A 220-kDa Activator Complex of the 26 S Proteasome in Insects and Humans
1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês
10.1074/jbc.274.36.25691
ISSN1083-351X
AutoresRichard A. Hastings, Ignacio Eyheralde, Simon Dawson, Gail Walker, Stuart E. Reynolds, Michael A. Billett, R. John Mayer,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe S10b (SUG2) ATPase cDNA has been cloned by reverse transcription-polymerase chain reaction/rapid amplification of cDNA ends from mRNA of intersegmental muscles of the tobacco horn moth (Manduca sexta). The S10b ATPase is a component of the 26 S proteasome, and its concentration and that of its mRNA increase dramatically during development in a manner similar to other ATPases of the 19 S regulator of the 26 S proteasome. The S10b and S6′ (TBP1) ATPases are also present in a complex of ∼220 kDa in intersegmental muscles. The 220-kDa complex markedly activates (2–10-fold) the 26 S proteasome, even when bound to anti-S10b antibodies immobilized on Sepharose, and increases in concentration ∼5-fold like the 26 S proteasome in the intersegmental muscles in preparation for the programmed death of the muscle cells. A similar activator complex is present in human brain and placenta. Free activator complexes cross-activate: the Manduca complex activates rat skeletal muscle 26 S proteasomes, and the placental complex activates Manduca 26 S proteasomes. The placental activator complex contains S10b and S6′, but not p27. This 220-kDa activator complex has been evolutionarily conserved between species from insect to man and may have a fundamental role in proteasome regulation. The S10b (SUG2) ATPase cDNA has been cloned by reverse transcription-polymerase chain reaction/rapid amplification of cDNA ends from mRNA of intersegmental muscles of the tobacco horn moth (Manduca sexta). The S10b ATPase is a component of the 26 S proteasome, and its concentration and that of its mRNA increase dramatically during development in a manner similar to other ATPases of the 19 S regulator of the 26 S proteasome. The S10b and S6′ (TBP1) ATPases are also present in a complex of ∼220 kDa in intersegmental muscles. The 220-kDa complex markedly activates (2–10-fold) the 26 S proteasome, even when bound to anti-S10b antibodies immobilized on Sepharose, and increases in concentration ∼5-fold like the 26 S proteasome in the intersegmental muscles in preparation for the programmed death of the muscle cells. A similar activator complex is present in human brain and placenta. Free activator complexes cross-activate: the Manduca complex activates rat skeletal muscle 26 S proteasomes, and the placental complex activates Manduca 26 S proteasomes. The placental activator complex contains S10b and S6′, but not p27. This 220-kDa activator complex has been evolutionarily conserved between species from insect to man and may have a fundamental role in proteasome regulation. abdominal intersegmental muscle(s) polymerase chain reaction rapid amplification of cDNA ends Type II programmed neuromuscular cell death is a feature of some abdominal motor neurons and intersegmental muscles (ISM)1 at eclosion in the tobacco horn moth (Manduca sexta) (2Truman J.W. J. Comp. Neurol. 1983; 216: 445-452Crossref PubMed Scopus (103) Google Scholar, 3Schwartz L.M. Truman J.W. Dev. Biol. 1983; 99: 103-114Crossref PubMed Scopus (190) Google Scholar, 4Stocker R.F. Edwards J.S. Truman J.W. Cell Tissue Res. 1978; 191: 317-331Crossref PubMed Scopus (33) Google Scholar). After emergence, these cells die within 24–36 h in response to changes in circulating levels of ecdysteroid hormone. Previous studies have shown that eclosion is preceded in ISM by a massive increase in polyubiquitin gene expression (5Schwartz L.M. Myer A. Kosz L. Engelstein M. Maier C. Neuron. 1990; 5: 411-419Abstract Full Text PDF PubMed Scopus (185) Google Scholar) and a large increase in ubiquitinylated proteins (6Haas A.L. Baboshina O. Williams B. Schwartz L.M. J. Biol. Chem. 1995; 270: 9407-9421Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and in the levels of proteasomes (7Jones M.E.E. Haire M.F. Kloetzel P.-M. Mykles D.L. Schwartz L.M. Dev. Biol. 1995; 169: 436-447Crossref PubMed Scopus (83) Google Scholar, 8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In the muscles of larvae at stage 0, before the hormone-dependent changes in gene expression activate the death process, proteasomes appear to be depleted in some regulatory ATPases (e.g. S4 and S7). 2The ATPase subunits of the 19 S regulatory complex of the 26 S proteasome have been named according to Ref. 1Richmond C. Gorbea C. Rechsteiner M. J. Biol. Chem. 1997; 272: 13403-13411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar; they are S4 (mts2), S6 (TBP7/MS73), S6′ (TBP1), S7 (MSS1), S8 (SUG1), and S10b (SUG2). Marked increases in several ATPases (S4, S6, and S7) then occur, so that by stage 8, just before cell death, the 26 S proteasomes prepared from muscle contain at least four of the regulatory ATPases (S4, S6, S6′, and S7). The simplest explanation is that proteasomes are increased in number and equipped with the ATPases needed to degrade the accumulating multi-ubiquitinylated proteins during the programmed elimination of the muscles (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The first observations that the ubiquitin/26 S proteasome system is involved in programmed cell death in M. sexta (5Schwartz L.M. Myer A. Kosz L. Engelstein M. Maier C. Neuron. 1990; 5: 411-419Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 6Haas A.L. Baboshina O. Williams B. Schwartz L.M. J. Biol. Chem. 1995; 270: 9407-9421Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Jones M.E.E. Haire M.F. Kloetzel P.-M. Mykles D.L. Schwartz L.M. Dev. Biol. 1995; 169: 436-447Crossref PubMed Scopus (83) Google Scholar, 8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) have been followed by other data that implicate proteasomes in the decisions that favor either cell death or survival (9Grimm L.M. Goldberg A.L. Poirer G.G. Schwartz L.M. Osborne B.A. EMBO J. 1996; 15: 3835-3844Crossref PubMed Scopus (300) Google Scholar, 10Sadoul R. Fernandez P.-A. Quiquerez A.-L. Martinou I. Maki M. Schroter M. Becherer J.D. Irmler M. Tschopp J. Martinou J.-C. EMBO J. 1996; 15: 3845-3852Crossref PubMed Scopus (253) Google Scholar, 11Drexler H.C.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 855-860Crossref PubMed Scopus (444) Google Scholar, 12King R.W. Deshaies R.J. Peters J.-M. Kirscner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar, 13Cui H.L. Matsui K. Omura S. Schauer S.L. Matulka R.A. Sonenshein G.E. Ju S.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7515-7520Crossref PubMed Scopus (76) Google Scholar, 14Hirsch T. Dallaporta B. Zamzami N. Susin S.A. Ravagnan L. Marzo I. Brenner C. Kroemer G. J. Immunol. 1998; 161: 35-40PubMed Google Scholar, 15Ward C. Chilvers E.R. Lawson M.F. Pryde J.G. Fujihara S. Farrow S.N. Haslett C. Rossi A.G. J. Biol. Chem. 1999; 274: 4309-4318Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 16Lin K.I. Baraban J.M. Ratan R.R. Cell Death Differ. 1998; 5: 577-583Crossref PubMed Scopus (72) Google Scholar). There are six ATPase molecules found in the 19 S regulator of the 26 S proteasome (1Richmond C. Gorbea C. Rechsteiner M. J. Biol. Chem. 1997; 272: 13403-13411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). As an extension of studies on these ATPases in programmed cell death in ISM during eclosion in M. sexta (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), we have cloned a second S10b (SUG2) ATPase and shown that this ATPase is not only associated with 26 S proteasomes, but is also found in a much smaller complex, of ∼220 kDa, with the S6′ (TBP1) ATPase. The concentration of the 220-kDa complex increases in ISM during programmed cell death at the same time as that of the 26 S proteasome, suggesting a role for the 220-kDa complex in muscle cell death. A modulator complex containing the S10b and S6′ ATPases and a p27 protein has been previously described (17DeMartino G.N. Proske R.J. Moomaw C.R. Strong A.A. Song X. Hisamatsu H. Tanaka K. Slaughter C.A. J. Biol. Chem. 1996; 271: 3112-3118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Complexes similar to the Manduca 220-kDa complex are present in human brain and placenta and contain the S10b and S6′ ATPases, but not p27. The 220-kDa complexes can activate preparations of 26 S proteasomes across species barriers, indicating the evolutionary conservation of these proteasomal activators. Their potential roles in the proteolytic mechanisms of the 26 S proteasome and other possible functions of the S10b, S6′, and other ATPases in the cell are discussed. The insects were raised, the stages of pre-ecdysial development recognized, and muscles collected as described previously (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Degenerate primers matching the ATPase boxes A and B (18Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar) were used to clone several different ATPase sequences from Manduca genomic DNA. The two primers TAYGGNCCNCCNGGNACNGGNA (corresponding to the protein sequence YGPPGTG) and GGNGCRTCRATYTCRTCRATRAA (antisense strand for the protein sequence FIDEIDA) were used at a concentration of 0.5 μm in a 50-μl PCR containing 50 μm dNTPs, 200 ng of genomic DNA, and 2.5 units of Taq DNA polymerase (Roche Molecular Biochemicals, Lewes, United Kingdom). A 200-base pair product was cloned into the Eco RV site of the pSK− plasmid (Stratagene, Cambridge, UK) and manually sequenced by the dideoxynucleotide method. This sequence information was used to design oligodeoxynucleotides for subsequent reverse transcription-PCR. PCR/3′-RACE was used to obtain the 3′-end of a clone that showed good homology to other proteasomal ATPases, but appeared to represent a distinct gene (“AAA (ATPases associated with diverse cellular activities) clone”). RNA (1 μg) obtained from stage 7 Manduca ISM (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar) was reverse-transcribed (37 °C, 2 h) in the following 20-μl reaction: 100 units of SuperScript II reverse transcriptase (Life Technologies, Inc., Paisley, UK), 50 mm Tris-HCl (pH 8.3), 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol, 0.125 mm dNTPs, 20 units of RNase inhibitor (Amersham Pharmacia Biotech, St. Albans, UK), and 0.5 μg of T17 adapter primer (GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT). Next, 2 μl of the reaction was used in a 50-μl PCR as described above, but with 25 pmol of adapter primer (GACTCGAGTCGACATCGA) and 25 pmol of AAA clone-specific primer (AACTACGCGCGCGACCACCAG) (annealing temperature of 60 °C, 35 cycles). A 650-base pair product was cloned into pSK− as described above and sequenced. PCR/5′-RACE was used to obtain the remaining sequence of the AAA clone transcript. Poly(A)+ RNA was isolated from 50–100 μg of total RNA using the PolyATtract kit (Promega, Southampton, UK). Reverse transcription was performed as described above using 20 ng of poly(A)+ RNA and 0.5 μg of an oligo(dT) primer. Excess primers were removed with an S-400 microspin column (Amersham Pharmacia Biotech), and after concentration, the 3′-end of the cDNA was tailed (37 °C, 20 min) using 20 units of terminal deoxynucleotidyltransferase (Life Technologies, Inc.) and 100 pmol of dGTP. The tailing reaction was halted by addition of EDTA and NaCl. The tailed cDNA was pelleted using cetyltrimethylammonium bromide, washed with 70% ethanol, resuspended in 10 μl of 1 mNaCl, and then repelleted by centrifugation following addition of ethanol and 0.25 μg of glycogen. The pellet was resuspended in 20 μl of 10 mm Tris-HCl and 1 mm EDTA (pH 8.0), and PCR was performed as with 3′-RACE, but with the addition of 25 pmol of C17 adapter primer (GACTCGAGTCGACATCGACCCCCCCCCCCCCCCCC) and another AAA clone-specific primer (CAGACTATTATTATTATTACAATGC). Following the initial reaction, a second round of PCR was performed with 2 μl of the initial reaction product, the adapter primer, and a second AAA clone-specific primer (AAATCTATGAAGTAATTACAGAT). A 1350-base pair PCR product was ligated into the Eco RV site of the pSK− plasmid and sequenced. Finally, the full-length coding sequence of the Manduca homologue of S10b was amplified (annealing temperature of 55 °C, 35 cycles) with 2.5 units of the proofreading Pfu DNA polymerase (Stratagene), 500 μm dNTPs, and 25 pmol of primers ATGCCTGCCGGACCTTCC and AAATCTATGAAGTAATTACAGAT using reverse-transcribed RNA obtained from stage 7 ISM; a 1200-base pair product was cloned into the Eco RV site of pSK−and named pSK.S10b.cds. Frozen (−70 °C) ISM (500–1000 mg) taken from different developmental stages of M. sexta was homogenized in 4 volumes of homogenization buffer (20 mm Tris-HCl, 2 mm ATP, 10 mmMgCl2, 1 mm dithiothreitol, and 10% glycerol (pH 7.5)) using a Polytron homogenizer. The homogenate was centrifuged twice at 15,000 × g for 10 min at 4 °C, and the supernatant was collected (S2, soluble muscle extract). Human placental extract was prepared as described for ISM. Homogenized human brain was similarly processed except, that one centrifugation at 8000 ×g for 20 min was carried out. Supernatants (S2) were taken for gradient fractionation. Fraction II was prepared from human erythrocytes essentially as described (17DeMartino G.N. Proske R.J. Moomaw C.R. Strong A.A. Song X. Hisamatsu H. Tanaka K. Slaughter C.A. J. Biol. Chem. 1996; 271: 3112-3118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 20Chu-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). Proteins bound to DE52 were eluted with buffer containing 0.25 m NaCl and then 0.5m NaCl, pooled, and used directly for glycerol gradient centrifugation. Soluble ISM extract (5 mg of protein) was loaded onto 14-ml 10–40% (v/v) glycerol gradients containing the same Tris-HCl, ATP, and MgCl2 concentrations as in homogenization buffer. Samples were centrifuged at 24,000 rpm in an SW 6 × 16.5 rotor for 20–22 h (Rav = 70,000 × g) at 4 °C. Fractions of 0.5 ml were collected by displacement with Maxidens (Nycomed, Oslo, Norway). Human placental (9 mg), brain (9.6 mg), and erythrocyte (10 mg) soluble proteins were fractionated in the same way. Fractions were assayed for peptidase activity (insect muscle, 5 μl; and human tissue extracts, 10 μl) (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and used for Western analyses. ISM extract (from ∼200 μg of tissue) was filtered through a 0.2-μm filter. Soluble proteins (up to 200 μl) were applied to a Superose 12 column (Amersham Pharmacia Biotech) and eluted (flow rate of 400 μl/min) using homogenization buffer. Thirty-six fractions (200 μl) were collected (from 6.8- to 14-ml elution volume). For activation assays, glycerol gradient fractions containing activator complex (fractions 3–5) or Superose fractions (150–250 kDa) were incubated with 26 S proteasomes from the glycerol gradients (fractions 12–15) at 37 °C for 30 min before carrying out chymotrypsin assays. The Manduca S10b coding sequence was cut from pSK.S10b.cds, using Eco RI and Hin dIII restriction enzymes, and ligated into pRSET.C (Invitrogen, San Diego, CA). His-tagged S10b fusion protein was prepared by transforming the plasmid construct into Escherichia coli strain BL21(DE3) (Invitrogen) and inducing expression with isopropyl-β-d-thiogalactopyranoside (0.1 mm) for 3 h at 37 °C. Harvested cells were resuspended in 0.2 volumes of 6 m guanidine hydrochloride, 20 mm sodium phosphate, and 500 mm NaCl (pH 7.8) and sonicated. After removal of cell debris by centrifugation, the His-tagged ATPase was purified by chromatography on nickel-charged chelating Sepharose Fast Flow (Amersham Pharmacia Biotech) at room temperature (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Eluted protein was dialyzed against 10 mmTris-HCl (pH 8.0) and 0.1% Triton X-100 overnight at 4 °C, freeze-dried, checked for purity by SDS-polyacrylamide gel electrophoresis, and then used for immunization of New Zealand White rabbits. Antiserum to S10b ATPase was tested against the expressed fusion protein by Western analysis of whole cell extract from isopropyl-β-d-thiogalactopyranoside-treated E. coli BL21(DE3) cells transformed with pSK.S10b.cds. The anti-S6 antibody was raised against the recombinant Manduca protein expressed from pSMS73c (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Anti-S6′ and anti-S10b antibodies was raised against His-tagged human S6′ and S10b expressed from pET recombinant plasmids (Novagen, Abingdon, UK) as described above. Antisera to Manduca and human S10b and human S6′ were affinity-purified by binding to purified His-tagged recombinant antigen protein, coupling to CNBr-activated Sepharose (Sigma, Poole, UK), and eluting with 1 m glycine (pH 2.5). Antiserum to a control (irrelevant) peptide (AG93, a fragment of pro-islet amyloid precursor protein) was similarly affinity-purified on immobilized AG93 peptide. Monoclonal antibodies to human S10b, S6′, S7, and S8 ATPases and the 20 S subunits HC2/MCP20 and Z/MCP205 were kindly provided by Dr. Klavs Hendil (August Krogh Institute, University of Copenhagen). Polyclonal antiserum to human recombinant p27 was from Prof. Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, Japan). Western analyses were performed as described (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Affinity-purified Manduca anti-S10b and human anti-S10b (10 ml) and anti-AG93 (equivalent to 2.5 ml of antiserum) antibodies were coupled to CNBr-activated Sepharose 4B (1 ml). Activator complexes from glycerol gradient fractions 3–5 were bound to the matrices by inversion for 1–2 h at room temperature. After repeated washing with homogenization buffer, activator complexes were eluted from the immunoaffinity matrices for Western analyses with 1 m glycine (pH 2.5), and residual bound material was released by boiling in urea/SDS loading buffer (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). As part of the characterization of all proteasomal regulatory ATPases from Manduca, multiple DNA sequences encoding the region between the two elements of the Walker ATP-binding motifs were amplified and cloned. Sequencing of individual clones revealed five different sequences, some of which bore very close homology to previously identified proteasomal ATPases. One clone that bore less homology to known proteasomal ATPases was chosen for further study, and full-length cDNA sequences were obtained using PCR/RACE. The DNA and predicted protein sequences of the ATPase shown in Fig.1 are closely homologous to the 19 S regulatory S10b (SUG2) ATPase (1Richmond C. Gorbea C. Rechsteiner M. J. Biol. Chem. 1997; 272: 13403-13411Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 3The nucleotide sequence shown in Fig. 1 has been scanned against the GenBankTM/EBI Data Bank, and there are scores of related sequences, including human S10b (accession numberD78275), yeast S10b (accession number U43720), and squirrel CADp44 (accession number U36395). As is the case for many other members of this subgroup of the AAA superfamily, the S10b ATPase has a putative coiled-coil motif, the Walker A and B nucleotide-binding motifs, and putative DNA helicase motifs. The S10b ATPase mRNA and protein increase ∼5-fold in concentration in the muscles in preparation for cell death (data not shown). Again, these changes are similar to those observed for Manduca S4, S6, and S7 ATPases during the same period (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The S10b ATPase is a component of the 26 S proteasome as shown by glycerol gradient centrifugation (Fig.2); this analysis also demonstrates that the increase in the tissue content of the S10b ATPase is mainly associated with the 26 S proteasome.Figure 2Glycerol gradient analysis of 26 S proteasome catalytic activities and S10b ATPase content at developmental stages 0 and 7. Soluble protein (5 mg) from stage 0 (A) and stage 7 (B) ISM was fractionated by centrifugation through a 10–40% glycerol gradient. Protein and chymotrypsin activity were determined and immunoassay of M. sexta S10b ATPase in the fractions was carried out as described under “Experimental Procedures.” Western analysis results shown in the inset are from 2-min exposures of the transfers. The stage 7 Western analysis in B was exposed for 30 min (C). ■, protein concentration (mg/ml); ⋄, chymotrypsin activity (arbitrary units).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overexposure of the enhanced chemiluminescence signals in the Western analysis of the glycerol gradient fractionation of stage 7 ISM (Fig. 2 B) shows that the S10b ATPase is not only present in the 26 S proteasome, but is also present in fractions (3Schwartz L.M. Truman J.W. Dev. Biol. 1983; 99: 103-114Crossref PubMed Scopus (190) Google Scholar, 4Stocker R.F. Edwards J.S. Truman J.W. Cell Tissue Res. 1978; 191: 317-331Crossref PubMed Scopus (33) Google Scholar, 5Schwartz L.M. Myer A. Kosz L. Engelstein M. Maier C. Neuron. 1990; 5: 411-419Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 6Haas A.L. Baboshina O. Williams B. Schwartz L.M. J. Biol. Chem. 1995; 270: 9407-9421Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) containing lower molecular mass complexes (Fig. 2 C). The nature of these complexes was further investigated by gel filtration chromatography on Superose 12. Western analysis of the fractions after gel filtration shows that both the S10b and S6′ ATPases elute as if present in complexes of ∼220 kDa (Fig.3, A, B, and D). The relatively weak immunoreactivity of S6′ is probably due to the fact that the affinity-purified anti-S6′ antibody was raised against the human S6′ protein. The S6 ATPase is not enriched in complexes of this size, with the antigen detected in this region probably representing the trailing edge of proteasomal antigen spread by diffusion during chromatography (Fig. 3, E and F). The 220-kDa complex is found in muscles at the beginning (stage 0) and toward the end (stage 7) of the period of preparation for muscle cell death. The volumes of the stage 7 fractions analyzed by Western blotting were five times less than those of stage 0 since the total ATPase content of ISM increases ∼5-fold during pupal development. The similar intensities of the S10b bands in the 220-kDa complex from stages 0 and 7 (Figs. 2 and 3) therefore demonstrate that, like the proteasomal ATPases (8Dawson S.P. Arnold J.E. Mayer N.J. Reynolds S.E. Billett M.A. Gordon C. Colleaux L. Kloetzel P.M. Tanaka K. Mayer R.J. J. Biol. Chem. 1995; 270: 1850-1858Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), the concentration of the complexes containing the S10b ATPase increases ∼5-fold in the muscle cells during preparation for cell death. The amounts of the ATPases in the 220-kDa complex do not change when soluble extracts are prepared from muscles in the absence of ATP, indicating that the complex containing the S10b ATPase is unlikely to be produced by dissociation of 26 S proteasomes on homogenization (data not shown). The fact that the S6 ATPase is not in the 220-kDa complex (Fig. 3, E and F) also supports the notion that the complex is not produced by dissociation of ATPases from the 26 S proteasome. A careful analysis of the relative amounts of the S10b ATPase in the 26 S proteasome compared with the 220-kDa activator indicates there is ∼3–5% of the S10b ATPase in the activator relative to the 26 S proteasome (data not shown). Glycerol gradient fractions containing the 220-kDa complex from stage 7 (fractions 1–8) were incubated with similarly prepared 26 S proteasomes to investigate whether the 220-kDa complex was able to directly influence the peptidase activity of the proteasome (Fig. 4). Fractions enriched in the 220-kDa complex (fractions 3–6) cause considerable activation of the chymotrypsin activity of the 26 S proteasome in a concentration-dependent manner. The Manduca activator was further characterized to determine whether it is related to the modulator complex isolated from human erythrocytes, which stimulates assembly of 26 S proteasomes from 20 S cores and 19 S regulators (17DeMartino G.N. Proske R.J. Moomaw C.R. Strong A.A. Song X. Hisamatsu H. Tanaka K. Slaughter C.A. J. Biol. Chem. 1996; 271: 3112-3118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 22Adams G.M. Falke S. Goldberg A.L. Slaughter C.A. DeMartino G.N. Gogol E.P. J. Mol. Biol. 1997; 273: 646-657Crossref PubMed Scopus (59) Google Scholar, 23Adams G.M. Crotchett B. Slaughter C.A. DeMartino G.N. Gogol E.P. Biochemistry. 1998; 37: 12927-12932Crossref PubMed Scopus (45) Google Scholar). These experiments are summarized in TableI.Table IProperties of the Manduca 220-kDa activatorGlycerol gradient fractionsActivation345%Proteasomes Exp. A26 S160.1184.9127.020 S<0<0 10-fold (Fig.4) to ∼2-fold (Table I). This presumably reflects variation both in the activator complex itself and in the capacity of the 26 S proteasome to respond in different experiments. Activation capacity does not appear to depend on the proportions of 20 S and singly or doubly capped 26 S proteasomes in the preparation since the glycerol gradient fractions showing peak peptidase activity were activated by the 220-kDa complex to a greater extent than leading or trailing edges of the proteasome peak (Table I, Experiment B). If the activator was simply stimulating the ass
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