and ITP all supported assembly, with half-maximal reconstitution of ATPase ... U.S.C. Section 1734 solely to indicate this fact. F,-ATPase might seem to be ...
Vol. 263, No. 12, Issue of April 25, pp. 5569-5573. 1988
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and MolecularBiology, Inc.
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I / S. A.
Trinitrophenyl-ATP and-ADP Bind to a Single Nucleotide Site on Isolated @-Subunit of Escherichia coliF1-ATPase IN VITRO ASSEMBLYOFF1-SUBUNITSREQUIRES OCCUPANCY OFTHENUCLEOTIDE-BINDINGSITE ON p-SUBUNIT BY NUCLEOSIDE TRIPHOSPHATE* (Received for publication, October 19, 1987)
Rajini Rao, Marwan K. Al-Shawi, and Alan E. Senior From the Department of Biochemistry, University of Rochester Medical Center, Rochester, N e w York 14642
The stoichiometry of nucleotide binding to the isolated a- and &subunits of Escherichia coli F,-ATPase was investigated using two experimental techniques: ( a )titration withfluorescent trinitrophenyl (TNP)derivatives of AMP, ADP, and ATP and ( b ) the centrifuge column procedure using the particularconditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, 2. (1985)FEBS Lett. 178, 10-14).Both procedures showed that a-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol &subunit, consistent with previous equilibrium dialysis studies which showed isolated 0subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984)Biochemistry 23, 6591-6595). However, binding of only -0.1 molof ATP or ADP per mol of &subunit was detected using centrifuge columns. Our resultsare consistent with the conclusion that each of the a- and &subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is a3f13y6c, this can account for the location of the six knownnucleotide-binding sites inE. coli F1-ATPase. Studies of in vitroassembly of isolated a-,8-, and ysubunits intoan activeATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 PM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supportedassembly and TNP-ADP did not. The results demonstrate that (a)the nucleotidebinding site on &subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the a-subunit nucleotide-binding site is not required, and ( b ) that enzyme assembly requires nucleoside triphosphate.
F,-ATPase might seem to be straightforward. The enzyme subunitstructureis (Bragg andHou, 1975; Fosterand Fillingame, 1982). There is evidence from direct binding studies that isolated a-subunit contains one nucleotide-binding site (Dunn and Futai, 1980; Senda et al., 1983; Perlin et al., 1984) and that isolated P-subunit contains one nucleotidebinding site (Issartel and Vignais, 1984). This suggests that in F1 three of the six sites are on a-subunits and three are on P-subunits. However, there arealso reports thatisolated P-subunit from Rhodospirillum rubrum F,-ATPase can be shown under par2 mol of ADP or ATP ticular experimental conditions to bind per mol (Gromet-ElhananandKhananshvili, 1984; Khananshvili and Gromet-Elhanan,1984, 1985). R. rubrum and E. coli p-subunitsare very similar in amino acid sequence (Walker et al., 1985). They have identicalamino acids in about 73% of their sequence, they are of almost equal length, and homology extends throughout the two sequences. They are likely to be very similar structurally, andso the apparent discrepancy in nucleotide-binding capacityis surprising. Wefelt,therefore,thatit was important to investigate further the nucleotide-binding properties of E. coli isolated F1-P-subunit. In this paper we report studies of nucleotidebinding to isolated E. coli a- and P-subunitsusing the particular experimental conditions specified by Khananshvili and Gromet-Elhanan (1984, 1985), and we also describe binding studies utilizing the fluorescent trinitrophenyl derivatives of AMP, ADP, and ATP. In 1980, Dunn and Futai showed that in vitro repolymerization of isolated E. coli F1-subunits to form a catalytically competent a3P3y oligomer was nucleotide-dependent. From the data they were not ableat that time deduce to the location of the nucleotide sites involved. We have pursued this question further in this paper to demonstrate which nucleotide sites are involved and to define the nucleotide specificity for enzyme assembly.
Escherichia coli F,-ATPase contains six nucleotide-binding MATERIALSANDMETHODS sites (Wise et al., 1983). Three sites exchange with medium Purification of Soluble E. coli F,-ATPase-Purification was done nucleotides slowly, are specific for adenine nucleotides, and as described by Wise et al. (1981) and modified by Duncan and Senior are therefore noncatalytic sites; whereas the other three sites exchange rapidly with medium nucleotides, are able to bind (1985). Strain AN1460 (unc’, Downie et al., 1980) or strain SWMl was used as a source of enzyme. Strain SWMl has the genotype adenine,guanine,andinosine nucleotides,show apparent pAN45 (unc+)/unc+,argH, pyrE, entA, rec+ and was constructed by negative cooperativity of binding, and are therefore potential transforming strain AN1339 (Perlin et al., 1983)to CmR withplasmid catalyticsites(Wise et al., 1983; Perlin et al., 1984). The pAN45 prepared from strain AN1460. Strain SWMl was found to subunit location of the six nucleotide-binding sites inE. coli yield 1.2 times more purified F, per liter of culture than strain
* This work wassupported by National Institutes of Health Grants GM25349 and GM29805. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
AN1460. Purification of Zsolated a-, p-, and y-Subunits from F,-This was done as described by Dunn and Futai (1980). All preparations of isolated subunits were shown to be fully competent in repolymerizaoligomer and in reconstitution of ATPase activity. tion of Repolymerization of Zsolated a-, p-, and ?-Subunits to an Active
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F1-ATPmeAssembly Nucleotide-binding and Sites
ATPase-a-, @-, and y-subunits were mixed in polypropylene tubes in a molar ratio of 3:3:1 and ata final concentration of 0.15 mg/ml. The buffer was 50 mM succinate Tris, pH 6.0, 10% (v/v) glycerol, 0.1 mM dithiothreitol, 0.25 mM EDTA, and 2 mM MgC12. Nucleotide concentrations were adjusted as required, and then the tubes were incubated overnight a t 23 "C, before assaying for ATPase activity colorimetrically (Taussky and Shorr, 1953; Wise et al., 1981). Measurement of Binding of ADP, ATP, and AMP-PNP'to Isolated a- and @-Subunits by the Method of Khananshvili and Gromet-Elhanun-This method was the centrifuge column technique and was carried out here exactly as described by Khananshvili and GrometElhanan (1984, 1985). Measurement of Endogenous Nucleotide Content of Isolated a- and @-Subunits-The luciferin-luciferase assay was used as described by Kironde and Cross (1986). Measurement of Binding of TNP Nucleotides to Isolated a- and @Subunits-TNP-ATP, TNP-ADP,andTNP-AMP were obtained from Molecular Probes, Inc., and the purity was checked on TLC using polyethyleneimine plates eluted with 2 M formic acid plus 0.5 M LiCl. TNP-AMP was pure and was used without further treatment, TNP-ADP and TNP-ATP were further purified as described by Grubmeyer and Penefsky (1981). T N P nucleotide concentrations were determined in 50 mM Tris-S04, pH8.0, using the molar absorption coefficients published by Hiratsuka (1982). Fluorescence measurements were made at 23 "C inaHitachi-Perkin-ElmerMPF3 spectrofluorometer in a total volume of2.5 ml in stirred 1 X 1-cm cuvettes. Addition of T N P nucleotide was in 5 pl or smaller incrementa. Absorption was a t 436 nm and emission a t 550 nm. Inner filter effects and volume changes were controlled for. Fluorescence enhancement calibrations were obtained by mixing solutions of isolated subunit with stoichiometric amounts of nucleotide as described by Grubmeyer and Penefsky (1981). Values of n and Kd were calculated in two ways: 1) from the direct plots of n versus total TNP nucleotide concentration, using nonlinear regression analysis (Duggleby, 1981); 2) a correction was applied to overcome the slight systematic error in the fluorescence enhancement calibration caused by relatively low binding affinity. The approximate Kd and n values, calculated as above, were used to iteratively correct the fluorescence enhancement calibrationusing the following equation:
TABLE I Binding of nucleotide to isolated a- and @-subunitsfrom E. coli F,-ATPase under the conditions of Khananshvili and Gromet-Elhanun (1984, 1985) Isolated subunit (0.5 mg/ml) was incubated for 1 h at 23 "C in buffer containing 50 mM Tricine-NaOH, pH 8.0, 20% glycerol, 5 mM MgClzwith 4 mM nucleotide. Then themixture was cooledfor 10 min at 4 "C and subjected to centrifuge column elution at 0-4 "C exactly as described by Khananshvili and Gromet-Elhanan (1984, 1985). Results presented are means of duplicate determinations, which agreed closely. Bound nucleotide P-Subunit a-Subunit
mollmol
0.14 [cY-~'P]ATP (4 mM) 0.05 [3H]ADP(4 mM) 0.2 (4 mM) [3H]AMP-PNP
1.04 0.56 0.3
the method of additions, and recoveries of "spiked" nucleotide were close to 100%. Therefore, the binding stoichiometries reported here are not affected by endogenous nucleotide. Binding of TNP Nucleotides to Isolated a- and @-Subunits of E. coli Fl-The results of experiments with @-subunit are shown in Fig. 1and Table11. With either TNP-ADPor TNPATP up to 50 pM concentration, in the presence or absence ofMg2+ ions, the maximal binding seen to p-subunit was approximately 1 mol/mol. NO binding of TNP-AMP to psubunit was noted either in the presence or absence of Mg2+ ions. We performed an experiment to test whether the binding of TNP-ADP and TNP-ATP was to two independent sites on p-subunit. Using the conditions of the experiment shown in Fig. 1B (i.e. M$+ present), TNP-ADP was titrated up to 40 p~ total concentration, giving maximal enhancement of fluorescence. Then TNP-ATP (final concentration = 10 WM) [TNP nucleotide] n= was added. No additional fluorescence enhancement was seen, Kd + [TNP nucleotide] demonstrating that no further binding of TNP nucleotide = maximal number of sites,n = TNP nucleotide bound, where I(d = dissociation constant, and [TNP nucleotide] = concentration occurred in excess of 1 mol/mol p-subunit. It was also important to show that TNP nucleotides comof free TNP nucleotide. peted for binding to p-subunit with the natural ligands ATP and ADP. The experiments shown in Fig. 1, B andD (TNPRESULTS ADP or TNP-ATP binding in the presence ofM$'), were Direct Binding of ADP, AMP-PNP, andATP to Isolated a - therefore repeated except that MgATP or MgADP (final of E. coli F1Using the ExperinentalConditions concentration = 4 mM) was added before additions of TNP and @-Subunits of Khanunshvili and Gromet-Elhanan-The method of Khan- nucleotide werebegun. MgATP and MgADP both caused anshvili and Gromet-Elhanan (1984,1985) involves centrifuge marked suppression of binding of either TNP-ATP or TNPcolumn elution of a preincubated, precooled mixture of 8- ADP. With 5 p~ TNP nucleotide added, in the presence of subunit, nucleotide, and buffer, with high concentration of either MgADP or MgATP, the amount of TNP-ADP or TNPMg2' ions present during the centrifuge column elution, and ATP bound was
