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The α and β Subunits of the GA-binding Protein Form a Stable Heterodimer in Solution

2000; Elsevier BV; Volume: 275; Issue: 11 Linguagem: Inglês

10.1074/jbc.275.11.7749

ISSN

1083-351X

Autores

Yurii Chinenov, Michael T. Henzl, Mark Martin,

Tópico(s)

Immune Cell Function and Interaction

Resumo

We have studied the assembly of GA-binding protein (GABP) in solution and established the role of DNA in the assembly of the transcriptionally active GABPα2β2 heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the αβ heterodimer complex, but readily binds as the GABPα2β2 heterotetramer complex on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These observations suggest that GABPαβ heterodimers are the predominant molecular species in solution and that DNA containing two PEA3/EBSs promotes formation of the GABPα2β2heterotetrameric complex. We analyzed the assembly of GABPα2β2 heteromeric complexes in solution by analytical ultracentrifugation. GABPα exists as a monomer in solution while GABPβ exists in a monomer-dimer equilibrium (K d = 1.8 ± 0.27 μm). In equimolar mixtures of the two subunits, GABPα and GABPβ formed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak transcriptional activator. Assembly of the transcriptionally active GABPα2β2 heterotetramer complex requires the presence of specific DNA containing at least two PEA3/EBSs. We have studied the assembly of GA-binding protein (GABP) in solution and established the role of DNA in the assembly of the transcriptionally active GABPα2β2 heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the αβ heterodimer complex, but readily binds as the GABPα2β2 heterotetramer complex on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These observations suggest that GABPαβ heterodimers are the predominant molecular species in solution and that DNA containing two PEA3/EBSs promotes formation of the GABPα2β2heterotetrameric complex. We analyzed the assembly of GABPα2β2 heteromeric complexes in solution by analytical ultracentrifugation. GABPα exists as a monomer in solution while GABPβ exists in a monomer-dimer equilibrium (K d = 1.8 ± 0.27 μm). In equimolar mixtures of the two subunits, GABPα and GABPβ formed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak transcriptional activator. Assembly of the transcriptionally active GABPα2β2 heterotetramer complex requires the presence of specific DNA containing at least two PEA3/EBSs. GA-binding protein polyomavirus enhancer A factor 3 ets-binding site recombinant GABP protein histidine tag fused to rGABP proteins derived from the bacterial expression vector, pET15b electrophoretic mobility shift assay basic leucine zipper The GA-binding protein (GABP)1 is a heteromeric transcription factor that binds to GA-rich sequences ((A/C)GGAAG) in DNA, and contains two unrelated subunits belonging to the Ets (GABPα) and the Notch-Ankyrin repeat (GABPβ) families of proteins (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 3.de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar, 4.Seth A. Ascione R. Fisher R.J. Mavrothalassitis G.J. Bhat N.K. Papas T.S. Cell Growth Differ. 1992; 3: 327-334PubMed Google Scholar, 5.Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (91) Google Scholar). GABP has been implicated in the regulation of several eukaryotic genes encoding proteins involved in oxidative phosphorylation (cytochrome oxidase subunits IV, V, and VII and ATP synthase β subunit) (6.Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1991; 11: 5631-5638Crossref PubMed Scopus (125) Google Scholar, 7.Virbasius J.V. Scarpulla R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1309-1313Crossref PubMed Scopus (640) Google Scholar, 8.Virbasius J.V. Virbasius C.C. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (245) Google Scholar, 9.Carter R.S. Avadhani N.G. J. Biol. Chem. 1994; 269: 4381-4387Abstract Full Text PDF PubMed Google Scholar, 10.Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 11.Bachman N.J. Yang T.L. Dasen J.S. Ernst R.E. Lomax M.I. Arch. Biochem. Biophys. 1996; 333: 152-162Crossref PubMed Scopus (23) Google Scholar, 12.Villena J.A. Vinas O. Mampel T. Iglesias R. Giralt M. Villarroya F. Biochem. J. 1998; 331: 121-127Crossref PubMed Scopus (46) Google Scholar) and the immune and inflammatory response (CD18, interleukin-2, γc chain of interleukin receptors) (13.Bottinger E.P. Shelley C.S. Farokhzad O.C. Arnaout M.A. Mol. Cell. Biol. 1994; 14: 2604-2615Crossref PubMed Google Scholar, 14.Rosmarin A.G. Luo M. Caprio D.G. Shang J. Simkevich C.P. J. Biol. Chem. 1998; 273: 13097-13103Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 15.Hoffmeyer A. Avost A. Flory A. Weber C.K. Serfling E. Rapp U.R. J. Biol. Chem. 1998; 273: 10112-10119Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16.Bannert N. Avots A. Baier M. Serfling E. Kurth R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1541-1546Crossref PubMed Scopus (58) Google Scholar, 17.Markiewicz S. Bosseult R. Le Deist F. de Villartay J.-P Hivroz C. Ghysdael J. Fisher A. de Saint Basile G. J. Biol. Chem. 1996; 271: 14849-14855Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Recently, an important role for GABP in the regulation of mitochondrial biogenesis in brown adipose tissue was demonstrated (12.Villena J.A. Vinas O. Mampel T. Iglesias R. Giralt M. Villarroya F. Biochem. J. 1998; 331: 121-127Crossref PubMed Scopus (46) Google Scholar). The GABP-binding site first identified in the herpes simplex virus immediate early promoter contains two tandemly arranged PEA3/Ets-binding sites (EBS) (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 18.LaMarco K.L. McKnight S.L. Genes Dev. 1989; 3: 1372-1382Crossref PubMed Scopus (84) Google Scholar, 19.Martin M.E. Piette J. Yaniv M. Tang W-J. Folk W.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5839-5843Crossref PubMed Scopus (151) Google Scholar). Early analysis of GABP binding to the herpes simplex virus immediate early promoter indicated that GABP forms predominantly a heterotetrameric complex composed of two α and two β subunits (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar). The results of gel filtration and preparative ultracentrifugation of highly purified recombinant GABPα and GABPβ proteins were further interpreted to support a model depicting GABP as a stable heterotetrameric complex in solution, suggesting that this complex binds as a single unit to DNA (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar). The ability to form the heterotetrameric complex has been shown to be necessary for GABP-dependent transcription (20.Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 21.Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar, 22.Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). Thus, modulation of GABP tetramer formation represents a potentially important means of regulating GABP-dependent transcription. We have previously shown that GABP-dependent transcription initiator activity required two PEA3/EBSs, and the highest activity was achieved with two sites positioned on the same face of the DNA helix (23.Yu M. Yang X.-Y. Schmidt T. Chinenov Y. Martin M.E. J. Biol. Chem. 1997; 272: 29060-29067Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24.Sucharov C. Basu A. Carter R.S. Avadhani N.G. Gene Exp. 1995; 5: 93-111PubMed Google Scholar, 25.Yoo W. Martin M.E. Folk W.R. J. Virol. 1991; 65: 5391-5400Crossref PubMed Google Scholar). Since the ability to activate transcription was previously shown to be dependent on heterotetramer complex formation, these results favored the stable GABP heterotetramer complex model. However, electrophoretic mobility shift assays (EMSA) with DNA probes containing only a single PEA3/EBS detected a complex consistent with the mobility of the GABPαβ heterodimer (23.Yu M. Yang X.-Y. Schmidt T. Chinenov Y. Martin M.E. J. Biol. Chem. 1997; 272: 29060-29067Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26.Martin M.E. Chinenov Y., Yu, M. Schmidt T.K. Yang X.-Y. J. Biol. Chem. 1996; 271: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27.Chinenov Y. Schmidt T. Yang X.-Y. Martin M.E. J. Biol. Chem. 1998; 273: 6203-6209Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In contrast, a probe with two PEA3/EBSs formed two complexes, one co-migrating with the GABPαβ complex observed on a single PEA3/EBS and a slower migrating complex identified as GABPα2β2heterotetramer. These observations are consistent with the notion that GABP binds to a single PEA3/EBS only as a heterodimer, and requires two or more PEA3/EBSs to bind DNA as a heterotetramer. Therefore, if GABP forms a stable heterotetrameric complex in solution, then dissociation of this complex would be required upon binding to a single PEA3/EBS. Alternatively, GABP may form a stable heterodimer, or may exist in heterodimer-heterotetramer equilibrium that, at concentrations typically found in EMSA assays, favors the GABPαβ heterodimer. Therefore, prior interaction of a GABPαβ heterodimer complex with DNA containing two PEA3/EBS may be required for successful assembly of a transcriptionally competent GABPα2β2heterotetramer. To address the mechanism of GABP assembly and the role of DNA binding in this process, we have utilized EMSAs and analytical ultracentrifugation. In this report we show that GABP exists exclusively as a stable heterodimeric complex (GABPαβ). Even at exceedingly high concentrations little or no significant quantities of GABP heterotetramer complex is observed. These observations support the hypothesis that GABP exists as a stable heterodimer in solution which assembles into the heterotetrameric complex upon binding to target DNA containing two PEA3/EBSs. We further demonstrate that heterotetramer DNA binding is stabilized by positioning the two PEA3/EBSs on the same face of the DNA helix, and that the two motifs can be separated by up to three helical turns. These results are consistent with our earlier results demonstrating that GABP-dependent initiators were most efficient when two PEA3/EBSs were positioned on the same face of the DNA helix, and confirms the importance of the GABP heterotetramer complex in transcription initiator and activator activities of this important regulatory factor (23.Yu M. Yang X.-Y. Schmidt T. Chinenov Y. Martin M.E. J. Biol. Chem. 1997; 272: 29060-29067Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). DNA-modifying enzymes were purchased from New England Biolabs. Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen. Talon Metal Affinity Resin for cobalt immobilized metal affinity chromatography was purchased fromCLONTECH. DNA oligonucleotides were synthesized by the University of Missouri DNA Core Facility on an Applied Biosystems DNA Synthesizer, Model 380B. [α-32P]dGTP, [α-32P]dCTP, and [γ-32P]ATP were purchased from NEN Life Science Products Inc. All other reagents were obtained from Sigma or Fisher Scientific. DNAs encoding GABPα and GABPβ proteins were amplified by polymerase chain reaction from cDNAs kindly provided by C. C. Thompson and cloned into pET15b (Novagene) as described previously (26.Martin M.E. Chinenov Y., Yu, M. Schmidt T.K. Yang X.-Y. J. Biol. Chem. 1996; 271: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The individual recombinant (rGABP) His6-tagged proteins (Fig. 1 A) were expressed in E. coli BL21 strain and recovered from cell extracts by nickel chelating or cobalt immobilized affinity chromatography under denaturing conditions in the presence of 6 m urea. rGABPα and rGABPβ proteins were precipitated with ammonium sulfate at 20 and 14% saturation, respectively. Precipitated material was resuspended in 50 mm potassium phosphate buffer, pH 7.4, containing 100 mm potassium chloride, 6 m urea, and 5 mm dithiothreitol and subjected to several steps of dialysis in the same buffer with progressively lower concentrations of urea (5 m, 3 m, etc.). The extinction coefficients of rGABPα (ε280 = 62,796m−1 cm−1), rGABPβ (ε280 = 18,262 m−1cm−1), rGABPαcQ (ε280 = 26,008m−1 cm−1), and rGABPβ334 (ε280 = 15,422m−1 cm−1) proteins were calculated from their amino acid compositions. The rGABPαcQ protein containing four Cys-Ser substitutions, described previously (27.Chinenov Y. Schmidt T. Yang X.-Y. Martin M.E. J. Biol. Chem. 1998; 273: 6203-6209Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), was expressed at high levels in E. coli and was less prone to aggregation than the wild-type GABPαc protein. For EMSA analysis, double-stranded oligonucleotide probes were end-labeled with [α-32P]dGTP and [α-32P]dCTP by incubation at 25 °C with the Klenow fragment of E. coliDNA polymerase I (New England Biolabs). Alternatively, double-stranded oligonucleotide probes were end-labeled with [γ-32P]ATP by incubation with T4 polynucleotide kinase. Radiolabeled probes were purified using the Mermaid Kit (BIO101). The sequences of one stand of each probe, with the PEA3/EBSs underlined, are as follows: PEA324, 5′-TCGAGCACCTTGAGGAAGTCTCGA; dPEA3-0, 5′-TCGAGCAGGAAGAGGAAGTCTCGA; dPEA3–10, 5′-TCGAGCAGGAAGTCGAGCTCGCAGGAAGTC; dPEA3–16, 5′-TCGAGCAGGAAGTCGAGCTCGTCGAGGCAGGAAGTC; dPEA3–22, 5′-TCGAGCAGGAAGTCTCGAGCACCAAGTCTCGAGCAGGAAGTC; dPEA3–26, 5′-TCGAGCAGGAAGTCTCGAGCACCATCGAAGTCTCGAGCAGGAA- GTC. rGABPβ (0.02–0.04 μg) was mixed with a slight excess of rGABPα in 20 μl of EMSA buffer (20 mm HEPES, pH 8.0, 50 mm KCl, 1 mm benzamidine, 20% glycerol, 5 μg/ml poly(dI-dC)-poly(dI-dC), and 0.2 mg/ml bovine serum albumin). After 10 min incubation at 25 °C, 0.5 ng of 32P-labeled probe was added and binding reactions were incubated for an additional 10 min at 25 °C. DNA-protein complexes were fractionated on a 5.0% nondenaturing polyacrylamide gel, dried, and subjected to autoradiography as described previously (25.Yoo W. Martin M.E. Folk W.R. J. Virol. 1991; 65: 5391-5400Crossref PubMed Google Scholar, 26.Martin M.E. Chinenov Y., Yu, M. Schmidt T.K. Yang X.-Y. J. Biol. Chem. 1996; 271: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27.Chinenov Y. Schmidt T. Yang X.-Y. Martin M.E. J. Biol. Chem. 1998; 273: 6203-6209Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Prior to sedimentation analysis, the samples were clarified by centrifugation at 45,000 rpm for 30 min in a TL-100 centrifuge (Beckman Instruments, Palo Alto, CA). Equilibrium sedimentation experiments with His6-tagged rGABPα and rGABPβ were performed in a Beckman Optima XL-I ultracentrifuge equipped with AN50ti rotor (Beckman Instruments, Palo Alto, CA). Six-channel cells with 12-mm optical path length were used. Data were collected at 20 °C at rotor speeds between 6,000 and 28,000 rpm. Sample loading concentrations ranged between 2 and 13 μm in 50 mm phosphate buffer, pH 7.4, containing 100 mm KCl, 10% glycerol, and 1 mm dithiothreitol. The distribution of solutes in the cell was monitored by absorbance at 280 nm. The resulting distributions at equilibrium were subjected to global least-square analysis employing several models: a single exponential (monomeric ideal, Equation 1), two exponential (dimeric ideal, Equation 2), or two exponential (associative, Equation 3) (28.Laue T.M. Stanfford III, W.F. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 75-100Crossref PubMed Scopus (240) Google Scholar) using the program Origin 5.0 (MicroCal Software, Inc. MA), C(r)=C10×EXPω2×(1−v¯×ρ)×M1×(r2−r02)2×RT+CxEquation 1 C(r)−C10×EXPω2×(1−v¯×ρ)×M1×(r2−r02)2×RT+C20×EXPω2×(1−v¯×ρ)×M1×(r2−r02)2×RT+CxEquation 2 C(r)=Cm×EXPω2×(1−v¯×ρ)×Mm×(r2−r02)2×RT+Cm2×K×EXPω2×(1−v¯×ρ)×2×Mm×(r2−r02)2×RT+CxEquation 3 Where C 10, C 20, and C m are the absorbance of the first, second, and monomeric species, respectively, at a reference point.M 1, M 2, andM m are the molecular weights of the first, second, and monomeric species, respectively. The angular velocity, ω, was calculated from the rotor speed. The value for partial volume,v, at 20 °C was calculated from amino acid composition using SEDNTERP software. R is the universal gas constant.T is the absolute temperature equal to 293.15 K. The parameter C x is the baseline that was either determined from the absorbance near the meniscus after sedimentation of the samples at 20,000 rpm for 12 h or from global least-square analysis. To account for nonspecific aggregation at high protein concentrations or for deviations from stoichiometry in multi-component mixtures, a third term was introduced into equation III, producing Equation4. C(r)=Cm×EXPω2×(1−v¯×ρ)×Mm×(r2−r02)2×RT+Cm2×K×EXPω2×(1−v¯×ρ)×Mm×(r2−r02)2×RT+C20×EXPω2×(1−v¯×ρ)×M2×(r2−r02)2×RT+CxEquation 4 The GABP-binding site originally identified in the HSV IE promoter contained two tandemly arranged PEA3/EBSs (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 18.LaMarco K.L. McKnight S.L. Genes Dev. 1989; 3: 1372-1382Crossref PubMed Scopus (84) Google Scholar). When two PEA3/EBSs (dPEA3-0) are present on the target DNA, approximately equal amounts of both heterodimeric and heterotetrameric complexes are readily observed (Fig. 1 B). However, on a probe containing only a single PEA3/EBS (PEA324), GABP forms exclusively the heterodimeric complex (Fig. 1 B). No heterotetrameric complex was observed on the PEA324 probe containing a single PEA3/EBS even at 10-fold higher GABP protein concentrations. 2Y. Chinenov and M. E. Martin, unpublished observations. These results indicate that at the concentration used in these EMSA experiments (∼10 nm), GABP requires two PEA3/EBSs to efficiently assemble into a heterotetrameric complex on DNA. Previous studies indicated that an NH2-terminal truncation mutant of the GABPα protein (GABPαcQ) exhibited enhanced heterotetramer complex DNA in the presence of GABPβ protein (26.Martin M.E. Chinenov Y., Yu, M. Schmidt T.K. Yang X.-Y. J. Biol. Chem. 1996; 271: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27.Chinenov Y. Schmidt T. Yang X.-Y. Martin M.E. J. Biol. Chem. 1998; 273: 6203-6209Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). GABPαcQ protein (Fig. 1 A) readily forms both heterodimeric and heterotetrameric complexes with GABPβ protein on DNA containing a single PEA3/EBS (Fig. 1 B). When two PEA3/EBSs are present on the target DNA, only the heterotetrameric complex is observed. Thus, enhanced GABP heterotetramer complex DNA binding is achieved by deletion of the NH2-terminal two thirds of the GABPα protein. Enhanced DNA binding of the heterotetramer complex requires the COOH-terminal leucine zipper-like domain in GABPβ. Deletion of this region in the GABPβ334 mutant abolishes the formation of detectable heterotetrameric complex with either full-length GABPα or GABPαcQ proteins on probes containing either one or two PEA3/EBSs. We have previously shown that two PEA3/EBSs function as a GABP-dependent initiator element, and that maximal initiator activity occurs on templates containing two PEA3/EBSs on the same face of the DNA helix (23.Yu M. Yang X.-Y. Schmidt T. Chinenov Y. Martin M.E. J. Biol. Chem. 1997; 272: 29060-29067Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In addition, GABP activation has been shown previously to depend on the ability of GABP to form the heterotetrameric complex (20.Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 21.Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar, 22.Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). Therefore, we sought to determine whether the preference of GABP-dependent initiator elements for templates containing two PEA3/EBS on the same face of the DNA helix correlates with the ability of GABP heterotetrameric complex to form on these initiator elements. We performed EMSA analysis of GABP binding to DNA probes containing two PEA3/EBSs separated by a 0, 10-, 16-, 22-, or 26-base pair linker DNA, corresponding to 0.5, 1.5, 2.0, 2.5, and 3.0 helical turns between PEA3/EBS. Approximately equivalent amounts of heterotetramer complex formed on each of the DNAs analyzed (Fig.2 A), suggesting that the ability to assemble into a heterotetrameric complex is not affected by the helical spacing between two PEA3/EBS elements. Analysis of heterotetramer complex DNA binding revealed that GABP heterotetramer complexes bound to DNA containing two PEA3/EBSs on the same face of the DNA helix were significantly more stable than complexes bound to DNA containing two PEA3/EBSs on the opposite sides of the DNA helix (Fig. 2 B). GABP heterotetramer complexes bound to radiolabeled probes were challenged with an excess of unlabeled competitor DNA and the rate of heterotetramer complex decay measured over time. Heterotetramer complexes bound to probes containing two PEA3/EBSs separated by 2.0 and 3.0 helical turns were substantially more resistant to challenge by the unlabeled competitor DNA than were complexes bound to probes containing two PEA3/EBSs separated by 0.5, 1.5, and 2.5 helical turns. Heterotetramer complexes bound to all probes were substantially more stable than the heterodimer complex bound to DNA containing a single PEA3/EBS. Thus, the ability to assemble into a heterotetrameric complex during DNA binding is independent of helical spacing or distance (up to 4 helical turns),2 but complex stability is enhanced when two PEA3/EBSs are positioned on the same face of the DNA helix. These observations explain, in part, our previous results correlating helical spacing between PEA3/EBSs with GABP initiator activity. The association states of GABPα, GABPβ, and the various mutant proteins (Fig. 1) were analyzed by sedimentation equilibrium. The distribution of GABPα (Fig.3 A) is satisfactorily described by a single species model ("Experimental Procedures," Equation 1), in which the molecular weight is set to the calculated value of 53,500 for the His6-tagged GABPα protein. We attribute the discrepancies between the calculated and experimental values observed at the highest protein concentration and rotor speeds, to nonspecific aggregation of GABPα. These data suggest that GABPα exists as a monomeric species under our experimental conditions. GABPαcQ protein behaved similarly (Fig.4 B), although no aggregation was observed. The radial distribution for GABPαcQ (Fig.4 B) is accommodated by an ideal single species model in which the molecular mass is fixed at 18,551 Da, the calculated molecular mass of His6-tagged GABPαcQ. In contrast to the full-length GABPα protein, we observe no indication of higher molecular weight aggregates.Figure 4GABP exists as a stable heterodimer in solution in the absence of DNA. A, equimolar mixtures of GABPα and GABPβ were centrifuged at 6,000 and 12,000 rpm in a Beckman Model XL-I ultracentrifuge at 20 °C, with initial protein concentrations between 0.4 and 1.3 A 280 nm. The concentration distribution data analyzed as described under "Experimental Procedures" were fitted to a two-species ideal model (Equation 2). The molecular mass of the first species was equal to the sum of the molecular mass of the His6-tagged GABPα and GABPβ proteins, and the molecular mass of the second species converged at 619,573 ± 13.597 Da. B, concentration distributions of the GABPα-GABPβ equimolar mixture were fitted to two alternative models: a single-species model (left panel), with a molecular weight equal to that of His6-tagged GABPα2β2 heterotetramer, and a heterodimer-to-heterotetramer associative model (right panel). The residuals are provided in the inset for each fitting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to GABPα, we see evidence for dimerization, albeit weak, of GABPβ. The distribution of GABPβ in sedimentation equilibrium experiments is consistent with a monomer-dimer equilibrium associative model (Equation3) (Fig. 3 C). The mass of the monomeric species was fixed at 43,422 Da, equal to that of His6-tagged GABPβ, and the dissociation constant was treated as a variable parameter. The optimal value for the homodimer dissociation constant,K dβ, was determined to be 1.8 ± 0.27 μm, indicating that GABPβ associates only weakly under these conditions. At physiologically relevant concentrations (0.1–10 nm), GABPβ would be almost exclusively monomeric. Deletion of the COOH-terminal portion of GABPβ (GABPβ334), which includes the leucine zipper-like domain, eliminates the tendency to self-associate. The radial distribution of GABPβ334 is consistent with an ideal two-species model (Equation 2) with the molecular mass of the first species equal to that of His6-tagged GABPβ334protein (37,691 Da), and the molecular mass of the second species equal to 1,417,503 ± 69,435 Da (Fig. 3 D). GABPβ334 protein tends to precipitate at high protein concentrations suggesting that the second high molecular weight species may be attributed to nonspecific aggregation of the GABPβ334 protein at high concentrations. Consistent with this assumption, the molecular mass of the second species varied from preparation to preparation but was consistently higher than 600 kDa. These results are consistent with the previously proposed role of the COOH-terminal leucine zipper-like domain of GABPβ in self-association (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 3.de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar, 20.Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 29.Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (239) Google Scholar). The virtual absence of heterotetrameric GABP bound to a single PEA3/EBS in EMSAs suggests that any heterotetrameric complex formed in solution must have a low affinity for a single PEA3/EBS. Alternatively, GABP heterodimers may have very little tendency to assemble into the heterotetrameric complex on DNA containing a single PEA3/EBS. To distinguish between these possibilities, we performed analytical ultracentrifugation to determine the relative amounts of GABP heterodimer and heterotetramer complexes in solution in the absence of DNA binding. When stoichiometric amounts of GABPα and GABPβ are combined and centrifuged to equilibrium, the resulting radial distribution is consistent with exclusive heterodimer formation. Optimal agreement between the calculated and observed values was obtained with an ideal two species model (Equation 2), in which the molecular mass for the major species was fixed at 96,949 Da (the sum of molecular masses of GABPα and GABPβ). The mass for the second species was allowed to vary and converged to 619,573 ± 13,597 Da (Fig. 4 A). The presence of this material, which accounted for less than 5% of the total protein, is attributed to nonspecific aggregation. We were unable to fit the concentration distributions to a hetero-associative model, suggesting a very strong GABPα-GABPβ interaction. Based on the instrument detection limit, the K d for the GABP αβ heterodimer was estimated to be below 10−8m. This estimated K d for the GABP αβ heterodimer is consistent with the reported K d(7.8 ± 0.63 × 10−10m) as measured by surface plasmon resonance (30.Suzuki F. Goto M. Sawa C. Ito S. Watanabe H. Sawada J. Handa H. J. Biol. Chem. 1998; 273: 29302-29308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These results demonstrate that GABP under the experimental conditions and concentration range used in these experiments, exists exclusively as a stable heterodimer. Attempts to fit the data to a "stable heterotetramer" model (Fig. 4 B, left panel) or to a two-species associative model (Fig. 4 B, right panel) were unsuccessful. Although our data indicate that GABPβ weakly dimerizes in solution, the GABPαβ heterodimer exhibits no tendency to associate, even at concentrations above 2.5 μm. To resolve this apparent contradiction, we studied the association of GABPαcQ and GABPβ in solution. In EMSA experiments (Fig. 1), GABPαcQ and GABPβ predominantly form heterotetrameric complex when incubated with probe containing two PEA3/EBSs and displays a limited tendency to bind as a heterotetramer to DNA containing a single PEA3/EBS. To determine the associative state of GABPαcQ and GABPβ when free in solution, a slight excess of GABPαcQ protein was combined with GABPβ protein and centrifuged to equilibrium. The solute distribution was satisfactorily described by a heterodimer-heterotetramer equilibrium plus a third nonassociating species (Fig. 5, Equation 4). The molecular mass of the heterodimer was fixed at 61,525 Da, equal to the molecular mass of the His6-tagged GABPαcQ·GABPβ complex. The molecular mass of the third species was fixed at 18,551 Da, equal to that of the His6-tagged GABPαcQ monomer, which was present in excess. Least-squares minimization yielded a dissociation constant for GABPαcQ-GABPβ dimer-tetramer equilibrium equal to 0.17 ± 0.045 μm, significantly lower than that obtained for GABPβ alone. These results suggest: 1) the COOH terminus of GABPα promotes GABPβ dimerization and 2) that the NH2 terminus of GABPα may interfere with heterotetramer complex assembly in solution. The latter conclusion explains, in part, the failure of the full-length GABPα protein to form heterotetrameric complex with GABPβ protein in solution or bound to DNA containing a single PEA3/EBS. Since the COOH-terminal leucine zipper-like domain has been shown previously to be essential for GABPβ homodimerization and for heterotetramer complex DNA binding (Fig. 1) (2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 3.de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar, 18.LaMarco K.L. McKnight S.L. Genes Dev. 1989; 3: 1372-1382Crossref PubMed Scopus (84) Google Scholar, 29.Brown T.A. McKnight S.L. Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (239) Google Scholar), we studied the association of GABPβ334, lacking the COOH-terminal homodimerization domain, with full-length GABPα and GABPαcQ in solution. The solute distribution in a GABPαcQ/GABPβ334 mixture could be accommodated by an ideal single-species model (Equation 1) with a molecular mass fixed at 56,241 Da, which equals the sum of the His6-tagged GABPαcQ and GABPβ334 molecular masses (Fig.6 A). Similar results were obtained when the concentration distribution of a mixture of full-length GABPα and GABPβ334 proteins was analyzed (Fig. 6 B). These data are consistent with an ideal two-species model (Equation 2) with a molecular mass of one species fixed at 91,218 Da (His6-tagged GABPα + His6-tagged GABPβ334) and the other fixed at 37,691 Da (uncomplexed His6-tagged GABPβ334). The apparent absence of tetrameric species in these two experiments suggests that the COOH-terminal leucine zipper-like domain of GABPβ is obligatory for formation of GABP heterotetramer complexes. Since the discovery of GABP (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 18.LaMarco K.L. McKnight S.L. Genes Dev. 1989; 3: 1372-1382Crossref PubMed Scopus (84) Google Scholar), it has been widely accepted that GABP exists in solution predominantly as a heterotetramer (α2β2), due to stable β-β interactions (2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 3.de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar). This model was supported by subsequent observations that heterotetramer formation was required for full transcriptional transactivation by GABP (20.Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 21.Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar). Although attractive, this model is not consistent with the absence of heterotetramer complex in EMSA experiments performed with a DNA probe containing a single PEA3/EBS (Fig. 1). DNA containing two PEA3/EBSs is required to observe significant amounts of heterotetramer complex in EMSA experiments, although equivalent amounts of heterodimer complex is observed in these experiments. These results are inconsistent with a stable GABP heterotetramer complex binding as a single unit to DNA containing one or two PEA3/EBSs. Cellular proteins such as bcl3 can promote GABP heterotetramer formation in EMSA experiments (31.Shiio Y. Sawada J.-i. Handa H. Yamamoto T. Inoue J. Oncogene. 1996; 12: 1837-1845PubMed Google Scholar), suggesting that the absence of the heterotetrameric complex does not merely reflect its instability under EMSA conditions, but that its formation may be a regulated process. Our observations suggest that the GABPαβ heterodimer is the major species found in solution and that efficient heterotetramer formation requires specific DNA for assembly. To directly address the associative behavior of GABP in solution, we have performed sedimentation equilibrium analysis of purified GABPα, GABPβ, and several deletion mutants in various combinations (27.Chinenov Y. Schmidt T. Yang X.-Y. Martin M.E. J. Biol. Chem. 1998; 273: 6203-6209Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 32.Hansen J.C. Lebowitz J. Demeler B. Biochemistry. 1994; 33: 13155-13163Crossref PubMed Scopus (113) Google Scholar,33.Minton A.P. Anal. Biochem. 1990; 190: 1-6Crossref PubMed Scopus (34) Google Scholar). Our observation that GABPβ exists in a "monomer to dimer" equilibrium (K d = 1.8 ± 0.4 μm), is incompatible with the "stable heterotetramer" model previously suggested to be required for heterotetramerization (1.LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 2.Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (321) Google Scholar, 3.de la Brousse F.C. Birkenmeier E.H. King D.S. Rowe L.B. McKnight S.L. Genes Dev. 1994; 8: 1853-1865Crossref PubMed Scopus (65) Google Scholar). Concentration distributions of an equimolar mixture of GABPα and GABPβ, however, best fit to a two-species model consisting of the GABPαβ heterodimer and a 619,573 Da species, which we attribute to the aforementioned nonspecific aggregation of GABPα. We have been unable to satisfactorily model the GABPαβ concentration distribution with an α + β ↔ αβ ↔ (αβ)2equilibrium model. These results suggest (i) that GABP α and β subunits form a stable heterodimer in solution with an estimatedK d less than 10−8m, and (ii) that, under our experimental conditions, no GABPα2β2 heterotetramer is observed in solution. Our estimation for a GABPαβ dissociation constant is consistent with the apparent K d determined by Suzukiet al. (30.Suzuki F. Goto M. Sawa C. Ito S. Watanabe H. Sawada J. Handa H. J. Biol. Chem. 1998; 273: 29302-29308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) using surface plasmon resonance (K d = 7.8 ± 0.63 × 10−10m). Our results are also consistent with EMSA experiments suggesting that efficient heterotetramer formation in the absence of any exogenous regulatory influences occurs only on DNA with two or more PEA3/EBSs (Fig. 1). GABPαβ heterodimers assemble into the heterotetramer complex on DNA containing two PEA3/EBSs separated by as many as 4.0 helical turns (Fig. 2).2 The stability of the heterotetramer complex is greatly affected by the spacing between PEA3/EBSs such that, stability is enhanced on DNAs containing two PEA3/EBSs positioned on the same face of the DNA helix. These results are consistent with our previous data showing that GABP-dependent initiator activity is enhanced when two PEA3/EBSs are positioned on the same face of the DNA helix (23.Yu M. Yang X.-Y. Schmidt T. Chinenov Y. Martin M.E. J. Biol. Chem. 1997; 272: 29060-29067Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Similarly, GABPβ isoforms or mutants lacking the COOH-terminal homodimerization domain are reportedly unable to promote heterotetramer complex formation and fail to activate transcription of linked promoters (20.Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 21.Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar, 22.Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J.-i. Handa H. Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar). The demonstration that heterotetramer complex stability correlates with GABP-dependent initiator activity further advances the notion that the heterotetramer complex is the functionally active form of GABP. The NH2-terminal deletion mutant of GABPα (GABPαcQ) forms significant amounts of heterotetramer complex even on DNA with a single PEA3/EBS. This finding suggests that the NH2 terminus of GABPα may interfere with heterotetramer complex formation. In agreement with this observation, the concentration distribution of a GABPαcQ/GABPβ equimolar mixture best fits a two-species associative model (K d = 0.17 ± 0.045 μm) with the molecular weight of the monomeric species equal to that of the His6-tagged GABPαcQ/GABPβ dimer. Therefore, we have demonstrated that GABP exists as a stable heterodimer, and that multiple PEA3/EBSs are required to promote formation of the heterotetramer complex. Our observation that the NH2-terminal portion of GABPα subunit exerts an inhibitory effect on GABPα2β2heterotetramer formation, and the observations of Shiijo et al. (31.Shiio Y. Sawada J.-i. Handa H. Yamamoto T. Inoue J. Oncogene. 1996; 12: 1837-1845PubMed Google Scholar) demonstrating the bcl3 enhances GABPα2β2 heterotetramer DNA binding, suggest that heterotetramer assembly is likely a regulated process that depends on specific promoter organization and/or specific regulatory proteins. Homo- and heterodimerization is often used by DNA-binding proteins in order to increase specificity and affinity for a cognate binding site. Although preassembled multiprotein complexes may possess higher overall affinity, they also will have higher nonspecific affinity for DNA, and at limiting concentrations, a protein complex could become kinetically trapped at random positions in the genome (34.Kohler J.J. Metallo S.J. Schneider T.L. Schepatz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11735-11739Crossref PubMed Scopus (94) Google Scholar, 35.Pomerantz J.L. Wolfe S.A. Pabo C.O. Biochemistry. 1998; 37: 965-970Crossref PubMed Scopus (56) Google Scholar). Therefore, a high "off-rate" of a monomeric protein, compared with the fully assembled complex, will ensure, that only high affinity sites will be occupied for a sufficient amount of time to enucleate the assembly of a higher order protein-DNA complex. This mechanism, designated as the "monomeric pathway," has been proposed for the basic helix-loop-helix protein max, the bZIP transcription factor ATF-2, and the Arc repressor (34.Kohler J.J. Metallo S.J. Schneider T.L. Schepatz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11735-11739Crossref PubMed Scopus (94) Google Scholar, 36.Reintzeperis D. Johnson T. Sauer R.T. Nat. Struct. Biol. 1999; 6: 569-573Crossref PubMed Scopus (54) Google Scholar). Although the GABPα subunit is capable of DNA binding, in the presence of sufficient GABPβ, a highly stable heterodimer complex is formed. Thus, the GABPαβ heterodimer serves as a functional "monomeric" DNA binding entity. We have demonstrated that the presence of two PEA3/EBSs promotes further high affinity multimerization, suggesting that GABP assembly may proceed along the monomeric pathway. We thank Dr. Jeffrey Hansen for helpful comments and suggestions during the completion of these studies. We are grateful to Matt Stanley for help in preparing the manuscript and figures for publication.

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