An Easily Dissociated 26 S Proteasome Catalyzes an Essential Ubiquitin-mediated Protein Degradation Pathway in Trypanosoma brucei
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m109029200
ISSN1083-351X
AutoresZiyin Li, Chun-Bin Zou, Yi Yao, Martin A. Hoyt, Stephen McDonough, Zachary B. Mackey, Philip Coffino, Ching C. Wang,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoThe 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the sixT. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. bruceicells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei. The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the sixT. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. bruceicells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei. succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amido Pro-Phe-Arg-MCA benzyloxycarbonyl-Gly-Gly-Arg-MCA dithiothreitol dimethyl 3,3′-dithiobispropionimide RNA interference double-stranded RNA reverse transcription 5-fluoroorotic acid adenosine 5′-O-(thiotriphosphate) The proteasome performs a central and ubiquitous biological function as follows: the degradation of intrinsically short lived regulatory proteins, such as those that control the cell cycle and transcription, as well as the disposal of potentially toxic denatured or misfolded proteins. Protein substrates are generally marked for degradation by their attachment to multiubiquitin chains catalyzed by a series of enzymes (1Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 2Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1600) Google Scholar). The ubiquitinated protein is then degraded by the 26 S proteasome in an ATP-dependent manner. The latter is a complex between a cylindrical 20 S proteasome and two 19 S regulatory complexes attached to each end of the cylinder (3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar). The 19 S complex consists of a characteristic set of some 17 heterogeneous protein subunits classified into two subgroups. One subgroup contains six structurally related ATPases, designated Rpt1 to 6 inSaccharomyces cerevisiae, which are encoded by a unique multigene family well conserved during evolution (3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar) and perform the presumed role of unfolding and translocating the proteins targeted for proteasome degradation. Another subgroup of some 11 non-ATPase subunit proteins, designated the Rpns in S. cerevisiae, are mostly structurally unrelated to one another (3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar). The 20 S proteasomes have been universally identified among the eukaryotes as well as some of the archaea and eubacteria (3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar). There has not been any 26 S proteasome identified in either Thermoplasma acidophilum, Rhodococcus erythropolis, or any other prokaryote. But an archaebacterial ATPase, homologous to the ATPases in eukaryotic 26 S proteasome, was recently identified inMethanococcus jannaschii and found to activate protein breakdown by bacterial 20 S proteasomes (4Ng P. Zwickl D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The eukaryotic 20 S proteasome has a similar structure as that of prokaryotes, and high resolution crystal structures have been reported for the 20 S proteasomes of T. acidophilum and S. cerevisiaeshowing remarkable structural similarities (5Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1379) Google Scholar, 6Groll M. Ditzel L. Löwe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1944) Google Scholar). Trypanosoma brucei is a parasitic protozoan and one of the causative agents of African trypanosomiasis. Recently, we have identified, purified, and characterized the 20 S proteasome from this organism (7To S.-B. Hua W.Y. Nguyen T.T. Wong M.L. Wang C.C. Mol. Biochem. Parasitol. 1996; 78: 33-46Crossref PubMed Scopus (63) Google Scholar), and we cloned full-length cDNAs encoding each of the seven α- and seven β-subunits of this complex. 1The GenBankTM database accession numbers of the DNA sequences encoding the 14 subunits of T. brucei 20 S proteasome are as follows: α1, AF198386; α2,AF148125; α3, AF198387; α4, AF169652; α5, AF140353; α6,AJ131148; α7, AF169651; β1, AJ131043; β2, AJ130820; β3,AF169653; β4, AF226673; β5, AF226674; β6, AF148124; and β7,AF290945. The trypanosome 20 S proteasome exhibits striking morphological similarities to the rat 20 S proteasome under electron microscopy (7To S.-B. Hua W.Y. Nguyen T.T. Wong M.L. Wang C.C. Mol. Biochem. Parasitol. 1996; 78: 33-46Crossref PubMed Scopus (63) Google Scholar). An activated form of the trypanosomal 20 S proteasome was identified and found to contain an additional protein of 26 kDa (PA26). Association with the PA26 heptamer ring confers enhanced peptidase activities on the trypanosomal 20 S proteasome (8To W.Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar, 9Yao Y. Huang L. Krutchinsky A. Wong M.L. Standing K.G. Burlingame A.L. Wang C.C. J. Biol. Chem. 1999; 274: 33921-33930Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A functionally similar but structurally diverged protein PA28 has been described previously in vertebrates (10Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar). Despite the many elements of similarity between the proteasomal systems of T. brucei and other eukaryotes, our efforts to identify the 26 S proteasome in T. brucei have been unsuccessful (8To W.Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar). There could be two causes of this failure as follows: either the 26 S proteasome in T. brucei is unstable and falls apart when cell lysates are processed by conventional means, or there may be no 26 S proteasome in T. brucei. The second possibility suggests a need for an alternative means of activating the 20 S proteasome in T. brucei. The PA26-activated 20 S proteasome in T. brucei, which has not yet been identified in any other eukaryotic microorganism includingS. cerevisiae, could fulfill such a need. However, the PA26-activated 20 S proteasome exhibits only peptidase activity, not protease activity. Furthermore, although poly-ubiquitin genes (11Wong S. Elgort M.G. Gottesdiener K. Campbell D.A. Mol. Biochem. Parasitol. 1992; 55: 187-195Crossref PubMed Scopus (10) Google Scholar) and ubiquitinated proteins are found in T. brucei (12Lowrie Jr., D.J. Giffin B.F. Ventullo R.M. Am. J. Trop. Med. Hyg. 1993; 49: 545-551Crossref PubMed Scopus (9) Google Scholar), these are not digested by the PA26-activated 20 S proteasome. 2Z. Li, C.-B. Zou, Y. Yao, M. A. Hoyt, S. McDonough, Z. B. Mackey, P. Coffino, and C. C. Wang, unpublished data. In this report, we demonstrate that a 26 S proteasome species is indeed present inT. brucei, but it dissociates into the 19 S complex and 20 S proteasome upon cell lysis. By using RNA interference (RNAi) inT. brucei (13Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), we also show that its function is essential for degradation of ubiquitinated proteins and cell viability. T. brucei strain 427 procyclic form cells were cultivated and harvested as described previously (7To S.-B. Hua W.Y. Nguyen T.T. Wong M.L. Wang C.C. Mol. Biochem. Parasitol. 1996; 78: 33-46Crossref PubMed Scopus (63) Google Scholar). The procyclic form T. brucei strain 29-13, which contains the genes expressing T7 RNA polymerase and tetracycline repressor (14Wirtz E. Leal S. Ochatt C. Cross G.A.M. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1115) Google Scholar), was a gift from Dr. Paul T. Englund of The Johns Hopkins University School of Medicine. The fluorogenic peptides LLVY-MCA,3 PFR-MCA, and GGR-MCA used in the peptidase assay, the cell-permeable bifunctional cross-linking reagent dimethyl 3,3′-dithiobispropionimidate (DTBP), rabbit anti-ubiquitin antiserum, [methyl-14C]casein, and apyrase were purchased from Sigma. Horseradish peroxidase-conjugated donkey antiserum against rabbit IgG and [35S]methionine were from Amersham Biosciences. Horseradish peroxidase-conjugated goat antiserum against mouse IgG and protease inhibitor mixtures were from Roche Molecular Biochemicals. Rabbit antisera against human Rpt1, Rpt2, Rpn8, and yeast Rpt2 were from Affinity Research Products. The S. cerevisiaestrain RJD 1171 in which Rpt1 was fused to a FLAG His6 tag was obtained from Verma et al. (15Verma R. Chen S. Feldman R. Schieltz D. Yates J. Dohmen J. Deshaies R.J. Mol. Biol. Cell. 2000; 11: 3425-3439Crossref PubMed Scopus (461) Google Scholar). Lactacystin was purchased from Professor E. J. Corey, Harvard University. All the other chemicals used in the current study were of the highest purity commercially available. A mixture of 20 S and activated 20 S proteasomes of T. brucei was purified from procyclic form cells by centrifugation of the crude cell lysate in a 15–50% glycerol gradient as previously described (8To W.Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar). For gel filtration, T. brucei lysate was fractionated using FPLC Superose 6 HR 10/30 chromatography (Amersham Biosciences) in a buffer containing 50 mm Tris-HCl, pH 7.0, 100 mm KCl, 10 mm NaCl, 1.1 mm MgCl2, 0.1 mm EDTA, and 10% glycerol. Further purification of 20 S proteasome was accomplished by an additional DEAE-cellulose column chromatography step (8To W.Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar). Isolation of 26 S proteasome from rat red blood cells was described previously (7To S.-B. Hua W.Y. Nguyen T.T. Wong M.L. Wang C.C. Mol. Biochem. Parasitol. 1996; 78: 33-46Crossref PubMed Scopus (63) Google Scholar). The FLAG His6-tagged 19 S proteasome regulatory complex fromS. cerevisiae was purified as described (15Verma R. Chen S. Feldman R. Schieltz D. Yates J. Dohmen J. Deshaies R.J. Mol. Biol. Cell. 2000; 11: 3425-3439Crossref PubMed Scopus (461) Google Scholar) using anti-FLAG M2 agarose beads from Sigma. For in vitrodegradation of [methyl-14C]casein (16Rock 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 (2197) Google Scholar), samples of purified 26 S proteasome from rat red blood cells (5 μg), rat red blood cell crude lysate (200 μg), a purified mixture ofT. brucei 20 S and activated 20 S proteasome (10 μg), and T. brucei crude lysate (200 μg), each containing a protease inhibitor mixture, were tested. Each sample was preincubated in a buffer containing 50 mm Tris-HCl, pH 7.25, 5 mm MgCl2, 1 mm dithiothreitol (DTT), 2 mm ATP (2 mm ATPγS or 2 units of apyrase), and 1% dimethyl sulfoxide (Me2SO) for 30 min on ice. [methyl-14C]Casein (3 μg) was then added to make a final volume of 20 μl. After incubation at 37 °C for 60 min, radioactivity in the trichloroacetic acid-soluble fraction was determined by scintillation counting. T. brucei procyclic form cells, suspended in methionine-depleted Cunningham's medium at a density of 5 × 107cells/ml, were pulse-labeled with [35S]methionine (50 μCi/ml, Amersham Biosciences) at 26 °C for 45 min. The radiolabeled methionine was then replaced by 1.3 mmunlabeled methionine to continue the incubation. Cell samples (4 × 107 cells) were taken periodically and lysed by sonication in 400 μl of TBS buffer (20 mm Tris-HCl, pH 7.9, 150 mm NaCl) including a protease inhibitor mixture. The lysate (containing 500 μg of protein) was first preabsorbed with preimmune rabbit serum and protein A-Sepharose (17Yeung S.J. Chen S.H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (170) Google Scholar). Ubiquitinated proteins were precipitated using anti-ubiquitin antiserum and protein A-Sepharose. The immunocomplexes were washed three times with phosphate-buffered saline and once with buffer A (30 mmTris-HCl, pH 7.5, 5 mm MgCl2, 2 mmDTT, 5 mm ATP, 1 mm phenylmethylsulfonyl fluoride, and 1 mm tosyl-lysine chloromethyl ketone). Immunoprecipitates were fractionated by 10% SDS-PAGE, autoradiographed, and immunostained using anti-ubiquitin antiserum. With DNA sequence information available from the data base of the TIGR Trypanosome Genome Project (www.tigr.org/tdb/mdb/tbdb/index.shtml), 14 cDNA fragments with their sequences closely similar to those in the six proteasome ATPases designated Rpt1 to 6 from other organisms were identified. Four cDNA fragments, 49F12.TR, 13G6.TR, 100G1.TR, and 49F12.TF, that appeared to code for an Rpt2 homologue were assembled by Dr. Colin D. Robertson of the University of Glasgow. By using reverse transcription-polymerase chain reactions (RT-PCR) with oligo(dT)30 (forward reaction) and the spliced leader (18Campbell D.A. Thornton D.A. Boothroyd J.C. Nature. 1984; 311: 350-355Crossref PubMed Scopus (109) Google Scholar) (TTAGAACAGTTTCTGTACTATATTG; reverse reaction) as primers, we were able to clone the full-length cDNA encoding an Rpt2 homologue (GenBankTM accession number AF227500). For the other five Rpt homologues, we designed five specific forward and five specific reverse primers (sequences available upon request) based on the 10 TIGR partial cDNA sequences (clones 43D4.TR, 49F12.TR, 13G6.TR, 100G1.TR, 49F12.TF, 24C21.TF, 17C3.TF, 38C11.TF, 10F8.TR, and 18H6.TR) and carried out RT-PCR with oligo(dT)30 and the spliced leader as the primers for synthesizing full-length cDNA. The five full-length cDNAs were each cloned and sequenced (GenBankTM accession numbers: Rpt1, AF227499; Rpt3,AF227501; Rpt4, AF227502; Rpt5, AF227503; Rpt6, AF227504). Pairwise alignments of the open reading frames in the six full-length cDNAs with those of the corresponding Rpts from human and S. cerevisiae were accomplished using the AlignX program in the vector NTI 5.5 program suite (InforMax Inc.). The results indicate sequence identities of 54–69% and similarities of 73–81% betweenT. brucei and human Rpts and 51–66% identities and 67–80% similarities between T. brucei and S. cerevisiae Rpts, respectively (Table I in Supplemental Data). The six full-length T. brucei Rpt cDNAs were each amplified by PCR and expressed inEscherichia coli M15 from the pQE30 vector (Qiagen), following the manufacturer's protocol. The recombinant Rpt proteins, expressed mostly as inclusion bodies in the transformed E. coli cell lysate, were each dissolved in 6 m guanidine HCl and purified through a Ni2+-agarose column (Qiagen) by the manufacturer's instructions. The purified recombinant T. brucei Rpt proteins (see the SDS-PAGE in Fig. I of Supplemental Data) were each used to produce rabbit antibodies (Animal Pharm Services, Inc., Healdsburg, CA). The coding regions of the six T. brucei Rpt full-length cDNAs were each amplified by PCR (primer sequences available upon request) and integrated into the yeast expression vector Dp22 by homologous recombination in yeast using the PCR-gap repair method (19Papa F.R. Amerik A.Y. Hochstresser M. Mol. Biol. Cell. 1999; 10: 741-756Crossref PubMed Scopus (106) Google Scholar). The Dp22 plasmid is a low copy (CEN/ARS), LEU2-marked vector into which a multiple cloning site has been inserted between the 5′ and 3′ regions of the S. cerevisiae RPT1 gene (20Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). PCR primers were designed to append 5′ or 3′ sequences of the S. cerevisiae RPT1 untranslated regions to the corresponding ends of theT. brucei coding regions. Following PCR-gap repair, the resulting expression vectors contained the T. brucei Rpt1 coding regions inserted between the two yeast RPT1untranslated regions and under the control of the yeastRPT1 promoter. Yeast manipulations were carried out using standard procedures (21Guthrie C. Fink G.R. Methods in Enzymology. 194. Acad. Press, San Diego1991Google Scholar), unless otherwise noted. Haploid yeast strains bearing HIS3-marked chromosomal insertion-deletions of individual RPT genes and containing the corresponding wild type gene on a low copy,URA3-marked plasmid were obtained from Daniel Finley, Harvard Medical School, and have been described previously (20Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). Yeast cells were transformed with individual T. brucei Rpt expression vectors by the lithium acetate method (22St-Gietz D. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar), and the transformants were selected on synthetic minimal (SM, Bio 101, Inc.) medium lacking histidine, leucine, and uracil. For selection on 5-fluoroorotic acid (FOA), the SM medium was supplemented with 0.1% FOA and 50 μg/ml uracil. The loss of URA3-marked plasmids following FOA selection was confirmed by the failure of the reverted strains to grow in medium lacking uracil. The [35S]methionine pulse-labeled T. brucei procyclic form cells, described previously, were suspended in 10 volumes of HEDS buffer (25 mm HEPES, pH 7.8, 1 mm EDTA, 0.25 msucrose, and 50 mm KOAc) plus 5 mm of the membrane-permeable chemical cross-linker DTBP and incubated at 26 °C for 30 min. The cells were then washed twice with the HEDS buffer and lysed in the lysis buffer (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% SDS, 1% Nonidet P-40 plus a protease inhibitor mixture) after 10 min at 4 °C. The lysate, preabsorbed with rabbit preimmune serum, was incubated with rabbit antiserum at room temperature for 1 h and precipitated with protein A-Sepharose. The precipitate was heated at 95 °C for 3 min, and the supernatant was boiled in an equal volume of SDS sample buffer plus 5 mm DTT to break the disulfide bond in DTBP prior to SDS-PAGE, which was followed by autoradiography. Partial cDNA fragments of the seven α- and seven β-subunits of 20 S proteasome, the six Rpt homologues, and PA26 were each amplified in RT-PCR using gene-specific primers with XhoI and HindIII linkers (primer sequences available upon request). The PCR fragments were each cloned into a pGEM-T easy vector and then subcloned into theXhoI/HindIII linearized pZJM vector flanked with tetracycline operators and T7 promoters (13Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The 5′ end cDNA segment (∼300–500 nucleotides) of each of the targeted mRNA was chosen for constructing the transfecting plasmid (see Table II in Supplemental Data) due to the highly diversified sequences in this particular region among the 20 cDNAs, thus achieving specific interference of gene expression. For the control plasmid construction, the linearized vector was blunt-ended with T4 DNA polymerase and self-ligated. The T. brucei procyclic form cells, expressing T7 RNA polymerase and tetracycline repressor, were grown in Cunningham's medium (23Cunningham I. J. Protozool. 1979; 24: 325-329Crossref Scopus (351) Google Scholar) supplemented with 10% fetal bovine serum plus 15 μg/ml G418 and 50 μg/ml hygromycin B for maintaining intracellular stability of the T7 RNA polymerase and tetracycline repressor DNA constructs, respectively. Transfection of T. brucei cells with DNA constructs by electroporation was essentially as described (13Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Briefly, 109 cells were harvested, washed once with cytomix buffer (24Van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Crossref PubMed Scopus (383) Google Scholar), and suspended in 0.5 ml of the same buffer containing 20 μg of thephleo-containing pZJM DNA construct linearized withNotI so that it can be integrated into the rDNA spacer region of the chromosome in T. brucei. Electroporation was carried out in a 2-mm cuvette using the Gene Pulser (Bio-Rad) with parameters set as follows: 1.6 kV voltage, 400 ohms resistance, and 25 microfarads capacitance. The cells were transferred to 10 ml of the antibiotics-supplemented Cunningham's medium immediately after electroporation and incubated at 26 °C for 24 h. The transfectants were then selected under 2.5 μg/ml phleomycin until stable cell lines were grown up after about 3 weeks of continuous incubation. The transfected cells were cloned by limiting dilution. To induce synthesis of the ∼300–500-bp dsRNA, the transfected cells were further incubated in the presence of 1.0 μg/ml tetracycline to induce the two tetracycline-inducible T7 promoters flanking the cDNA insert in the integrated plasmid construct. Cell number in the time sample was counted using a hemocytometer. The T. brucei expression vector pTSO-HYG4 (25Sommer J.M. Li S.-B. Hua F. Gottesdiener K.M. Wang C.C. Mol. Biochem. Parasitol. 1996; 76: 83-89Crossref PubMed Scopus (15) Google Scholar), which utilizes the poly(ADP-ribose) polymerase promoter and replicates extrachromosomally by virtue of a minicircle origin of replication, was used for expressing the yeast RPT2 gene in T. brucei. By site-directed mutagenesis, a BglII restriction site was introduced upstream of the hygromycin phosphotransferase gene (hyg) in the vector to produce the plasmid pTSO-HYG4m. A puromycin-N-acetyltransferase gene (pac) was PCR-amplified from the plasmid pγG-GFP (26Singer S.M. Yee J. Nash T.E. Mol. Biochem. Parasitol. 1998; 92: 59-69Crossref PubMed Scopus (111) Google Scholar) using primers with BglII and BseAI linkers. The PCR fragment was cloned into a pGEM-T easy vector and subcloned into the BglII/BseAI-linearized pTSO-HYG4m to replace the hyg gene with pac gene, thus generating the plasmid pTSO-PAC. The entire coding region of yeast RPT2 was then PCR-amplified from yeast genomic DNA using primers withSalI and BamHI linkers (primer sequences available upon request), cloned into a pGEM-T easy vector, and then inserted into the SalI/BamHI sites of pTSO-PAC. The resulting plasmid, designated pScRPT2-PAC, was used to transfect the T. brucei cells already harboring a pZJM-TbRPT2 RNAi construct. The transfectants, selected under 1.0 μg/ml puromycin, were grown in the presence of puromycin, phleomycin, hygromycin B, and G418 in culture medium. Synthesis of the dsRNA, encoding a portion of T. brucei Rpt2, was then induced in the transfectant by adding tetracycline (1.0 μg/ml) to the medium to disrupt the expression of T. brucei Rpt2 through RNAi. 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 in a 20× SSC (150 mm NaCl and 0.15 mm sodium citrate) solution. Northern hybridization was carried out overnight at 42 °C in 50% formamide, 6× SSC, 0.5% SDS, 1× Denhardt's solution with 0.1 mg/ml salmon sperm DNA. After stripping the probes, the same blots were re-hybridized with an α-tubulin gene fragment as a loading control. RT-PCR was performed using gene-specific primers and first strand cDNAs as templates. Instability of the 26 S proteasome could have frustrated prior efforts to purify it from T. brucei (8To W.Y. Wang C.C. FEBS Lett. 1997; 404: 253-262Crossref PubMed Scopus (35) Google Scholar). To test this possibility, we monitored 26 S proteasome-like activity in the crude lysate using [methyl-14C]casein as a substrate (16Rock 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 (2197) Google Scholar). An appreciable fraction of the radioactivity appeared in the trichloroacetic acid-soluble fraction (TableI). This apparent degradation of casein was inhibited when ATP was replaced with the inactive analogue ATPγS or when the lysate was pretreated to remove existing ATP. Addition of 5 μm lactacystin, a specific inhibitor of the 26 S proteasome (27Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1501) Google Scholar) and of the 20 S proteasome peptidase activity inT. brucei (28To M.C. Mutomba W.Y. Hyun W.C. Wang C.C. Mol. Biochem. Parasitol. 1997; 90: 491-504Crossref PubMed Scopus (69) Google Scholar), also blocked degradation (Table I). These characteristics are those expected of the 26 S proteasome. The activity is, however, less than one-third that in rat erythrocyte lysate on an equal protein weight basis (Table I) and suggests a relatively weak 26 S proteasome-like function in T. bruceilysate.Table IATP-dependent and lactacystin-sensitive proteolysis of [14C]casein by T. brucei lysate10% TCA1-aTCA, trichloroacetic acid.-soluble radioactivitycpmRat erythrocyte crude lysate2 mmATP7475.3 ± 180.42 mmATPγS107.5 ± 6.42 units apyrase87.3 ± 7.2Rat 26 S proteasome2 mm ATP7581.0 ± 479.52 mm ATPγS125.5 ± 10.52 units apyrase122.1 ± 6.7T. brucei crude lysate2 mm ATP2418.0 ± 184.02 mmATPγS576.0 ± 8.52 units apyrase384.8 ± 15.7T. brucei-activated 20 S proteasome2 mmATP381.5 ± 35.92 mm ATPγS170.6 ± 67.22 units apyrase145.3 ± 13.21-a TCA, trichloroacetic acid. Open table in a new tab We next examined the turnover of ubiquitinated proteins in intact T. brucei cells. T. bruceiprocyclic form cells were pulse-labeled with [35S]methionine and chased with unlabeled methionine, and the radiolabeled ubiquitinated proteins were immunoprecipitated and analyzed by SDS-PAGE via autoradiography or immunostaining using the anti-ubiquitin antiserum. The amount of [35S]methionine-labeled ubiquitinated proteins inT. brucei gradually decreased over a 30-h chase period (Fig.1A). The half-life of the ubiquitinated protein is about 10 h. Lactacystin treatment prevented the turnover for the duration of the chase period (Fig.1A). Thus, there is an apparent proteasome-mediated turnover of ubiquitinated protein in T. brucei, albeit at a relatively slow rate. When the
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