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• W1980378067 abstract "The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of ∼300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A.(1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity. The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of ∼300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A.(1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity. The proteasome is a 700,000-Da multicatalytic protease that participates in a number of proteolytically mediated intracellular processes, including the constitutive turnover of many intracellular proteins(1.Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2171) Google Scholar), the rapid elimination of proteins with abnormal structures(2.DeMartino G.N. McCullough M.L. Reckelhoff J.F. Croall D.E. Ciechanover A. McGuire M.J. Biochim. Biophys. Acta. 1991; 1073: 299-308Crossref PubMed Scopus (14) Google Scholar, 3.Heinemeyer W. Kleinschmidt J.A. Saidowsky J. Escher C. Wolf D.H. EMBO J. 1991; 10: 555-562Crossref PubMed Scopus (357) Google Scholar), the temporal reduction in levels of critical regulatory proteins for control of the cell cycle and transcription (4.Murray A. Cell. 1995; 81: 149-152Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 5.Chen P. Hochstrasser M. EMBO J. 1995; 14: 2620-2630Crossref PubMed Scopus (98) Google Scholar, 6.Kominami K. DeMartino G.N. Moomaw C.R. Slaughter C.A. Shimbara N. Fujimuro M. Yokosawa H. Hisamatsu H. Tanahashi N. Shimizu Y. Tanaka K. Toh-e A. EMBO J. 1995; 14: 3105-3115Crossref PubMed Scopus (93) Google Scholar, 7.Gridley T. Jaenisch R. Gendron-Maguire M. Genomics. 1995; 11: 501-507Crossref Scopus (18) Google Scholar), the proteolytic activation of the transcription factor NF-κB (8.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1908) Google Scholar), and the processing of antigens for presentation by class I major histocompatibility complex proteins(9.Goldberg A.L. Rock K.L. Nature. 1992; 357: 375-379Crossref PubMed Scopus (502) Google Scholar, 10.Peters J.-M. Franke W.W. Kleinschmidt J.A. J. Biol. Chem. 1994; 269: 7709-7718Abstract Full Text PDF PubMed Google Scholar). Despite the important role of the proteasome in these various processes, the mechanisms by which its action is controlled remain unclear. Several lines of evidence indicate that proteasome function is controlled by specific regulatory proteins. First, the proteasome can be isolated as part of a larger protein complex (Mr≥ 1,500,000) referred to as the “26 S protease”(11.Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar). This complex displays catalytic and regulatory properties that differ considerably from those of the purified 20 S proteasome, most likely because of regulatory influences exerted by the non-proteasome components of the complex. Second, individual proteasome regulatory proteins have been identified and purified. One of these proteins, which we call PA700 and which has been independently described in several laboratories, appears to represent the major non-proteasome component of the 26 S protease(10.Peters J.-M. Franke W.W. Kleinschmidt J.A. J. Biol. Chem. 1994; 269: 7709-7718Abstract Full Text PDF PubMed Google Scholar, 12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar, 13.Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar, 14.Udvardy A. J. Biol. Chem. 1993; 268: 9055-9062Abstract Full Text PDF PubMed Google Scholar). PA700 is a 700,000-Da multisubunit ATP-dependent proteasome activator. It forms a complex with the proteasome by a mechanism that requires ATP hydrolysis. The proteasome-PA700 complex has physical properties, such as molecular weight, and catalytic properties, such ATP-dependent degradation of ubiquitinated proteins, that are characteristic of the purified 26 S protease. At least four of the ∼20 electrophoretically distinct subunits of PA700 are homologous to one another and are members of a large protein family that contains a consensus sequence for ATP binding(15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 16.Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar, 17.Confalonieri F. Duguet M. BioEssays. 1995; 17: 639-650Crossref PubMed Scopus (313) Google Scholar). Some of these same proteins have been identified as components of the purified 26 S protease, providing additional strong evidence that the proteasome-PA700 complex is similar, if not identical, to the 26 S protease(13.Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar, 15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 16.Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar). One or more of these ATP-binding proteins may be responsible for the function of ATP in proteasome activation. In fact, purified PA700 expresses ATPase activity. Surprisingly, many of these “ATPase” subunits of PA700 have been identified independently as proteins involved in processes with no obvious relationships to proteasome function(17.Confalonieri F. Duguet M. BioEssays. 1995; 17: 639-650Crossref PubMed Scopus (313) Google Scholar). These findings may be explained by new and unexpected roles for the proteasome or may indicate that a given ATPase protein has multiple cellular functions. During the course of our continuing characterization of the function of PA700, we have identified a new protein complex that functions as a PA700-dependent activator of the proteasome. This report describes the identification, purification, and initial structural and functional characterization of this protein, which contains two members of the ATPase protein family. Furthermore, the same two proteins are identified here as new subunits of PA700, raising the number of family members in this complex to six. The 20 S proteasome and PA700 were purified from bovine red blood cells as described previously(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar, 18.McGuire M.J. DeMartino G.N. Biochem. Biophys. Res. Commun. 1989; 160: 911-916Crossref PubMed Scopus (33) Google Scholar). Proteasome activity was measured by the hydrolysis of the synthetic peptide succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin, also as described previously(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). The production of free 7-amino-4-methylcoumarin was monitored continuously at 380 nm (excitation) and 460 nm (emission), and initial steady-state rates were assessed. One unit of proteasome activity is defined as a change in 7-amino-4-methylcoumarin concentration of 1.0 nM/min under standard assay conditions. PA700 activity was assessed by measuring the proteasome activity after preincubation with pure PA700. The preincubation contained 45 mM Tris-HCl, pH 8.0, 5.6 mM dithiothreitol, 200 μM ATP, and 10 mM MgCl2 in a final volume of 50 μl and was carried out for 45 min at 37°C. This solution was then added to 1.0 ml of substrate solution for the measurement of proteasome activity as described above(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). The identification, purification, and characterization of the PA700-dependent activator (modulator) was carried out with a variation of the PA700 assay. The modulator sample to be tested was mixed with the purified proteasome and PA700 and then preincubated under the same conditions as the normal PA700 assay. Modulator activity is expressed as the increase in the PA700-dependent proteasome activity caused by the modulator. One unit of modulator activity is defined as an increase of 1 unit of PA700 activity. Bovine red blood cells were collected, washed, and lysed as described previously(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). Fraction II from the soluble lysate was prepared using DEAE-cellulose (DE52, Whatman). Fraction II was dialyzed against Buffer X (20 mM Tris-HCl, pH 7.6, 20 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 20% glycerol) for 16 h. After dialysis, solid ammonium sulfate was added to the sample to 38% saturation. The precipitated proteins were collected by centrifugation, resuspended in a large volume of Buffer X containing ammonium sulfate to 38% saturation, and collected again by centrifugation. The resulting pellet was dissolved in Buffer X containing 100 mM NaCl and dialyzed against 4000 ml of the same buffer for 8 h. After dialysis, any undissolved material was removed by centrifugation, and the soluble sample was chromatographed on a 5 × 100-cm column of Sephacryl S-300. The column was equilibrated and eluted with buffer consisting of 50 mM Tris-HCl, pH 7.6, 5 mM β-mercaptoethanol, 100 mM NaCl, and 20% glycerol. The column fractions were assayed for PA700 activity and for modulator activity as described above and in the legend to Fig. 1. The fractions containing PA700-dependent activator activity were pooled, dialyzed against column buffer containing 50 mM NaCl, and applied to a 2.5 × 5-cm column of DEAE-Fractogel. The bound proteins were eluted with a 500-ml linear gradient of NaCl (50-300 mM) in the same buffer. The fractions were assayed for PA700-dependent activator activity as described above and in the legend to Fig. 2. The fractions with this activity were pooled and dialyzed for 16 h against 20 mM potassium phosphate buffer, pH 7.6, 1 mM β-mercaptoethanol, and 20% glycerol and then applied to a 2.5-cm diameter column containing 6 g of hydroxylapatite equilibrated in the same buffer. The bound proteins were eluted with a 300-ml linear gradient of phosphate (20-100 mM). The fractions with peak activity were pooled, dialyzed against Buffer X containing 5% glycerol, and concentrated to 100-500 μg/ml protein. This material was stored at −70°C, where it was stable for at least 6 months.Figure 2:Ion-exchange chromatography of the PA700-dependent proteasome activator (modulator). Column fractions from the Sephacryl-S300 column (see Fig. 1) containing modulator activity (fractions 110-125) were pooled and subjected to ion-exchange chromatography on DEAE-Fractogel as described under “Materials and Methods.” Column fractions were assayed for PA700-dependent activator (modulator) activity. Samples (5 μl) of the column fractions were assayed in the presence of the purified exogenous proteasome (0.25 μg) and PA700 (0.67 μg, 8.8 units).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Complexes formed by the proteasome with PA700 and PA700/modulator were assessed by glycerol density gradient centrifugation. Linear gradients of 10-40% glycerol were prepared in 50 mM Tris-HCl, pH 7.6, 1 mM β-mercaptoethanol, 200 mM ATP, and 300 mM MgCl2 in a final volume of 4.55 ml. Samples in a volume of 200 μl were applied per tube and were centrifuged at 4°C for 16 h at 30,000 rpm in a Beckman SW 50.1 rotor. 24 samples of 200 μl were collected and subjected to assays as described in the text below. Separate tubes contained marker proteins including thyroglobulin and aldolase. Protein was determined by the method of Bradford (19.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211925) Google Scholar) using reagents purchased from Bio-Rad. Bovine serum albumin was used as a standard. Subunits of purified PA700 or the modulator were isolated by reverse-phase chromatography using an HPLC ( 1The abbreviations used are: HPLChigh performance liquid chromatographyPAGEpolyacrylamide gel electrophoresis.) system from Waters Chromatography (Milford, MA) equipped with a 6 × 150-mm Shodex RS Pak D4-613 column. Chromatography was conducted at a flow rate of 0.75 ml/min and a column temperature of 50°C. Two solvents were employed: solvent A containing 0.06% (v/v) trifluoroacetic acid in water and solvent B containing 0.05% trifluoroacetic acid in 70% acetonitrile, 30% water (v/v/v). The sample was initially injected onto a column equilibrated with a solution composed of 63% solvent A, 36% solvent B. After 10 min of isocratic flow under initial conditions, elution proceeded with a 130-min linear gradient from 36 to 70% solvent B. Eluant absorption was monitored at 214 nm. Peaks were collected manually, dried in a Savant Instruments Speed-Vac concentrator, redissolved in SDS sample buffer, and heated at 95°C for 4 min. Subunits in each pool were then further resolved by SDS-PAGE in 12.5% polyacrylamide gels as described previously(15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar). high performance liquid chromatography polyacrylamide gel electrophoresis. PA700 or modulator subunits, subjected to the two-dimensional isolation procedure described above, were electroblotted from SDS gels to Immobilon PSQ paper (Millipore Corp., Bedford, MA) by the method of Matsudaira(20.Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Blots were stained with Coomassie Blue R-250, and the bands were excised individually. Immobilized protein was digested in situ with sequencing-grade trypsin or Lys-C protease (Boehringer Mannheim). Released peptides were purified by reverse-phase chromatography using a Model 130A HPLC system from Applied Biosystems Division of Perkin-Elmer (Foster City, CA) with a 2.1 × 100-mm RP 300 column, also from Applied Biosystems. Separations were conducted at 50 μl/min in 0.1% (v/v) trifluoroacetic acid or 0.1% (w/v) ammonium acetate in water. Peptides were eluted with a 100-min linear gradient of 0-70% acetonitrile. NH2-terminal sequence analysis of Lys-C and of tryptic peptides was performed by automated Edman degradation with a Model 477A sequencer from Applied Biosystems using the manufacturer's standard programing and chemicals. Polyclonal antibodies against human TBP1 were prepared in rabbits against the protein expressed in Escherichia coli as described previously(6.Kominami K. DeMartino G.N. Moomaw C.R. Slaughter C.A. Shimbara N. Fujimuro M. Yokosawa H. Hisamatsu H. Tanahashi N. Shimizu Y. Tanaka K. Toh-e A. EMBO J. 1995; 14: 3105-3115Crossref PubMed Scopus (93) Google Scholar). Western blotting was conducted with an ECL Western blotting kit (Amersham Corp.) following the manufacturer's directions. As part of a search for proteins that regulate proteasome function, we have been screening cell extracts for proteins that can influence one or more activities of the purified 20 S proteasome. A number of such proteins have now been identified, and they include both activators and inhibitors. Because it seemed reasonable to assume that some proteasome regulatory proteins might act in concert or might themselves be regulated by other proteins, we also have designed assays to test the effects of cell extracts on the proteasome in the presence of previously identified regulators. In the experiments described here, we tested the ability of a red blood cell extract, fractionated by ammonium sulfate precipitation and gel filtration chromatography on Sephacryl S-300, to affect the proteolytic activity of the exogenous 20 S proteasome in the presence of purified PA700, an ATP-dependent proteasome activator(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). When column fractions were added to assays containing the purified proteasome and PA700, two peaks were identified that contained more activity (3-8-fold in various preparations) than that accounted for by the purified proteasome and PA700 alone (Fig. 1). One of these peaks was coincident with the elution position of PA700 endogenous to those fractions. Therefore, this peak probably resulted from the concentration-dependent increase in proteasome activity from increased PA700 in the assay. The second peak of enhanced proteasome activity was identified in fractions with an apparent Mr of ∼300,000. Control assays indicated that this peak did not result from endogenous proteasome activity, which was very low in these column fractions (<1.5 units/assay) and was not observed unless exogenous proteasome, PA700, and MgATP were present in the assays (see further characterization below). The fractions containing the PA700-dependent proteasome-activating activity were pooled and subjected to further purification by ion-exchange chromatography on DEAE-Fractogel as described under “Materials and Methods.” The PA700-dependent proteasome-activating activity bound to this resin and was eluted as a single peak at a position corresponding to ∼100 mM NaCl (Fig. 2). The fractions containing the peak activities were pooled and subjected to hydroxylapatite column chromatography. The PA700-dependent proteasome-activating activity eluted from this resin as a single peak (Fig. 3). SDS-PAGE analysis of the fractions from the hydroxylapatite column showed that three major proteins (denoted with arrows in Fig. 3) had elution profiles indistinguishable from one another and were coincident with PA700-dependent proteasome activation. These proteins had apparent Mr values of 50,000, 42,000, and 27,000. Retrospective analysis of the column fractions from the Fractogel ion-exchange chromatography by SDS-PAGE also showed that these three proteins coeluted with one another and with modulator activity (data not shown). The activity from the hydroxylapatite chromatography was subjected to a second Sephacryl S-300 chromatography step. The activity eluted at a position corresponding to its originally estimated Mr of 300,000 and was coincident with the three proteins described above (data not shown). Therefore, we conclude that red blood cell extracts contain a PA700-dependent proteasome activator composed of protein subunits with Mr values of 50,000, 42,000, and 27,000. The purified PA700-dependent proteasome activator (for simplicity, hereafter termed modulator) was characterized with respect to its effect on proteasome activity. In the absence of PA700, the modulator had no effect on proteasome activity (Fig. 4). However, the modulator stimulated PA700-dependent proteasome activity up to 8-fold. The magnitude of this effect was deceased at very high concentrations of PA700 (Fig. 4; see “Discussion”). We previously showed that PA700 activation of the proteasome required preincubation of both proteins in the presence of ATP(12.Chu-Ping M. Vu J.H. Proske R.J. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1994; 269: 3539-3547Abstract Full Text PDF PubMed Google Scholar). The modulator's effect on proteasome activity required the simultaneous preincubation of all three proteins with ATP. Preincubation of any individual protein or various combinations of any two proteins, followed by the addition of the other(s) immediately prior to the addition of the substrate for assay of proteasome activity, did not result in activated rates of PA700-dependent proteasome activity (data not shown). The modulator did not affect the rate of proteasome activation compared with that promoted by PA700 alone (data not shown). To determine possible mechanisms of action of the modulator, proteasome/PA700-containing complexes formed in the presence and absence of the modulator were isolated by glycerol density gradient centrifugation. As reported previously, the proteasome and PA700 form a complex that is activated with respect to proteasome and that is much larger than either individual protein. Preincubation of all three proteins resulted in a complex that was 2-4-fold more active and significantly larger than the complex formed after preincubation of the proteasome and PA700 alone (Fig. 5). To learn about the structural basis of modulator function, we subjected each of its subunit proteins to amino acid sequence analysis. The p50, p42, and p27 proteins were isolated by HPLC and digested with trypsin or Lys-C protease. The resulting peptides were isolated by HPLC, and selected peptides were subjected to automated Edman degradation as described under “Materials and Methods.” Sequences were obtained for 12 peptides of the p50 subunit; the sequences contained 211 amino acids. Comparison of these sequences with those in current data bases showed that they exactly matched the sequence of a previously described human protein, TBP1 (human immunodeficiency virus Tat-binding protein) (21.Nelbock P. Dillion P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Crossref PubMed Scopus (195) Google Scholar) (Fig. 6). TBP1 is a member of a large protein family that contains a consensus sequence for nucleotide binding (see below). Sequences were obtained for five peptides of the p42 subunit, containing 88 amino acids. Comparison of these sequences with proteins in current data bases indicated that p42 had significant sequence similarity to TBP1 (p50) as well as to other members of this protein family (Fig. 7 and data not shown). It is not possible from these partial data to determine whether p42 is the clear homolog of any one member of the family. Eight tryptic peptides of the p27 subunit of the modulator were isolated and sequenced. 90 amino acids were identified, and these sequences had no significant similarity to those of any protein listed in current data bases (data not shown).Figure 7:The p42 subunits of the modulator and PA700 are identical to one another and are homologous to p50 (TBP1). The p42 subunit of the modulator was isolated by HPLC and SDS-PAGE and subjected to amino acid sequencing as described under “Materials and Methods.” The sequences of five peptides produced by Lys-C digestion were obtained and are shown aligned with peptides from p50. A dash denotes an identical amino acid at a given position. The p42 subunit of PA700 (isolated as described under “Materials and Methods”) was subjected to amino acid sequencing, and three peptides (denoted by asterisks) had identical sequences to the corresponding peptides from p42 of the modulator.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Four members of a protein family containing a consensus sequence for ATP binding have been identified as components of PA700 or of the 26 S protease, a large proteasome-containing complex that contains PA700 as its major, if not its only, non-proteasome component. These proteins include S4(22.Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar), MSS1(23.Dubiel W. Ferrell K. Rechsteiner M. FEBS Lett. 1993; 323: 276-278Crossref PubMed Scopus (77) Google Scholar, 24.Shibuya H. Irie K. Ninomiya-Tsuji J. Goebll M. Taniguchi T. Matsumoto K. Nature. 1992; 357: 700-702Crossref PubMed Scopus (141) Google Scholar), TBP7(15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 25.Dubiel W. Ferrell K. Rechsteiner M. Biol. Chem. Hoppe-Seyler. 1994; 375: 237-240Crossref PubMed Scopus (37) Google Scholar), and p45(15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 26.Akiyama K. Yokota K. Kagawa S. Shimbara N. DeMartino G.N. Slaughter C.A. Noda C. Tanaka K. FEBS Lett. 1995; 363: 151-156Crossref PubMed Scopus (59) Google Scholar). Although TBP1, a member of this family originally identified as a human immunodeficiency virus Tat-binding protein(21.Nelbock P. Dillion P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Crossref PubMed Scopus (195) Google Scholar), has not been identified as a component of PA700 by direct sequencing, an antibody prepared against human TBP1 was shown to cross-react with a 50,000-Da subunit of PA700 from rat liver (6.Kominami K. DeMartino G.N. Moomaw C.R. Slaughter C.A. Shimbara N. Fujimuro M. Yokosawa H. Hisamatsu H. Tanahashi N. Shimizu Y. Tanaka K. Toh-e A. EMBO J. 1995; 14: 3105-3115Crossref PubMed Scopus (93) Google Scholar). We used this antibody to examine the bovine modulator and PA700 proteins. Fractions from the Sephacryl S-300 column on which PA700 and the modulator were first isolated contained a single immunoreactive band of 50,000 Da. This band was present in two peaks that where coincident with PA700 and the modulator, respectively (Fig. 1). Interestingly, more immunoreactive protein was present in the modulator peak than in the PA700 peak. The same immunoreactive band was observed in the purified PA700 and modulator proteins (Fig. 8). These results indicate that TBP1 (p50) is a subunit of each protein. To provide direct evidence for this conclusion, the PA700 subunit that reacted with the anti-TBP1 antibody was isolated by a two-dimensional procedure involving HPLC and SDS-PAGE(15.DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 199" @default.
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• W1980378067 title "Identification, Purification, and Characterization of a PA700-dependent Activator of the Proteasome" @default.
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