Functional Characterization of the 11 Non-ATPase Subunit Proteins in the Trypanosome 19 S Proteasomal Regulatory Complex
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207183200
ISSN1083-351X
Autores Tópico(s)Biochemical and Molecular Research
ResumoThe ubiquitin-proteasome pathway is responsible for selective degradation of short-lived and dysfunctional proteins in eukaryotes. The recently demonstrated presence of a functional 26 S proteasome in Trypanosoma brucei led to the identification and isolation of genes encoding all 11 non-ATPase (Rpn) subunit proteins in the trypanosome 19 S regulatory complex. Using the technique of RNA interference, expression of individual RPNgenes was disrupted in the procyclic form of T. brucei, resulting, in each case, in intracellular accumulation of polyubiquitinated protein, cell arrest at the G2/M phase, and eventual cell death. With the exception of Rpn10, depletion of individual Rpn proteins disrupted also trypanosome 19 S complex formation, with the complex virtually depleted in the cell lysate. This functional and structural essentiality of 10 of the 11 Rpn proteins in T. brucei differs significantly from that observed in other organisms. When Rpn10 was deficient in trypanosomes, a 19 S complex without Rpn10 was still formed, whereas cell growth was arrested. This structural dispensability but functional indispensability of Rpn10 may constitute another unique aspect of the proteasomes in T. brucei. The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived and dysfunctional proteins in eukaryotes. The recently demonstrated presence of a functional 26 S proteasome in Trypanosoma brucei led to the identification and isolation of genes encoding all 11 non-ATPase (Rpn) subunit proteins in the trypanosome 19 S regulatory complex. Using the technique of RNA interference, expression of individual RPNgenes was disrupted in the procyclic form of T. brucei, resulting, in each case, in intracellular accumulation of polyubiquitinated protein, cell arrest at the G2/M phase, and eventual cell death. With the exception of Rpn10, depletion of individual Rpn proteins disrupted also trypanosome 19 S complex formation, with the complex virtually depleted in the cell lysate. This functional and structural essentiality of 10 of the 11 Rpn proteins in T. brucei differs significantly from that observed in other organisms. When Rpn10 was deficient in trypanosomes, a 19 S complex without Rpn10 was still formed, whereas cell growth was arrested. This structural dispensability but functional indispensability of Rpn10 may constitute another unique aspect of the proteasomes in T. brucei. RNA interference rapid amplification of cDNA ends ubiquitin-interacting motif The ubiquitin-proteasome pathway is involved in eliminating short-lived and misfolded proteins in the cytosol and nucleus of eukaryotic cells. Proteasomal substrates are generally marked for destruction through covalent attachment of polyubiquitin chains catalyzed by a series of enzymes, the ubiquitin-activating enzyme, the ubiquitin-conjugating enzyme, and the ubiquitin ligase. The ubiquitinated protein is then degraded by the 26 S proteasome in an ATP-dependent manner (1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3375) Google Scholar, 2Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1601) Google Scholar). The 26 S proteasome is composed of the 20 S catalytic complex capped at one or both ends by the 19 S regulatory complex, which binds, unfolds, and translocates polyubiquitinated protein into the interior of the 20 S complex, where proteolysis occurs. The 20 S complex of eukaryotes is a barrel-shaped ring structure with seven distinct α-subunits forming the two outer α-rings and seven distinct β-subunits forming the two inner catalytic β-rings. The 19 S complex in Saccharomyces cerevisiae is composed of six distinct ATPase (Rpt (regulatory particle triple-A ATPase)) subunits and at least 11 non-ATPase (Rpn (regulatory particle non-ATPase)) subunits (3Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell. Biol. 1998; 18: 3149-3162Crossref PubMed Google Scholar). Unlike the 20 S complex, whose structure and catalytic properties have been well understood, much less is known about the structural and functional organization of the 19 S complex. Nevertheless, the relative arrangement of several 19 S subunits has been deduced through assaying the protein-protein interactions (4Ferrell K. Wilkinson C.R.M. Dubiel W. Gordon C. Trends Biochem. Sci. 2000; 25: 83-88Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 5Fu H. Reis N. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; 20: 7096-7107Crossref PubMed Scopus (212) Google Scholar). The six Rpt subunits are present in a hexamer ring structure lying on top of the α-ring and interacting with Rpn1, Rpn2, and Rpn10 to form the "base" subcomplex in S. cerevisiae, whereas the rest of the eight Rpn subunits form a "lid" subcomplex in the 19 S complex (6Glickman M.H. Rubin D.M. Coux O. Wefes I. Pfeifer G. Cjeka Z. Baumeister W. Fried V.A. Finley D. Cell. 1998; 94: 615-623Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). Arrangement of the eight Rpn subunits in the lid was tentatively mapped by a comparative alignment with the positions of their homologs in the COP9 signalosome (7Kapelari B. Bech-Otschir D. Hegerl R. Schade R. Dumdey R. Dubiel W. J. Mol. Biol. 2000; 300: 1169-1178Crossref PubMed Scopus (96) Google Scholar, 8Bech-Otschir D. Seeger M. Dubiel W. J. Cell Sci. 2002; 115: 467-473Crossref PubMed Google Scholar). A drafted structural map of the yeast 19 S complex was recently deduced from combined data on genetic interactions and protein-protein interactions among the subunits (5Fu H. Reis N. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; 20: 7096-7107Crossref PubMed Scopus (212) Google Scholar), although the process of assembly remains unknown. Specific functions have been assigned to only a few subunits in the yeast 19 S complex (2Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1601) Google Scholar). By gene deletion analysis in S. cerevisiae (9Burns N. Grimwade B. Ross-MacDonald P.B. Choi E.Y. Finberg K. Roeder G.S. Snyder M. Genes Dev. 1994; 8: 1087-1105Crossref PubMed Scopus (463) Google Scholar), most 19 S subunit genes were found to be essential for the yeast, with only two exceptions, RPN9 andRPN10. The Δrpn9 cells grow normally at the permissive temperature, but display a strong growth defect at the nonpermissive temperature, and are arrested at the G2/M phase of the cell cycle (3Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell. Biol. 1998; 18: 3149-3162Crossref PubMed Google Scholar, 10Takeuchi J. Fujimuro M. Yokosawa H. Tanaka K. Toh-e A. Mol. Cell. Biol. 1999; 19: 6575-6584Crossref PubMed Scopus (60) Google Scholar, 11Takeuchi J. Toh-e A. Biochimie (Paris). 2001; 83: 333-340Crossref PubMed Scopus (14) Google Scholar). The normally growing Δrpn10/Mcb1 mutant exhibits, however, a modest sensitivity to amino acid analogs and accumulates ubiquitin-protein conjugates, suggesting that this polyubiquitin-binding protein (Rpn10) may interact with only a subset of proteasomal substrates (12van Nocker S. Sadis S. Rubin R.M. Glickman M., Fu, H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (356) Google Scholar). Conditional yeast mutants of several other subunits of the 19 S complex display distinct cell cycle defects (13Ghislain M. Udvardy A. Mann C. Nature. 1993; 366: 358-361Crossref PubMed Scopus (372) Google Scholar, 14Bailly E. Reed S.I. Mol. Cell. Biol. 1999; 19: 6872-6890Crossref PubMed Scopus (39) Google Scholar, 15Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar, 16Kominami 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, 17Rinaldi T. Ricci C. Porro D. Bolotin-Fukuhara M. Frontali L. Mol. Biol. Cell. 1998; 9: 2917-2931Crossref PubMed Scopus (66) Google Scholar). Therpt1/Cim5-1 mutant as well as therpt6/Cim3-1 and rpn3-4 mutants are arrested at the G2/M phase with increased Cdc28 kinase activity (13Ghislain M. Udvardy A. Mann C. Nature. 1993; 366: 358-361Crossref PubMed Scopus (372) Google Scholar, 14Bailly E. Reed S.I. Mol. Cell. Biol. 1999; 19: 6872-6890Crossref PubMed Scopus (39) Google Scholar), whereas mutation in the ATP-binding site of Rpt1 results in a G1 delay (15Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). Therpn12-1/nin1-1 mutant is arrested at both the G1/S and G2/M boundaries with reduced Cdc28 kinase activity (16Kominami 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), whereas the rpn11/Mpr1-1mutant of S. cerevisiae displays a G2/M delay and altered mitochondrial morphology (17Rinaldi T. Ricci C. Porro D. Bolotin-Fukuhara M. Frontali L. Mol. Biol. Cell. 1998; 9: 2917-2931Crossref PubMed Scopus (66) Google Scholar). TheSchizosaccharomyces pombe Rpn11 homolog Pad1 was found to positively regulate Pap1-dependent transcription and was implicated in the maintenance of chromosome structure (18Shimanuki M. Saka Y. Yanagida M. Toda T. J. Cell Sci. 1995; 108: 569-579Crossref PubMed Google Scholar). Some of the Rpn proteins may thus have functions unrelated to the function of the 26 S proteasome, and the complete functional profile of the 19 S complex remains unclear. Trypanosoma brucei, a parasitic protozoan and a causative agent of African sleeping sickness, is generally regarded as a relatively primitive eukaryote farther removed from mammals than yeast (19Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (591) Google Scholar). Gene expression in this organism depends primarily on post-transcriptional regulation (20Berberof M. Vanhamme L. Tebabi P. Pays A. Jefferies D. Welburn S. Pays E. EMBO J. 1995; 14: 2925-2934Crossref PubMed Scopus (97) Google Scholar). Functions of the proteasomes inT. brucei differ significantly from those of other eukaryotic microorganisms (21Hua S.B., To, W.-Y. Nguyen T. Wong M.L. Wang C.C. Mol. Biochem. Parasitol. 1996; 78: 33-46Crossref PubMed Scopus (62) Google Scholar, 22To W.-Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar). For instance, trypanosomes have the activated 20 S proteasomal complex as the dominant proteasomal species (22To W.-Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar), which is absent in all other eukaryotic microbes investigated thus far, but finds a counterpart in mammals, playing an essential role in major histocompatibility complex class I antigen presentation (23Preckel T. Fung-Leung W.-P. Cai Z. Vitiello A. Salter-Cid C. Winqvist O. Wolfe T.G. Von Herrath M. Angulo A. Ghazal P. Lee J.D. Fourie A.M., Wu, Y. Pang J. Ngo K. Peterson P.A. Früh K. Yang Y. Science. 1999; 286: 2162-2165Crossref PubMed Scopus (153) Google Scholar). Unlike mammals, however, there are apparently only seven α- and seven β-subunits in its 20 S complex reservoir, without detectable heterogeneous isomeric forms (24Huang L. Jacob R.J. Pegg S.C.-H. Baldwin M.A. Wang C.C. Burlingame A.L. Babbitt P.C. J. Biol. Chem. 2001; 276: 28327-28339Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). There are only two catalytically active β-subunits (β2 and β5) in the trypanosome 20 S proteasome. 1C. C. Wang, unpublished data. The recently identified 26 S proteasome from trypanosomes consists of stable 20 S and 19 S complexes, but they are readily dissociated from each other upon cell lysis (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The six Rpt proteins in the 19 S complex are highly conserved and capable of complementing the corresponding yeast mutants, with the only exception of Rpt2 (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The down-regulated expression of each of the seven α-, seven β-, and six Rpt subunits in trypanosomes by RNA interference (RNAi)2 indicated that each protein plays an essential function in the growth and viability of this organism. Apparently, the depletion of each protein cannot be replaced by any other protein in the cell (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), suggesting a rather simple family of genes encoding the proteasomal complex without the isogenes, whose protein products may exchange with the existing subunits in mammalian proteasomes, resulting in a heterogeneous population (2Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1601) Google Scholar, 26Monaco J.J. Nandi D. Annu. Rev. Genet. 1995; 29: 729-754Crossref PubMed Scopus (107) Google Scholar,27Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (874) Google Scholar). The trypanosome proteasome exists in a largely homogeneous population (24Huang L. Jacob R.J. Pegg S.C.-H. Baldwin M.A. Wang C.C. Burlingame A.L. Babbitt P.C. J. Biol. Chem. 2001; 276: 28327-28339Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), which is also reflected by results from RNAi (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It has the potential of being a simple model like the one from yeast for further in-depth investigation. In this study, we have identified and isolated the 11 full-length Rpn subunit genes from T. brucei and disrupted their expression individually to determine the effects on cell cycle progression, cell viability, and assembly of the 19 S complex. In contrast to observations obtained with yeast, each Rpn protein in T. brucei was found to be essential for cell viability. With the only exception of Rpn10, depletion of each of the other 10 Rpn proteins in trypanosomes disrupted also formation of the 19 S complex. The procyclic form of T. brucei strain 29-13, which contains the genes expressing T7 RNA polymerase and the tetracycline repressor (28Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1126) Google Scholar), was a gift from Dr. Paul T. Englund (Johns Hopkins University School of Medicine). Fetal bovine serum was purchased from Atlanta Biologicals, Inc. Mouse monoclonal antibodies against α-tubulin and ubiquitin were purchased from Sigma and Zymed Laboratories Inc., respectively. Horseradish peroxidase-conjugated donkey antiserum against rabbit IgG, horseradish peroxidase-conjugated goat antiserum against mouse IgG, [35S]methionine, and protein A-Sepharose CL-4B were fromAmersham Biosciences. Protease inhibitor mixtures were purchased from Roche Molecular Biochemicals. All other chemicals used in this study were of the highest purity commercially available. Procyclic T. brucei cells were cultivated at 26 °C in Cunningham's medium supplemented with 10% fetal bovine serum. G418 (15 μg/ml) and hygromycin B (50 μg/ml) were added to the culture medium to maintain the T7 RNA polymerase and tetracycline repressor gene constructs within the cells. Partial genomic sequences of the 11 T. brucei Rpn homologs were identified in the TIGR Trypanosome Genome Project Sequence Databases 3Available at www.tigr.org/tdb/mdb/tbdb/index.shtml. by the BLAST search program using the yeast RPN genes as queries. Based on the partial sequence information, gene-specific primers 4Sequence information is available upon request. were designed and employed in reverse transcription-PCRs for rapid amplification of cDNA ends (RACE). First-strand cDNAs were generated from the total RNA of T. brucei with an oligo(dT)15-adaptor primer and Moloney murine leukemia virus reverse transcriptase. 5′-RACE was performed using a gene-specific antisense primer in combination with the splice leader sequence (TTAGAACAGTTTCTGTACTATATTG) known as the 5′-end of all mRNAs in T. brucei (29Campbell D.A. Thornton D.A. Boothroyd J.C. Nature. 1984; 311: 350-355Crossref PubMed Scopus (109) Google Scholar). 3′-RACE was carried out using a gene-specific sense primer in combination with the adaptor primer, which was introduced into cDNA during reverse transcription. The PCR fragments thus synthesized were cloned into the pGEM-T-easy vector (Promega) for sequencing. A pair of specific primers were then designed based on the sequence data and used to amplify the full-length cDNA, which was then sequenced for complete open reading frame identification. The 11 full-length T. brucei Rpn cDNAs were each amplified by PCR, and the encoded recombinant proteins were expressed with an added N-terminal His6 tag in Escherichia coli strain M15 cells using the pQE30 vector (QIAGEN Inc.) following the manufacturer's protocol. The recombinant Rpn proteins, expressed mostly as inclusion bodies in the transformedE. coli cell lysate, were each dissolved in 8 murea and purified through a Ni2+-agarose column (QIAGEN Inc.) following the manufacturer's instructions. The purified proteins, with their purity verified by SDS-PAGE (data not shown), were used to produce polyclonal antibodies in rabbit (Animal Pharm Services Inc., Healdsburg, CA). Only the antibodies against T. brucei Rpn10 and Rpn11 have now become available and were used in this investigation. Partial cDNA fragments (300∼500 bp in length) of the 11 T. brucei RPN genes were each amplified by PCR using gene-specific primers with XhoI andHindIII linkers4 and subcloned into the pZJM vector (30Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar) by replacing the α-tubulin stuffer. For the control plasmid, the pZJM vector was digested withXhoI/HindIII, end-blunted with T4 DNA polymerase, and self-ligated. The construct was linearized with NotI so that it could be integrated into the rDNA spacer region of the T. brucei chromosome. Transfection of T. brucei by electroporation was essentially performed according to our previous procedures (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The transfectants were selected with 2.5 μg/ml phleomycin and cloned by limiting dilution. For induction of RNAi, stable transfectants were cultured in the presence of 1.0 μg/ml tetracycline. Cell numbers were counted at different timed intervals using a hemocytometer. Total RNA was extracted from T. brucei cells using the TRIzol reagent (Amersham Biosciences). Thirty μg of total RNA was denatured, separated on 1.2% formaldehyde-agarose gel, and blotted onto nitrocellulose membranes. Northern hybridization was carried out overnight at 42 °C in 50% formamide, 6× SSC, 0.5% SDS, 1× Denhardt's solution, and 0.1 mg/ml salmon sperm DNA. After stripping the probes, the same blots were rehybridized with an α-tubulin gene fragment to ensure equal RNA sample loading. For immunoblotting, 12.5 or 8.5% acrylamide gels following SDS-PAGE were blotted and stained as previously described (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For immunoprecipitation, T. brucei cells were incubated in methionine-free medium for 1 h and labeled with [35S]methionine (50 μCi/ml) at 26 °C for 4 h. The labeled cells were washed twice with Tris-buffered saline (25 mm Tris-Cl, pH 7.6, and 100 mm NaCl) and incubated in lysis buffer (25 mmTris-Cl, pH 7.6, 100 mm NaCl, 1% Nonidet P-40, 1 mm dithiothreitol, and protease inhibitors) for 30 min on ice. The cleared lysate, pre-absorbed with rabbit preimmune serum and protein A-Sepharose beads, was incubated with rabbit antiserum against T. brucei Rpt3 (25Li Z. Zou C.-B. Yao Y. Hoyt M.A. McDonough S. Mackey Z.B. Coffino P. Wang C.C. J. Biol. Chem. 2002; 277: 15486-15498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) at 4 °C for 1 h and precipitated with protein A-Sepharose beads. The immunoprecipitates thus collected were fractionated by SDS-PAGE, and the dried gels were autoradiographed. T. brucei cells were lysed by sonication in buffer A (10 mm Tris-Cl, pH 7.4, 25 mm KCl, 10 mm NaCl, 1 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 2 mmATP, 1 mm N α-p-tosyl-l-lysine chloromethyl ketone, and 1 mm phenylmethylsulfonyl fluoride) containing 20% glycerol, and the lysate was cleared by centrifugation at 80,000 × g for 60 min. The proteasomal complexes, collected by an additional centrifugation at 100,000 × g for 60 min, were resuspended in buffer A containing 5% glycerol, with insoluble materials removed by centrifugation at 80,000 × g for 30 min. The cleared supernatant was overlaid on top of a stepwise gradient of 15∼50% glycerol and centrifuged in a Beckman TLS55 rotor at 35,000 rpm for 16 h. Fractions were collected dropwise from the bottom of the tube, separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and immunostained with the appropriate antibodies. The fluorescence-activated cell sorting analysis of propidium iodide-stained trypanosome cells was carried out as described previously (31Mutomba M.C., To, W.-Y. Hyun W.C. Wang C.C. Mol. Biochem. Parasitol. 1997; 90: 491-504Crossref PubMed Scopus (69) Google Scholar) with minor modifications. Briefly, T. bruceicells were washed twice with phosphate-buffered saline and fixed in ethanol at 4 °C for 1 h. The cells were washed twice and suspended in phosphate-buffered saline. DNase-free RNase (10 μg/ml) and propidium iodide (20 μg/ml) were added to the suspension and incubated for 30 min at room temperature. The DNA content of the propidium iodide-stained cells was analyzed with a FACScan analytical flow cytometer (BD Biosciences). The percentage of cells in the G1, S, and G2 phases of the cell cycle was determined using ModFitLT Version 3.0 Software (BD Biosciences). The partial nucleotide sequences encoding 11 distinct T. brucei Rpn homologs were identified in the TIGR Trypanosome Genome Project Sequence Databases by the BLAST program using the 11 yeastRPN gene sequences as queries. Based on these partial sequences, Rpn-specific primers were designed and employed in 5′- and 3′-RACE reactions for the remainder of the 5′- and 3′-sequences in each of the 11 RPN genes. A pair of primers encompassing the entire coding region were designed and used to amplify the full-length cDNA of each of the 11 Rpn homologs, resulting in the cloning and sequencing of each of the 11 full-length Rpn cDNAs. The deduced amino acid sequences from these cDNA clones were compared with those of the yeast counterparts and revealed sequence identities ranging from 46% between the Rpn11 proteins to 20% between the Rpn9 proteins (Table I). The yeast Rpn4 protein, a putative transcription factor only loosely associated with the yeast 26 S proteasome (32Fujimuro M. Tanaka K. Yokosawa H. Toh-e A. FEBS Lett. 1998; 423: 149-154Crossref PubMed Scopus (59) Google Scholar, 33Mannhaupt G. Schnall R. Karpov V. Vetter I. Feldmann H. FEBS Lett. 1999; 450: 27-34Crossref PubMed Scopus (269) Google Scholar, 34Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3056-3061Crossref PubMed Scopus (350) Google Scholar), was postulated to be a protein uninvolved in the yeast 19 S complex. We found no apparent homolog in the T. brucei genome sequence data bases. The 11RPN genes we have identified in T. brucei thus far may represent the complete profile of Rpn subunits in the T. brucei 19 S complex mimicking that observed in the yeast 19 S complex (3Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell. Biol. 1998; 18: 3149-3162Crossref PubMed Google Scholar).Table IThe non-ATPase subunits of the T. brucei 19 S proteasomal regulatory complexNon-ATPase subunitsT. bruceiIdentity (similarity)S. cerevisiaeaaaAmino acid.Molecular massDomainbThe domains were determined by sequence homology to yeast Rpn homologs. LRR, leucine-rich repeat.aaMolecular massDomainRefs.kDakDaRpn191199.9LRR32 (50)993109.4LRR2Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1601) Google Scholar,35Lupas A. Baumeister W. Hofmann K. Trends Biochem. Sci. 1997; 22: 195-196Abstract Full Text PDF PubMed Scopus (70) Google ScholarRpn2977106.6LRR31 (51)945104.3LRR2Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1601) Google Scholar,35Lupas A. Baumeister W. Hofmann K. Trends Biochem. Sci. 1997; 22: 195-196Abstract Full Text PDF PubMed Scopus (70) Google ScholarRpn333438.2PCI31 (42)52360.4PCI2Voges D. Zwickl P. Baumeister W. Annu. 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