli ML308-225 and etc-15. This work was supported by a Grant from the Medical Research Council of Canada and by the award of a Medical Research Council Studentship to P. L. Davies. REFERENCES 1. RSCKER, E. (1970) in Membranes of mitochondria and chloroplasts (Racker, E., ed.), p. 127, Van Nostrand Reinhold Co., New York. 2. BUTLIN, J. II., Cox, G. B., AND GIBSON, F. (1971) Biochem. J. 124, 75. 3. Is~r~~4w.4, s. (1970) J. Biochem. 67, 297. 4. SENIOR, A. E. AND BROOKS, J. C. (1971) FEBS Lett. 1’7, 327. 5. TZAGOLOFF, A., AND ME.\GHER, P. (1971) .I. Bid. Chem. 246, 7328. 6. FARRON, F. (1970) Biochemistry 9, 3823. 7. DIIVIES, P. L., AND BRAGG, P. L). (1972) Biochim. Biophys. Acta 266, 273. 8. MIRSKY, R., AND BI\RLOW, V. (1973) Biochim. Biophys. Acta 291,480. 0. SCIINEDLI, H. P., VATTER, A. E., .4x1 ABI<.\MS, A. (1970) J. Biol. Chem. 246, 1122. 10. MUNOZ, E., SALTOK, 11. Ii. J., NG, 11. II.,
.INII SCHOR, M. T. (1969) Eur. J. Biochevr. 7, 490. 11. ?rTr:rsoN, N., I)I:TISRS, I). W., NICLSON, H., ANI) li.w~u:~c, I<., (lQi3) J. Rior. Chenr. 248, 2049. 12. BIUGG, P. I). .ZNI) Hnu, C. (1972). Ph’US I,el!. 28, 309. 13. S.\LTON, M. It. J., .\su SCHOR, M. T. (1972)
Bioc,hcva.
Biophys.
Hes.
N., NELSON, (1972). J. Hid. Ch,em. 15. ~~~~~~~~~~~ B. I., .\ND FEBS Lett. 22, lQ7. 1-i.
16.
1;ELSW,
SIMONI,
It.
Il.,
AND
Conrmm.
Il.,
AND
247,
7657.
C~UTNICK,
49,
350.
I<;\(-~lsll,
li:.
(1972) 1248.
8~H.~I~L1~~N~I~:l~Gl~:l~,
I,.
(1972) hf.
K.
(lQ72) Proc. .Vaf. Acrid. Sci. USA 69, 2663. 17. HONG, J. S., AND KLILXK, H. R. (1972) Proc. .\:a/. Acad. Sci. I’SA 69, 3336. 18. B~i:\c:c;, P. I)., D.\vrw. P. L., :\SD HOE, C:.
Riophys.
Kes.
Con~trau~~.
47,
19. BR.\GG,
P. 1). .\SD Hou, (‘. (1972) Hiochiw, Acla 274, 17X. 20. K.\\v.tsarcr, T., S.\TOH, K., AND K.\Fc.\N, N. 0. (1964) Riochew. Hiophys. /?e,s. (‘ortut/ IO,. 17, 64X. 21. V.IX III,; ST.WT, I<. J., 111,: Bor.:~l, B. L., .\sI) V.\N I).\M, K. (1973) Hiochim. Hiophys. Hiophy,s.
Ada
22. B.\Kos, 1)
Hiochrtu
292,
33X.
.\NU AHR.\MS, A. (1971) J. Niol. 154‘. 23. K~PMN, N. 0. (1967) ir, Methods in enzymology (I’:stabrwok, R. W., and Pullman, M. E., eds.), Vol. 10, 1,. 317, Academic Press, New York. (‘hew.
C.,
246,
OF
Effect
HIOCHEMISTKY
ANI)
of Removal
BIOPRYSIVS
169,
66&670
or Modification
Coupling
Factor
and
Mg*+-Activated
and
of Subunit
Hydrolytic
P. D. BRAGG, of Biochemistry,
Received
of the
Triphosphatase
on the Ca2+ of
coli
P. L. DilVIES,
University
Polypeptides
Activities
Adenosine Escherichia
Deparlme,lt
(1973)
of Brifish August
AND
Columbia,
c.
HOU
Vancouver
8, B. C., Ca?larla
23, 1973
The effect of removal or modificat.ion of t.he polypeptide subunits (a, p, y, 6, and 6) of the Ca*+ and Mg2+-activated ATPase of Escherichia coli was investigated. Removal of the d-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of t,he r-polypeptide, as found in the ATPase of an energy transfer coupling mutant, (k-15), resulted in diminut.ion of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.
Calcium or magnesium ion-act,ivaDed adenosine triphosphatase (ATPase) has been implicat’ed in the coupling of electron transport to the formation or use of ATP in mitochondria (I), chloroplasts (l), and bacteria (2, 3). The molecular weight of the membrane-bound, Ca2+ or LIg2+-activat’ed ATPase solubilized from different organisms is remarkably similar. Thus, the molecular weight of the enzyme from rat liver and beef heart mitochondria is 360,000 (4), from yeast mitochondria, 340,000 (5), from spinach chloroplasts, 325,000 (6), from Escherichia coli, 365,000-390,000 (7), from Bacillus ~negateriunz, 379,000~410,000 (8), and from Streptococcus &ecu&, 385,000 (9). It is likely that the enzyme from d/icrococcus lysodeikticus has a molecular weight in the same range since it has a sedimentation const’ant of 1415 s (10). With the exception of the enzymr from 5’. faecalis (9) which is mado up of t,no nonidentical polypeptidos of RIW 33,000, the other enzymes show a similar polypeptide composit’ion. The mit,ochondrial ATPase from beef heart contains polyCopyright ;\I1 rights
@ 1973 by Academic Press, of reproduction in my form
Inc. reserved.
pept,idcs of NW 53,000, 50,000, 25,000, 12,500, and 7,500 (4), while that from chloroplasts has subunits of MW 59,000, 56,000, 37,000, 17,500, and 13,000 (11). The enzyme from E. coli also contains five polypcpt,ldes (av MW: 56,800; 51,800; 30,500; 21,000; 11,500) (12). Fewer polypeptides have been found in the ATPases of X. lysodeikticus (13) and B. megaterium (8). This is probably due to insufficient, protein having been applied to the gels to detect the smallrlr polypeptides since it is t,he two subunits of highest molecular weight which stain most st’rongly with coomassie blue. However, the major polypeptides of the enzyme from R. nzeguterium had molerular weights of 68,000 and 6.5,OOO (8), while those of the M. lysodeikticus ATPase were 62,000, 60,000, and 35,000 (13). Recently Nt>lson et al. (11, 14) haw rclport.ed on the function of the polypcptides of the chloroplast enzymes, but much still remains unknown. The similarity in fun<:tion and polypeptide composit’ion of the ATPase from the various sources raises the
E’. coli
Ca*+
AND
Mg2’
problem of whether t’he polypeptides also have an analogous funct’ion in the different enzymes. The availability of a number of mutants of E. coli in which the ATPase activity has been altered (2,15,16) prompted us to study the functions of the polypeptide subunits of the ATPasc from this organism. The initial result’s arc given in this paper. For convenience of identification of the five subunit polypeptides of the E. coli ATPasc we follow t,hr proccdurc of Nelson et al. (11) and rcfrr t’o them as the (Y,p, y, 6, and Epolypcptides in order of decreasing molecular wights. METHODS Escherichia coli strains NRC-482, ML308-225 (wild type) and it’s electron t,ransfer coupling mutant etc-15 (17), were grown with vigorous aeration on a minimal salts-glucose (O.l(yfi) medium containing 12 ).LM ferric citrate. The cells were harvest.ed in the 1at.e exponent.ial phase and rtrnverted to washed respiratory particles as described previously (18). ATPase was stripped from these particles by dialysis against 0.5 mM EDTA at low ionic strength, and purified to homogeneit,y by a combination of chromatography on DEAE-cellulose and Sepharose 6B, followed by sucrose density gradient centrifugation. The procedure followed the method used before (12). The st.ripped part.icles were suspended in 50 mM Tris-H&O4 buf?‘er, pH 7.8, containing 10 rn>f MgClz , and lyc bovine serum albumin, at a concentration of lo-20 mg protein/ml. Protein, Caz+-activated ATPase, aerobicand ATP-driven energy-dependent transhydrogenase activities were measured exactly as described previously (18) except that the final concentration of sucrose in the transhydrogenase assays was 0.133 M. ATPase aet,ivity was determined in the presence of 5 mM CaCll The activities of the energy-dependent t,ranshydrogenases were corrected for that of the energy-independent transhydrogenase. Enzyme activities were measured at 37°C. For reconstitution of transhydrogenase activity the stripped particles (50 ~1) in a cuvette with 1 ml Tris-HzSO, buffer, pH 7.8, containing 10 rnM bovine serum albumin, 0.1 rn~ MgCIz , O.lt>i dit,hiothreitol, and 0.16 hi sucrose, were preincuhat.ed for 5 min at, 37°C with various amounts of ATPase. Following preincubation, the transhydrogenase assay was carried out directly on the cont,ents of the cuvette (18). The final volllme of the complete assay mixture was 1.2 ml. Analytical disc gel electrophnresis was performed either on 5”; polyacrylamidc gels prepared
ACTIVATED
ATP~sE:
665
and run in 0.05 M Tris-glycine buffer, pH 8.7, or on 7.5y0 polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS). For the latter system the protein sample was depolymerized at 37°C for 1 hr, and then ate 100°C for 3 min, in 4 M urea-l% SDS-l(r, mercaptoethannl, and run in system 1 as described previously (19). Protein bands were stained with Coomassie blue. When the ATPase was purified on polyacrylamide gels a slab gel vertical clectrophoresis apparatus (E-C Apparatus Corp.) was used. Partially purified ATPase of E. coli NRC-4X2 (1.2 mg protein) obtained following chromatography on Sepharose 6B (12) was applied t,o a 4 X 100 mm slot in a 59> polyacrylamide gel made in 0.05 M Tris-glycine buffer, pH 8.7, which had been prerun to remove charged compounds. The buffer cornpartment,s contained the same buffer. I.;lectrophoresis was carried out at 300 V for 90 min at which time the bromophenol blue marker dye had migrated CR. 13 cm through the gel. During electrophoresis the gel was cooled by circldating water at. 10°C. Guide strips were cut from the gel slab aft,er electrophoresis and were stained for ATPase ac%ivity using the assay reagents normally used to quantitate the enzyme. The area containing t,he ATPase was cut out from the remainder of the gel and extracted with 0.05 M Tris-HCl buffer, pH 7.5, cont’aining0.5 mM EDTA, 0.1 m&f dithiothreitol, and 107, glycerol. The enzyme solution was concentrated immediately to about 0.7 mg protein/ml by ultrafiltration through a PM 10 membrane (Amico Corp.). The recovery of ATPase activit)y from the gel was about 357). RESULTS
&feet
of gel electrophoresis
on ATPase
of
E. coli LVCR-482. In experiment’s designed to purify ATPase the almost homogcnt>ous enzyme was separated on polyacrylamide geIs at pH 8.7. Although t.hc enzyme could be rcext’racted from the gels thercb \vas a considerable loss of clnzymc units. Furthcrmore, t)here was some decline in the specifk activity of the enzyme (Tablr I) even though the enzyme appeared to migrate as a single protein band on the gel. When the extracted enzyme was disaggregatcd into its const,ituent polypeptides by heating ivith 1 y0 SDS in the presence of 4 XI uwa and 1% mercaptoethanol and examined by c+ctrophoresix in SDS-containing gels, the 6polypeptide of the ATPaw was found to be missing (Fig. 1). Sinw thc>scgels n-erc ovcrloaded with sampleto show the minor bands, the a- and ,kpolypcq)tidcs haw not rwolwd
666
BRAGG, TABLE
HOMOGI.:NXOUS NTIV.411:D
PREPARATIONS
Final
OF
cb~moles,‘min/mg
purification
Sucrose gradient Gel electrophoresis Sucrose gradient Sucrose gradient protein.
HOU
CA*+COLI
.___-NRC-482 NRC-482 MT,308-225 elc-15
AND
I
ATPASl':IRoLATEDFRoME.
Strain
IlAVIES,
step
Specific activity& ..38.9 26.9 31.3 18.6
172
3
132
3
from one another on these gels. In other gels 4 (not, shown) where less sample was applied t,o the gel the (Y- and P-polypeptides were found to bc identical in the two prepara5 5 tions. In the gel shown in Fig. 1 there is a trace of another polypeptidc band migrating more slowly than the y-subunit of the ATPase obt’ained from t’he gel. It is not clear if t’his represents an impurity or whether this is dclrived from the missing polypeptide. Although the increased molecular size of this band over the &polypeptide could be FIG. 1. Ele&rophoresis of purified preparations explained by the formation of disulfide of the ATPase of E. culi NRC-482 purified in the bridges between two molecules of the 6- final step either by sucrose densit,y gradient, rerltrifugation (left gel) or by gel electrophoresis at polypept,idc, it seems unlikely that these gel). The samples were depolymbridges I\-ould not have been cleaved by the pH 8.7 (right as described in Met,hods and elertrodisaggregation technique used to prepare t,he erized phoresis was carried out in the presence of ().I”;, sample for analysis on the SDS-containing SDS. Bands l-5 indicate polypeptides ol-e. gels. Removal of ATPase from membrane pardependent transhydrogcnasc: activities than t’icles of E. coli results in loss of both aerobict.hc parent strain, 1lL308-225, lvhil(a the and ATP-driven transhydrogenase activities. nonenergy dependent transhydrogenasc, as These activit’ics can be restored to the st,ripped particles by incubating them in the measured by the reduction of Z-acetylpyridine-NAD+ by NADPH, was much less presence of the purified ATPase and magaffected. Moreover the specific act’ivit) nesium ions or salts (12). Figure 2 shows the of the membrane-bound Ca2+-activatcld restoration of these activities to stripped ATPase of the mut’ant’ was about half that of particles by an clectrophoretically homothe parent (Table II). In mitochondria th(a geneous ATPase purified on a sucrose gradienergy-dependent transhydrogenase enzyme cnt,. In contrast to this ATPase, the ATPase extracted from the polyacrylamide gel had appears t,o bc the same clnzyme t’hat functions in the rloncncrgg-dcpcndent reaction no ability to r&ore either of t,hc encrgy(20). Thus, the lower activity of the clncrgydependent transhydrogenase activities even dependent reactions suggested that, th(l aftc,r preincubation with mercaptoethanol supply of ctnergy to the transhydrogenast: or dithiothreit’ol. enzyme was impaired. This would agree wit,h A TPase of electron transfer coupling mutant. Preliminary experiments with E. coli the results of Hong and T
E. co7i CaZ+
AND
Mg2+
45
0
ACTIVATED
90
0
ATPass
667
45
Factor-p
FIG. 2. Effect of purified ATPase (“factor”) on the aerobic-driven (A) and ATP-driven (B) transhydrogenase activities of stripped particles of E. coli NRC-482. The final step in the purification of the ATPase of E. coli NRC-482 was either sucrose density gradient centrifugation (solid points) or gel electrophoresis (open points). Activity is expressed as nmoles NADPH formedjmin. Stripped particles, 0.94 mg protein. TABLE SPECIFIC
.\NU ATP-DRIVEN ICNERGY-NONDEPENDENT* OF WASHED
ML308-225 Particles
BND AND
II OF
ACTIVITIES~
ATPAsE,
AEROBIC-
TRINSHYDROGENMX, TRANSHYDKOGEN.ME STRIPPED
PARTICLES
OF
.IND
E.
COLZ
ETC-15
ATPase
AerobicTH
Washed ML308-225 100 etc.15 4x Stripped ML308-225 etc.15 0 nmoles/min/mg protein. h Assayed as in Ref. 23 as pyridine-NAD+ by NADPH.
ATPTH
NondependentTH
74 24
38 14
424 345
35 14
0 0
922 646
reduction
of 3.aretyl-
when compared to the wild type. In order to see if the energy defect n-as related to the lower specific activity of t’he ATPase lve purified this enzyme to homogcncity. The homogeneous enzyme had a lower specific activity than that from JIL308-225 (Table I) although the two enzymes comigrated on polyacrylamide gels at pH 8.7 (Fig. 3; left gel). Examination of the subunit st’ructure of the two enzymes on polyacrylamide gels in the prescncc of SDS revealed a distinct difference in the rate of migration of the ypolypeptidr of the ATPasc (Fig. 3; middle and right g&). The incwawd rate of migra-
tion of this polypeptide, although small, has been verified numwous times and appears to be a real difference between the two cnzymcs. No difference in Dherate of migration of the a, 0, 6, and E-polypeptides bctwen the mutant and t,he parent could be detected. The increased rate of migration of t)hc ysubunit’ in the enzyme from the mutant would bc consistent \vith the dclction of a small part of thr polypoptide chain. The ability of the clcctrophorctically homogeneous ATPascs from JIL308-‘225 and etc.15 to restore aerobic- and ATEdependent transhydrogmation t’o stripped particles of both skains is shown in Fig. 4. On a weight’ basis the mutant, ATPaw appoarcd to bc Iws &cient’ in restoring cncrgy-de pcndcnt transhydrogenation than the (‘nzyme from the parent, strain. Howwr, the final activity achieved with both c~nzgmw was similar. These results suggest that the mutant ATPasc may bind lesscfhcic,ntly to the stripped particles such that largw amounts are required for saturation of the binding sites. It is also clear from Fig. 4 that an atlditional defect t’o that present in th(, =\TPase must occur in thr mut,ant strain. Evcm at saturating levels of the ATPasr th(b cntrrgydependent t,ranshydrogcnasc act,ivitics \\-cre lowr than in the parent’ strain. It is probably this defect, and not that, in the ATPaw, n-hich :twounts for tlw drastic Io\vwirlg
668
Blli\GG,
l)AVIES,
AND
HOIJ
4. Effect of purified ATPases (“factor”) of coli ML308-225 (solid points) or etc.15 (open points) on t,he aerobic-drive (A, C) and ATPdriven (B, 11) transhydrogennse activities of stripped particles from ML3OH-225 (C, I)) or efc-16 (A, B). Activit,y is expressed as nmoles NADPH formed!min. The amounts of stripped particles of ML308-225 and eic-16 were 0.73 and 0.89 mg protein, respectively. FIG.
E.
FIG. 3. Electrophoresis of purified preparations of the ATPases of E. roli ML308-225 and e(c-15. The gels were run using the split gel technique (24) with the enzyme from strain ML308-225 applied to t,he left half of the top of each gel and with that. of e/c-15 to the right half. The middle and right-hand gels were run in the presence of O.ll,‘;, SDS following depolymerization of the enzymes as described in Methods. Smaller amounts of the two enzymes were applied to the right-hand gel to obtain resolution of the w and 8-polypeptides. The durations of electrophoresis for the middle and right-hand gels were 2 and 3.5 h, respectively. Bands l-5 indicate polypeptides a--t. The enzymes on the left-hand gel were not depolynerized an d were rlul in Tris-glycine bufl’er, PIT x.7.
of c,nc~rg~-dependent t’ranshydrogenasc and transport activitic,s which initially prompted examination of th(> ATPasc of etc-15. JIuch remains t,o bc discovered about the function of the individual subunit polypept,ides of the coupling factor ATI’ascs of mitochondria, chloroplasts, and the bacterial cytoplasmic membrane. Th(b most extcnsiw studies have been carried out with thca ATPaw from chloroplasts by Nelson et al. (11, 14). These aorkers observed that a pwparation having LY,p, and y polypeptides
was still active as a coupling factor for photophosphorylat,ion, and retained its ATPase activity (14). The pwcisc function of the cy, fi, and y subunits \vas not determined. However, antibodies against both cr and y polypeptidcs \I-cre required to inhibit the ATI’asc activit,y of the compl& coupling factor. Antibodies against /3 or 6 polypept,idcs \vcrc ineffectiw. This suggestc>d that tho LYand y subunits might have the active site for ATP hydrolysis (I 1). The E polypcptide appcsawd to have a regulatory function since: it \vould inhibit the AkT!‘as(b activity of the complete coupling factor (I 4). It seems to be analogous in function t)o the ATPasc inhibitor of mammalian mitochow dria which is b&vcd to control the back flow of csncrgy from ATE’ to t’hc mitochondrial electron- and ion-transport systerns (21). No function for th(b 6 subunit XV:W d&crmined. The onI>- previous information regarding th(l role of the subunits of ATPase in bact,crial systems has bwn provided by Salton and Schor (13). Thcsc workers solubilized the ATPasc from the mombrancs of MiwoCOCCUSlysodeikticus by but,anol trwtmcwt.
IS. coli
C;t2+
AN11
Mg *+ ACTIVATED
It, differed from another form of ATPase which was released by a washing procedure in that it had only the a and p polypeptides, t,hat it would not rebind to ATPase-depletod membranw, and that, it \\-as not stimulated by trypsin. Thus, for ATPasc act,ivity t)hr 01 and 0 subunits had to be present, whercas one (or more) of the other subunits was required for binding of the enzyme to tho mc>mbrancl and for the masking of the ATPasc activity. It is possib10 that one of bhrse other polypeptides might correspond to ncctin. Scctin is a protein (MW 37,000) which is not found as a subunit of the ATPaw of &~eptococcus fczecalis, as isolated, but is rcquircd for the binding of the enzyme to t,hc cytoplasmic membrane (22). The ATPases of Jif. Zysodeilcticus (13) and of E. coli (12) prepared by washing procedures do not’ require a dissociable binding protein like m&in for attachment to the mcmbranc, but a protein with this funct,ion is presumably a subunit of thcw ATPascs as isolated. The results described in the prcscnt paper extend the information given above particularly with regard to the coupling factor activity of the ATPasc in bacterial systems. Modification of the y-polypcptide as occurs in the ATPase of etc.15 is associated with a somewhat lower ATPase act,ivit’y. This agrrcs with the results for chloroplast ATPasc where the y-subunit has some involwment in ATPase activity. The coupling fact,or act’ivity is part’ly affected in that more of the enzyme from the mutant than from t!he parent is required to obtain full stimulation of the energy-dependent transhydrogenase. This might be due to impairment of binding of t’he ATPasc to the membrane but wc have no clear evidcncc for this yet’. Removal, or possibly modification, of the &subunit of thr ATPase by alkaline gel rlwtrophoresis has a much greater effect on coupling factor activity than on ATPase activity. Thus, although this result agrees with those of Salton and Schor (13) and of N&on et al. (14) which indicate t,his subunit is not directly involved in ATP hydrolysis, it diffws from the findings of the latter workws n-ith th(l chloroplast ATPase in that it. appears t,o be essential for energy coupling in I<. coli. If this diffcrcncc is substant,iated,
ATP.\sfi:
(X9
it, will show that, regardless of th(l apparently similar polypc’ptide subunit atruct’ure of ATI’ases from various sources, the function of the individual polypeptidw in thrw cxnzymclsmay not be the same. Thtl wason \vhy gel clcctrophorcsis appears to remove t’hc d-subunit from the ATPaxc of B. coli has not been complctcly de&mined. If the prowss simply involved dissociation of the subunit at the alkalino pH of t’hc gel then t’he 1iberatc.d subunit should be detectable on alkaline gels heavily loaded with the enzyme. However, MYhave not been able to detect t>he released subunit under these conditions. The reason for this is unclear, but this result may indicat,cl that the &subunit’ has undergone some modification on the gels. It is possible that the other subunits of the ATPase may have been modified also, but we have no evidence for this. Att’empts to duplicate the results of gel elcctrophoresis by taking the enzyme to pH 9 during the purificat)ion step on the sucrose gradient have at present been only partiall? successful in causing removal of the 6subunit. ACKNOWLEDGMENTS We are pleased to acknowledge the generosity of Dr. H. R. Kaback in providing us with cultures of E. coli ML308-225 and etc-15. This work was supported by a Grant from the Medical Research Council of Canada and by the award of a Medical Research Council Studentship to P. L. Davies. REFERENCES 1. RSCKER, E. (1970) in Membranes of mitochondria and chloroplasts (Racker, E., ed.), p. 127, Van Nostrand Reinhold Co., New York. 2. BUTLIN, J. II., Cox, G. B., AND GIBSON, F. (1971) Biochem. J. 124, 75. 3. Is~r~~4w.4, s. (1970) J. Biochem. 67, 297. 4. SENIOR, A. E. AND BROOKS, J. C. (1971) FEBS Lett. 1’7, 327. 5. TZAGOLOFF, A., AND ME.\GHER, P. (1971) .I. Bid. Chem. 246, 7328. 6. FARRON, F. (1970) Biochemistry 9, 3823. 7. DIIVIES, P. L., AND BRAGG, P. L). (1972) Biochim. Biophys. Acta 266, 273. 8. MIRSKY, R., AND BI\RLOW, V. (1973) Biochim. Biophys. Acta 291,480. 0. SCIINEDLI, H. P., VATTER, A. E., .4x1 ABI<.\MS, A. (1970) J. Biol. Chem. 246, 1122. 10. MUNOZ, E., SALTOK, 11. Ii. J., NG, 11. II.,
.INII SCHOR, M. T. (1969) Eur. J. Biochevr. 7, 490. 11. ?rTr:rsoN, N., I)I:TISRS, I). W., NICLSON, H., ANI) li.w~u:~c, I<., (lQi3) J. Rior. Chenr. 248, 2049. 12. BIUGG, P. I). .ZNI) Hnu, C. (1972). Ph’US I,el!. 28, 309. 13. S.\LTON, M. It. J., .\su SCHOR, M. T. (1972)
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Biophys.
Hes.
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li:.
(1972) 1248.
8~H.~I~L1~~N~I~:l~Gl~:l~,
I,.
(1972) hf.
K.
(lQ72) Proc. .Vaf. Acrid. Sci. USA 69, 2663. 17. HONG, J. S., AND KLILXK, H. R. (1972) Proc. .\:a/. Acad. Sci. I’SA 69, 3336. 18. B~i:\c:c;, P. I)., D.\vrw. P. L., :\SD HOE, C:.
Riophys.
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47,
19. BR.\GG,
P. 1). .\SD Hou, (‘. (1972) Hiochiw, Acla 274, 17X. 20. K.\\v.tsarcr, T., S.\TOH, K., AND K.\Fc.\N, N. 0. (1964) Riochew. Hiophys. /?e,s. (‘ortut/ IO,. 17, 64X. 21. V.IX III,; ST.WT, I<. J., 111,: Bor.:~l, B. L., .\sI) V.\N I).\M, K. (1973) Hiochim. Hiophys. Hiophy,s.
Ada
22. B.\Kos, 1)
Hiochrtu
292,
33X.
.\NU AHR.\MS, A. (1971) J. Niol. 154‘. 23. K~PMN, N. 0. (1967) ir, Methods in enzymology (I’:stabrwok, R. W., and Pullman, M. E., eds.), Vol. 10, 1,. 317, Academic Press, New York. (‘hew.
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