Identification of the 19S regulatory particle subunits from the rice 26S proteasome Tadashi Shibahara, Hiroshi Kawasaki and Hisashi Hirano Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Japan The 2 6S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes. In yeast, the RP contains s ix d ifferent A TPase s ubunits and, at least, 11 non-ATPase subunits. In t his study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library. The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt 4, OsRpt5 and OsRpt6, and five non-ATPase subunits , OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non-ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined. Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsR pt4 and OsRpt5, and three non- ATPase subunits, OsRpn1, OsRpn2 and OsRpn9,were found to be encoded by duplicated genes. The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S protea- some, and the subunit composition was determined. T he homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, inclu- ding the both products of duplicated genes by using elec- trospray ionization quadrupole time-of-flight mass spectrometry. Post-translational modifications and pro- cessing in rice RP subunits wer e also identified. Various types of RP complex with d ifferent subunit c ompositions are present in rice cells, suggesting t he multiple functions of rice proteasome. Keywords: proteasomes; rice; gene duplication ; purification; immunoaffinity ch romatography. All organisms possess highly selective proteolytic systems which are essential for cellular functions such as cell cycle progression and apoptosis and a lso r emove abnormal and unnecessary intracellular proteins. A major proteolytic system of them in both cytoplasm a nd nucleus of eukaryote is ubiquitin-proteasome pathway that involves the covalent attachment of polyubiquitins to substrate targeted for degradation. The ubiquitinated proteins are degraded by the 26S proteasome which is a multicatalytic protease complex. The 26S proteasome is composed of two major complexes, a 20S proteasome (700 kDa) and a pair of 1 9S regulatory particles (RP; 700 kDa). The 20S proteasome has a cylindrical structure, consisting of a and b rings stacked in the order of abba, and each ring contains seven structurally related a and b subunits, respectively. On the other hand, RP consists of six different ATPase subunits and, at least, 11 non-ATPase subunits, which were desig- nated as Rpt (RP t riple A-ATPases) and Rpn ( RP non- ATPases), respectively [1]. Eight of them, six Rpt subunits and t wo Rpn subunits, assemble in the base of RP that associates with the 20S proteasome [2]. The other nine Rpn subunits assemble in the lid of RP which c overs the base and provides the binding or recognition specificity for ubiqui- tinated substrates [2]. ATP hydrolysis i n RP i s indispensable to assemble the 26S holocomplex, to r ecognize appropriate substrates, a nd to translocate the substrates to the 20S proteasome for degradation. In plants, the 26S proteasome has been implicated in cell- cycle progression, photomorphogenesis, hormone respon- ses, leaf, floral and x ylem differentiation and pathogen resistance [3–11]. Although there are some reports on the purification and characterization of 26S proteasome sub- units in plants [12,13], the composition o f the RP subunits in plants remains unclear. In this study, we identified the rice homologs of the yeast RP subunit genes from t he rice EST library, determined their nucleotide sequences, and found that their products assembledinanRPcomplexofrice. EXPERIMENTAL PROCEDURES Identification of cDNAs EST clones encoding the rice RP subunits were identified from the GenBank EST database by TBLASTN in the BLAST program o f NCBI network service, with 17 protein sequences of yeast ( Saccharomyces cerevisiae) RP subunits as queries. The EST c lones were p rovided by t he DNA bank Correspondence to H. Kawasaki, Yokohama City University, Kihara Institute for Biological Research/Graduate Schoo l of In t egrated Science, Maioka 641-12, Totsuka-ku, Yokohama 244-0 813, Japan. Fax: + 81 45 820 1901, Te l.: + 81 45 820 1904, E-mail: kawasaki@yokohama-cu.ac.jp Abbreviations: AAA, ATPases a ssociated with variou s cellular activities; CTAB, cetyltrimethylammonium bromide; EST, expressed sequence tag; Q-TOF, quadrupole t ime-of-flight mass spectrometer; RP, regulatory particle; Rpt, regulatory particle triple-A ATPase; Rpn, regulatory particle non -ATPase. Enzymes: proteasome (EC 3.4.99.46); lysylen dopeptida se (EC 3.4.21.50). Note: The novel nucleotide sequences reported here have been sub- mitted to DDBJ w ith the accession numbers AB033535–AB033537, AB037149–AB037155, AB070252–AB070262 and AB071016. (Received 4 January 2002, accepted 17 January 2002) Eur. J. Biochem. 269, 1474–1483 (2002) Ó FEBS 2002 in the Ministry of Agriculture, Forestry and Fisheries (http://bank.dna.affrc.go.jp/), where the EST libraries were prepared from rice (Oryza sativa L., cv. Nipponbare) a t different developmental stages [14]. Rapid amplification of cDNA 5¢ ends (5¢ RACE) The upstream coding regions of the EST clones were obtained from Cap site cDNA of rice shoot (L16D8) by 5¢ RACE (Nippon Gene, Toyama, Japan). The amplified RACE products were cloned into a pT7Blue-T vector (Novagen, Madison, WI, USA). Sequence analysis All cycle sequence reactions of the E ST and 5 ¢ RACE clones were carried out using Amersham thermo sequence kits (Amersham Pharmacia Biotech, Uppsala, Sweden). Nuc- leotide sequences were determined by a DNA sequen cer (model 4000 L, LI-COR, Lincoln, NE, USA). The nucleo- tide sequence i nformation was analyzed by GENETYX (Software Development, Tokyo, Japan). Isolation and gel blot analysis of genomic DNA Genomic DNA was extracted from mature leaves o f rice (cv. Nipponbare) by the CTAB method [15]. The genomic DNAs (6 lg) digested with three restriction enzymes (BamHI, EcoRI and EcoRV, Takara, Otsu, Japan) were separated on a 0.8% (w/ v) agarose gel (SeaKem GTG agarose, FMC BioProducts, Rockland, ME, USA), and transferred onto nylon membranes (Hybond-N + ,Amer- sham Pharmacia B iotech, Uppsala, Sweden). Probe DNA fragments were amplified by PCR from the corresponding EST clones with vector specific primers. After purification by gel extraction, the DNA fragments were labeled with AlkPhos Direct Labeling M odule (Amersham Pharmacia Biotech, Uppsala, Sweden). All hybridization reactions were performed at 55 or 70 °C with AlkPhos Direct Labeling M odule. Chemiluminescent s ignals were generated with CDP-Star (Amersham Pharmacia Biotech, U ppsala, Sweden), and detected with Hyperfilm ECL (Amersham Pharmacia B iotech, U ppsala, Sweden) at room temperature for 1 h. Purification of 20S proteasome from rice bran The 20S proteasome was purified from the rice bran (400 g) by ammonium sulfate precipitation (40–80%), DEAE-Sepharose CL-6B chromatography, hydroxy- apatite chromatography, a nd FPLC with Poros HQ/L, RESOUCE-PHE (1 mL) and Mono Q HR5/5 chromato- graphy. The purification procedure has been described previously [16]. Preparation of polyclonal anti-(rice 20S proteasome) serum A portion of the purified rice 20S proteasome fraction (3 mg ) was dialyzed against 50 m M potassium phosphate buffer, pH 7.5, and t he protein s olution was co ncentrated to 1mgÆmL )1 . This solution was divided into three parts (1 mL · 3 tubes). A New Zealand White rabbit was immunized against the antigen three times every two weeks. Antiserum was collected after 10 days of immunization, and stored at )80 °C until use. Immobilization of antibody to Protein A column The anti-(rice 2 0S proteas ome) Ig a nd Pr otein A beads were cross-linked with disuccinimidyl s uberate (DSS) (Pierce, Rockford, IL, USA). Two milliliters (80 mg ÆmL )1 )ofthe anti-(rice 20S proteasome) serum were mixed with equal volume of binding/washing buffer containing 140 m M NaCl, 8 m M Na 2 PO 4 ,2m M potassium phosphate and 1m M KCl, pH 7.4, and loaded onto HiTrap r Protein A FF 1 m L (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with b inding/washing buffer. The flow-through was completely washed with binding/washing buffer. The DSS was dissolved in d imethylsulfoxide to a final concen- tration of 13 mgÆmL )1 . Six hundred microliters of the DSS solution were diluted with 600 lL of binding/washing buffer, and quickly loaded onto HiTrap rProtein A FF with Terumo syringe 2.5 mL (Terumo, Tokyo, Japan). Both ends of the column were t hen capped, and the column was kept a t r oom temperature f or 1 h. The nonreacted DSS was eluted with quenching/washing buffer containing 25 m M Tris and 0 .15 M NaCl, pH 7.2. The noncross-linked IgG was eluted with ImmunoPure IgG elution buffer (Pierce, Rockford, IL, USA). Finally, the column was neutralized with binding/washing buffer, a nd stored at 4 °C before use. Immunoaffinity purification of the RP All procedures were performed at 4 °C. The rice bran (40 g) was mixed with fifth volume of extraction buffer c ontaining 100 m M Tris/HCl, p H 7.5, 20% (v/v) glycerol, 1 0 m M ATP, 10 m M MgCl 2 ,10m M EGTA, 14 m M 2-mercaptoethanol and protease inhibitor cocktail t ablets EDTA free (Roche Molecular B iochemicals, Mannheim, Germany), and grin- ded with an ice-cold mortar and a pestle. The homogenate wasthenfiltratedwithanylonmesh,andcentrifugedat 12 000 g for 20 min The s upernatant w as centrifuged a gain, at 40 000 g for 2 0 min. The supernatant was ultracentri- fuged a t 80 000 g for 1 h, and t hen at 370 000 g for 5 h. The pellet was dissolved in a suitable v olume of the extraction buffer and the insoluble material was removed by centri- fugation at 12 000 g for 2 0 min. The 3-mL samples (300 m g of protein) were loaded onto a HiTrap rProtein A F F column containing immobilized anti-(rice 20S proteasome) Ig. The column was equilibrated with the extraction buffer containing no protease inhibitor before sample l oading. T he column was then washed with extraction buffer without protease inhibitor cocktail t ablets EDTA free and the RPs were eluted with a RP elution buffer containing 100 m M Tris/HCl, pH 7.5, 20% (v/v) glycerol, 10 m M EGTA, 14 m M 2-mercaptoethanol and 1 .0 M NaCl. The 20S protea- some was eluted 2 mL of ImmunoPure IgG elution buffer. SDS/PAGE The purified 20S proteasome and RP were analyzed by 15 and 12% (w/v) SDS/PAGE, respectively. The protein bands were visualized with Coomassie Brilliant Blue (CBB). Ó FEBS 2002 The 19S regulatory particle from rice (Eur. J. Biochem. 269) 1475 Protein assay The protein concentration of the sample was d etermined by the Bradford method [17] using c-globulin as a standard. Western blotting analysis The subunits of rice RP were separated by 15% (w/v) SDS/ PAGE and electroblotted onto poly(vinylidene difluoride) membranes (Fluorotrans, Pall BioSupport, PortWashing- ton, NY, USA). The blots were stained with Ponceau S. Following blocking with 0.5% skimmed milk in blocking buffer (20 m M Tris/HCl buffer, pH 7.5, 500 m M NaCl and 0.05% Tween 20), the blots were incubated o vernight with a primary antibody (rabbit polyclonal a nti-AtRpn6 Ig or rabbit polyclonal a nti-AtRpt5 Ig; Affinity Research Prod- ucts, Mamhead Castle, UK). T hese primary a ntibodies were then detected with alkaline phosphatase-conjugated goat anti-(rabbit IgG) Ig (Vector Laborato ries, Burlingame, CA, USA) using alkaline phosphatase substrate (Moss, Int., Asbach, German). In-gel digestion In-gel digestion was performed by the method of Hellman et al. [18]. The gel pieces were washed three times with a 70% (v/v) acetonitrile solution, dried c ompletely a nd then rehydrated with digestion buffer containing 100 m M Tris/ HCl, pH 9.0 and 1 n gÆlL )1 of lysylendopeptidase (Wako Pure Chemical Industries, Osaka, Japan). T he protein was digested at 37 °C for 18 h. After digestion, 1 lL of acetic acid was added to t he buffer to s top t he reaction. The buffer was collected in a tube and t he peptides in the gel piece were extracted twice with 60% (v/v) acetonitrile. The collected solution was concentrated to 20 lL. ESI-Q-TOF mass spectrometry Peptides generated by in-gel digestion with lysylendopept- idase were desalted and concentrated with Zip TipC 18 (Millipore, Bedford, MA, USA), and then eluted with 1% (v/v) formic acid/70% (v/v) acetonitrile. T he peptides were loaded into a borosilicate nanoflow tip ( Micromass, Man- chester, UK), and subjected to Q-TOF2 (Micromass, Manchester, UK) with positive ion detection mode. The MS and MS/MS spectra we re analyzed by MASSLYNX v3.4 software (Micromass, Manchester, UK). The MS spectra of each subunit and MS/MS spectra of each peptide fragment were processed by a maximum e ntropy data enhancement program, MAXENT 3, a component of MASSLYNX v3.4. The spectra o f MS and MS/MS deconvoluted with MAXENT 3 were used for the peptide mass fingerprinting and amino- acid sequencing, respectively. RESULTS Identification of rice homologs of genes encoding yeast 19S regulatory particle subunits A total of 24 EST clones were identified in the rice EST library based on the amino-acid sequences of the yeast RP subunits. Three of the EST clones, E20984, C2890 and C11294, had the entire coding regions structurally similar to those o f t he yeast Rpt subunit genes, ScRpt1, ScRpt2 and ScRpt5, r espectively. Similarly, five E ST clones , R3615, S13278, S13105, S4633 and E1287, contained the entire coding regions structurally si milar to t hose of t he yeast Rpn subunit genes, ScRpn7, ScRpn8, ScRpn10, ScRpn11 and ScRpn12, respectively. How ever, the other 16 EST clones, E61121, C2890, E0 641, C50126, R2695, E0746, R 1494, E40363, E50789, E1935, C10189, C10401, S20324, C1087, C50129 and R1547, had parts of the coding regions structurally similar to those of ScRpt and ScR pn genes (Table 1). T he partial sequence determination of six clones such as E0641, R2695, E40363, C10401, S20324 and C50129, which are the homologs of ScRpt3, ScRpt4, ScRpn1, ScRpn3, ScRpn5,andScRpn9, respectively, was complemented by the results of cDNA sequencing i n the rice genome research project (Table 1). On the o ther hand, in our study, the full length of homologs of ScRpt4 and ScRpt6 were cloned by 5¢ RACE using C 50126 and R1494 as primers from the rice shoot cDNA library to determine their nucleotide sequences. Thus, the complete nucleotide sequences of homologs of all the six Rpt and nine Rpn subunit genes, and the partial sequences (about 80%) of the homologs of two Rpn genes were determined to deduce the amino-acid sequences of the rice RP subunits. Designations of the RP subunits of rice were based on those of yeast [1], e.g. OsRpt1 represents Rpt1 subunit of Oryza sativa. Duplication of the RP gene homologs Gene duplication was found in seven g enes, OsRpt1, OsRpt2, OsRpt4, OsRpt5, OsRpn1, OsRpn2 and OsRpn9 (Table 1) . The nucleotide sequence identity of the open reading frame between the duplicated genes was 81–88%, but the identity of the deduced amino-acid sequences was over 95%. In the nucleotide sequences, t he similarity of 3 ¢ untranslated region of the duplicated genes pairs was relatively low (data not shown). In the present study, these duplicated genes are marked with the small alphabetical s uffixes, ÔaÕ and ÔbÕ,e.g. OsRpt1a and OsRpt1b asshowninTable1. Rice genomic DNA was prepared from a plant d eveloped from a seed of the inbred strain of rice (Oryza sativa cv. Nipponbare), and genomic Southern hybridization was conducted using OsRpt gene specific probes to estimate the copy number of the OsRpt genes. In hybridization under high-stringency conditions (70 °C), only one DNA frag- ment was detected in the genomic DNA digested with various restriction enzymes, except for OsRpt3, OsRpt5b and OsRp t6 in the EcoRV digests (Fig. 1A). These results indicated t hat e ach g ene has a s ingle c opy i n the genome. As OsRpt6 has an EcoRV site in the cDNA sequence deter- mined, the probe of OsRp t6 must hybridize to two bands o f the EcoRV digest (Fig. 1A). OsRpt3 and OsRpt5b have no EcoRV site in the cDNA sequence, but the p robes of each gene hybridized to two bands. This p robably means that OsRpt3 and OsRpt5b have an intron containing one EcoRV site (Fig. 1A). In hybridization under normal conditions (55 °C), a ll the gene-specific p robes, except OsRpt3, cross- hybridized weakly to the DNA fragments identical to those detected with the probes for another m ember of t he same type of OsRpt genes (Fig. 1B). No other fragments were found to cross-hybridize. Although only one rice EST clone encoding Rpt6 was identified from the EST database, 1476 T. Shibahara et al. (Eur. J. Biochem. 269) Ó FEBS 2002 several fragments were additionally detected in all three digests with the OsRpt6-specific probe under normal conditions (Fig. 1 A). This suggests that the rice genome has another gene closely related to OsRpt,suchasother members of OsRpt genes. All OsRpt subunits, except OsRpt3, have two closely related genes, each of which are encoded by a single copy of gene in the rice genome. Purification of the rice RP Approximately 3 mg of the rice 20S proteasome were purified from rice bran (Fig. 2A) and rabbit antiserum was raised against t he purified 20S proteasome. The rice RP was then purified by immunoaffinity chromatography (HiTrap rProtein A FF column) using the anti-(20S proteasome) Ig as a ligand. The purified RP subunits were separated by SDS/PAGE and stained with CBB (Fig. 2B). We found that the molecular masses of the RP subunits were between 32 000 and 110 000 D a. Identification of the rice RP subunit by ESI-Q-TOF mass spectrometry Prior to identification of the RP subunits by ESI-Q-TOF MS, we separated the subunits by SDS/PAGE, and detected the those that cross-reacted with antibodies raised against Arabidopsis R pt5 and R pn6 ( Fig. 3). I n this experiment, two bands that cross-reacted with the antibodies were detected on the g el, suggesting that t hese bands contain OsRpt5 and OsRpn6. The rice R P s ubunits were separated by S DS/PAGE, and 14 bands were detected on the gel (Fig. 2B). The gel piece containing each ban d was removed and soaked in the lysylendopeptidase digestion buffer to digest into p eptides. The resultant peptides were collected and subjected to ESI-Q-TOF MS and MS/MS analyses. In th is analysis, the products of all the rice RP genes obtained from the EST library were found in the RP complex p urified from rice bran (Tables 2 and 3). Two proteins contained in bands 2 and 3 showed no amino-acid sequence similarity to the RP subunits, suggesting that these proteins may be novel subunits of rice RP or proteins interacting with rice RP. In the ESI-Q-TOF MS spectra of band 5, we found a peptide with m/z ¼ 558.29 and a doubly charged state, which corresponds to the mass value of N-myristoylated N-terminal peptide of OsRpt2. This is confirmed by MS/MS analysis of the peptide (Fig. 4 and Table 3). We also identified an N-acetylated peptide of OsRpt6 (Table 3 ). InthericeRP,therelativemolecularmassesofthe OsRpt subunits were similar to the theoretical values calculated from the deduced amino-acid sequences. How- ever, the relative molecular masses of some OsRpn subunits were significantly different from t hose of theor- etical ones. The relative molecular masses o f OsRpn3 and OsRpn8 were determined as 45 and 42 kDa, respectively, whereas the deduced molecular masses were 55 000 and 34 900 Da, respectively (Fig. 2B, Table 1 and Table 2 ). The difference of the molecular masses observed in these subunits might be resulted from the post-translational modifications. Table 1. Description of genes encoding the rice 19S regulatory particle subunits. N D, not determined. Gene a Predicted peptide length (aa)/MW (kDa)/pI Representative EST Nucleotide length of EST (bp) Accession number Full length cDNA clone Name Length (bp) OsRpt1a 426/47.7/5.95 E20984 1481 AB033535 OsRpt1b ND E61121 887 AB070252 OsRpt2a 448/49.6/5.85 C2890 876 D17789 b OsRpt2b 450/49.7/5.85 E31171 1616 AB037154 OsRpt3 419/46.5/5.69 E0641 1375 AB070253 J023091H23 1517 OsRpt4a 400/44.6/7.25 C50126 1637 AB033536 OsRpt4b 401/44.6/8.47 R2695 1390 AB070254 J013074G13 1555 OsRpt5a 429/47.8/4.52 C11294 1510 AB037155 OsRpt5b 429/47.8/4.55 E0746 758 AB071016 OsRpt6 424/47.2/9.24 R1494 1625 AB033537 OsRpn1a 891/97.5/5.01 E40363 636 AB070255 J023092J12 3091 OsRpn1b ND E50789 1004 AB070256 OsRpn2a ND E1935 1065 AB070257 006-201-F07 1886 d OsRpn2b ND C10189 ND D22019 c OsRpn3 486/55.2/8.53 C10401 1458 AB070258 006-212-H08 1698 OsRpn5 443/50.6/7.52 S20324 1075 AB070259 J023081N11 1890 OsRpn6 ND C1087 385 AB070260 001-033-C10 568 d OsRpn7 389/44.1/5.57 R3615 1547 AB037149 OsRpn8 310/34.9/6.30 S13278 1308 AB037150 OsRpn9a ND C50129 707 AB070261 J023028J18 1517 OsRpn9b ND R1547 1239 AB070262 001-042-D11 1407 d OsRpn10 402/42.4/4.35 S13105 1536 AB037151 OsRpn11 307/34.4/6.14 S4633 1129 AB037152 OsRpn12 267/30.8/4.85 E1287 1063 AB037153 a Adapted from Finley et al. [1]. b EST accession number. c previously reported Suzuka et al. [19]. d Not encoded full length. Ó FEBS 2002 The 19S regulatory particle from rice (Eur. J. Biochem. 269) 1477 The i soforms o f OsRpt subunits ÔaÕ and ÔbÕ encoded by the duplicated genes were analyzed with ESI-Q-TOF MS and MS/MS. There are some differences in amino-acid sequen- ces between these isoforms. We i dentified peptides with different mass values derived from homologous region between these isoforms. By these MS and MS/MS analyses, we detected both products encoded by the duplicated genes of OsRpt2, OsRpt4 and OsRpt5 (Table 4). F or example, there were two pairs of peptides with a single amino-acid difference derived from homologous regions of OsRpt2a and OsRpt2b in the ESI MS spectrum of band 5 (Table 4 and Fig. 5A); these amino-acid sequences were confirmed by MS/MS analysis (Figs 5B,C). As the i onization efficiency of these two homologous peptides is assumed to be almost identical, it is considered that OsRpt2a and OsRpt2b are expressed equally in protein level based on the ratio o f peak heights between them (Fig. 5A). Therefore, we concluded that both translation products of these three duplicated genes were components within the RP of the rice 26S proteasome. As the amino-acid sequences of OsRpt1a and OsRpt1b are identical, we could not distinguish t he products of these genes by MS analysis. DISCUSSION In th e p resent study, we i dentified 24 rice genes encoding the homologs of all 17 yeast RP subunits from the rice EST library. The amino-acid sequences of the subunits encoded by these homologs were highly homologous to those of Arabidopsis Rpt (91–96%) and Rpn (64–93%) subunits. Three o f the homologs have iden tical sequences to TBPOs-2 (OsRpt2a), TBPOs-1 (OsRpt5a)andOsS5a (OsRpn10), which have been reported as homologs of rice proteasome subunit genes found in other organisms [19,20]. T he rice RP subunits possess the a mino-acid sequence m otifs commonly found in RP subunits of the o ther eukaryotes. F or example, the OsRpt subunits have the 200-amino-acid AAA cassette essential for Walker-type ATPases, consisting of eight domains [21], and OsRpn subunits have consensus sequence motifs such as the polyubiquitin binding site motif, PUbS1 and P UbS2 (OsRpn10), and Cys box ( OsRpn11) a s reported in other eukaryotes [22,23]. Duplicated genes of OsRpt1, OsRp t2, OsRpt4 and OsRpt5, which transcribe two different mRNAs with nucleotide sequence similarity (81–88%), were found in the EST library. Genomic Southern hybridization was carried out using OsRpt gene-specific probes f or the genomic DNA from a plant developed from a single inbred seed, and confirmed to be present in the rice genome (Fig. 1 ). Duplication of the genes encoding RP subunits has been reported in Arabidopsis thaliana and Trypanosoma cruzi [24,25]. It has been indicated that both transcripts of the duplicated genes are expressed in these organisms [24,25], but it has never been shown whether these duplicated gene products assemble in the proteasome complex. Fig. 1. Genomic DNA gel b lot analysis of genes encoding six O sRpt subunits of the rice 19S regulatory particle. The rice genomic DNA was isolated from an individual grown from single seed of inbred strain (Oryza stiva L. , cv. Nipponbare), digested with BamHI (B), EcoRI (I) or EcoRV (V). The digests w ere separated by agarose gel electrophoresis. E ach band marked by an a rrow represents a genomic D NA fragment whic h corresponds to the gene-specific probe used in the pa nel. (A) Hybridized under highly stringent condition (70 °C). (B) Hy bridized under normal condition ( 55 °C). 1478 T. Shibahara et al. (Eur. J. Biochem. 269) Ó FEBS 2002 To determine whether both of the duplicated gene products assemble in the rice proteasome complex, we analyzed the subunit composition of the purified RP complex. The R P complex from the rice bran was purified by immunoaffinity chromatography with a column immobilized antibo dy against the rice 20S proteasome. As R P attaches at t he end of 20S protea- some in an ATP-dependent manner, the column retains RP complex as the 26S proteasome in the presence of ATP, and RP complex can be s pecifically eluted by removal of ATP. This method allowed the effective purification of RP from rice bran by utilizing these two different affinities. In the purified RP complex, w e identified the products of all rice homologs obtained from the EST library, including both products of the duplicated OsRpt gen es. Each RP complex is thought to contain only one of the duplicated gene products. Therefore, a single rice cell is considered to contain several types of RP complex as a mixture, or different cells may contain respective types of RP complex as reported in the 20S proteasome of mammals [26,27]. In mammals, three 20S proteasome subunits are known to be replaced by c-interferon-inducible s ubunits, resulting in the immuno-proteasome [26]. Dahlmann et al. reported that there are five subtypes of the 20S proteasome in the rat skeletal m uscle, including immuno-proteasome. These subtypes exhibit different substrate specificities [27]. In rice, the different types o f RP c omplex, each of which contains only one of the products of duplicated OsRp t genes, may have s pecific functions. Fig. 3. Western blotting analysis of the rice 19S regulatory particle subunits. Purified rice RP resolved by SDS/PAGE was transferred to PVDF membranes. L ane 1, molecular m ass marker s tained with CB B; lane 2, detected with ant i-AtRpt5 Ig; lane 3, d etected w ith anti-AtRpn6 Ig; Lane 4, stained with CBB. Fig. 2. SDS/PAGE of the rice 20S proteasome and 19S regulatory particle. (A) Purified rice 20S proteasome resolved by SDS/PAGE, stained with C BB. Lane 1, m olecular mass marker; lane 2, purified rice 20S proteasome. (B) Purified rice RP resolved by SDS/PAGE and stained with CBB. Lane 1, mo lecular mass marker; l ane 2, purified rice RP. Table 2. Protein identification of t he rice 19S regulatory particle sub- units. Band no. Protein 1 OsRpn2 2* 3* 4 OsRpn1 5 OsRpt2 6 OsRpt3 7 OsRpt1,OsRpt5,OsRpn10 8 OsRpn3,OsRpn5,OsRpn6 9 OsRpt6 10 OsRpt4 11 OsRpn8,OsRpn9 12 OsRpn7 13 OsRpn11 14 OsRpn12 * Protein without sequence homology to RP subunits. Ó FEBS 2002 The 19S regulatory particle from rice (Eur. J. Biochem. 269) 1479 Table 3. Identification of the rice 19S regulatory particle subunits by ESI-Q-TOF MS. myri, N-myristoylaton; ac, N-acetylation. Protein Band Observed mass Charge Theory mass Amino-acid sequence a Mode OsRpn1 #4 767.95 +2 1533.89 (K)PLSVPVRVGQAVDVV(G) MS/MS 674.34 +2 1346.68 (R)NLAGEIAQEFQK(R) MS/MS 622.30 +2 1242.68 (K)EALQDIISNIK(L) MS/MS 639.32 +2 1276.61 (K)QESVEATAEVSK(T) MS/MS OsRpn2 #1 589.35 +2 1176.68 (K)LPTAILSTYAK(A) MS/MS 647.84 +2 1293.67 (K)FLEGGRYEPVK(L) MS/MS OsRpn3 #8 652.34 +2 1302.70 (K)EIASVIEAGSLSK(E) MS/MS OsRpn5 #8 519.64 +3 1555.91 (K)NLSEIPNFRLLLK(Y) MS 666.36 +2 1330.69 (K)ISPRVFDADPSK(E) MS 1083.08 +2 2164.13 (K)EGDNIVQEAPAEIPSLLELK(R) MS 511.99 +3 1532.94 (K)LRIIEHNILVVSK(Y) MS OsRpn6 #8 895.94 +2 1789.95 (K)TEAIFPATLETISNVGK(V) MS/MS OsRpn7 #12 408.23 +2 814.49 (K)LSRVIDL(-) MS/MS 528.26 +3 1581.84 (K)LFLLSHPDVDDLAK(V) MS/MS 606.32 +2 1210.67 (K)SLYFIRVGEK(E) MS/MS 662.38 +2 1322.78 (K)VVDAPEILAVIGK(V) MS/MS 773.37 +2 1544.79 (K)SFFAAFSGLTEQIK(L) MS/MS OsRpn8 #11 536.27 +2 1070.53 (K)AYYAVEEVK(E) MS/MS 611.12 +5 3050.62 (K)VFVHVPSEIAAHEVEEIGVEHLLRDVK(D) MS/MS 701.33 +2 1400.68 (K)AEDSKPTAIPTAIPSAAGS(-) MS/MS 733.87 +2 1465.75 (K)DTTISTLATEVTSK(L) MS/MS 840.44 +4 3357.81 (K)LRENDLDIHALFNNYVPNPVLVIIDVQPK(E) MS/MS OsRpn9 #11 573.79 +2 1145.61 (K)BQIAAINLEK(G) MS/MS 713.84 +2 1425.74 (K)LSISDVEYLLBK(S) MS/MS 933.48 +2 1864.98 (K)VHTTLLSVEAETPDLVAA(-) MS/MS OsRpn10 #7 721.43 +2 1440.83 (K)GVRVLVTPTSDLGK(I) MS/MS 745.35 +3 2233.04 (K)NNVALDIVDFGETDDDKPEK(L) MS/MS 898.94 +2 1795.81 (K)TQSNPENTVGVBTBAGK(G) MS/MS OsRpn11 #13 442.75 +2 883.49 (K)BLLNLHK(K) MS/MS 628.36 +2 1254.70 (K)LAIANVGRQDAK(K) MS/MS 1016.72 +3 3045.48 (K)HLEEHVSNLBSSNIVQTLGTBLDTVVF(-) MS/MS OsRpn12 #14 571.66 +3 1711.96 (K)IARDIYEHAVVLSVK(I) MS/MS 489.61 +3 1465.83 (K)LVEVTQLFSRFK(A) MS/MS 857.82 +3 2570.41 (K)EIPSLQVINQTLSYARELERIV(-) MS/MS OsRpt1 #7 417.73 +2 833.46 (K)FVVGLGDK(V) MS/MS 461.23 +2 920.46 (K)DFLDAVNK(V) MS/MS 598.85 +2 1195.70 (K)YQIQIPLPPK(I) MS/MS 643.82 +2 1285.66 (K)TYGLGPYSTSIK(K) MS/MS 972.93 +2 1943.91 (K)ESDTGLAPPSQWDLVSDK(Q) MS/MS OsRpt2 #5 558.29 +2 1114.61 myri-GQGTPGGBG(K) MS/MS 595.80 +2 1189.63 (K)GVILYGEPGTGK(T) MS/MS 679.03 +3 2034.13 (K)QIGIDPPRGVLLYGPPGTGK(T) MS/MS 710.99 +3 2130.08 (K)APLESYADIGGLDAQIQEIK(E) MS/MS OsRpt3 #6 944.41 +2 1886.86 (K)BTLADDVNLEEFVBTK(V) MS/MS 645.29 +2 1288.60 (K)KPETDFDFYK(-) MS/MS 457.59 +3 1369.77 (K)RELLRAQEEVK(R) MS/MS 501.80 +2 1001.60 (K)NRYVILPK(K) MS/MS OsRpt4 #10 473.27 +2 944.55 (K)IVSSAIIDK(H) MS/MS 579.83 +2 1157.64 (K)GVLLYGPPGTGK(T) MS/MS 611.01 +3 1830.04 (K)TLLARAIASNIDANFLK(I) MS/MS 630.32 +2 1258.62 (K)HGEIDYEAVVK(L) MS/MS OsRpt5 #7 579.82 +2 1157.64 (K)GVLLYGPPGTGK(T) MS/MS 622.32 +3 1864.01 (K)QIQELVEAIVLPBTHK(D) MS/MS 705.35 +3 2113.06 (K)DSYLILDTLPSEYDSRVK(A) MS/MS 830.93 +2 1659.87 (K)LAGPQLVQBFIGDGAK(L) MS/MS OsRpt6 #9 645.27 +3 1932.96 (K)IEFPNPNEDSRFDILK(I) MS/MS 733.00 +3 2196.07 ac-ATVABDISKPPPAAGGDEAAAAK(G) MS/MS a B and J were defined as oxidation of Met and acrylamidation of Cys, respectively. 1480 T. Shibahara et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Post-translational modifications of the p roteasome, such as phosphorylation, N-acetylation, glycosylation and pro- cessing of the proteasome, have been reported in various organisms [16,28–33]. The present study revealed two post-translational modifications of the rice RP; N-myris- toylation of OsRpt2 and N-acetylation of OsRtp6, as determined by MS analysis (Fig. 4 and T able 3). Rpt2 from various organisms such as human, yeast, Arabidopsis, Caenorhabditis and Drosophila, h ave the consensus motif (M-G-X-X-X-S/T-X-X-X) for N-myristoylation at the N-terminus [34]. A lthough the N-terminal sequence (M-G-Q-G-T-P-G-G-M) of OsRpt2a and OsRpt2b was slightly different from the consensus motif, the N-termini were also myristo ylated. This suggests that the rice N-myristoyltransferase has a substrate specificity different from that of the other organism. Protein N-myristoylation promotes reversible and weak protein–membrane and protein–protein interactions. Usually, myristate acts with other mechanisms to r egulate p rotein targeting and protein function. For example, some proteins (e.g. MARCKS, Src) employ Ômyristoyl-electrostatic switchesÕ where membrane association is promoted by the myristoyl moiety plus electrostatic interactions between positively charged protein side chains and negatively charged membrane phospho- lipids [35,36]. The function of N-myristoylation in Rpt2 is not yet clear, but it probably plays important role in the 26S proteasome. It is well known that the N-terminus of 80–90% of proteins in the cell is acetylated. Recently, Kimura et al. ([32]; Kimura, Y., Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Japan, personal communication) iden- tified N-acetylation of many of the yeast 26S proteasome subunits (a1, a2, a3, a4, a5, a6, a7, a3, a5, Rpt3, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, Rpn8 and Rpn11), and described the relationship between N-acetylation and the chymotrypsin-like activity of 20S proteasome. In the present s tudy, we detec ted the N-acetylation of OsRpt6 in the rice RP complex by MS. Other N-terminal modifica- tions have not been identified in the rice proteasome. Processing of proteins is important for their function. In the 20S proteasome, processing during assembly produces the active Thr residue at the N-termini of three a subunits, a1, a2anda5 [37,38]. We found differences between observed molecular masses a nd theoretical values i n t he rice RP subunits. O sRpn3 was i dentified as a 45-kDa protein by SDS/PAGE, while the molecular mass of OsRpn3, deduced from the nucleotide sequence, being 55 000 Da. Similarly, in carrot, the deduced molecular mass of t he carrot Rpn3 was 55 000 Da, but the observed mass was 45 000 Da [39]. It is likely t hat these differences in molecular masses may be due to the processing during maturation. The human-specific subunit of proteasome, S5b, has been copurified with RP complex from human erythro- cytes. This subunit in teracts with the N-terminal r egion of Rpt1 and with the C-terminal portion of Rpt2 [40]. However, no proteasome subunit specific to plants has been identified. In the analysis of rice RP subunits by SDS/ PAGE, two of the 14 bands were found to contain two proteins without sequence s imilarity to RP subunits. These Fig. 4. Determination of N-terminal modifica- tion of OsRpt2. The MS/MS spectrum deconvoluted with MaxEnt3 from dou bly charged ion at m/z 558.29 of Os Rpt2 digested with lysylendopeptidase. Major b-series and y-series ions are indicated bold spectra. M(ox) indicated oxidation of Met. Table 4. Identification of OsRpt subunits a and b b y ESI-Q-TOF. B, oxidation of Met. J, acrylamidation of Cys. Substituted am ino-acid residues between a and b are indicated in bold. Protein Observed mass Charge Theory mass Amino-acid sequence Mode OsRpt2a 795.92 +2 1589.89 (K)GPEAAARLPNVAPLSK(C) MS and MS/MS OsRpt2b 774.42 +2 1546.88 (K)GPEAAARLPAVAPLSK(C) MS and MS/MS OsRpt2a 783.64 +4 3130.69 (K)LVRELFRVADELSPSIVFIDEIDAVGTK(R) MS and MS/MS OsRpt2b 780.17 +4 3116.68 (K)LVRELFRVADDLSPSIVFIDEIDAVGTK(R) MS and MS/MS OsRpt4a 632.68 +3 1895.02 (K)IEIPLPNEQARBEVLK(I) MS OsRpt4b 638.03 +3 1911.01 (K)IEIPLPNEQSRBEVLK(I) MS OsRpt5a 862.42 +2 1722.84 (K)DELQRTNLEVESYK(E) MS and MS/MS OsRpt5b 861.43 +2 1720.86 (K)DELQRTNLELESFK(E) MS and MS/MS OsRpt5a 903.45 +2 1804.93 (K)SPJIIFIDEIDAIGTK(R) MS and MS/MS OsRpt5b 895.45 +2 1789.94 (K)APJIIFIDEIDAIGTK(R) MS and MS/MS Ó FEBS 2002 The 19S regulatory particle from rice (Eur. J. Biochem. 269) 1481 proteins may be novel components or regulatory factors of rice proteasome. In the present study, we found isoforms of RP subunits in rice, and indicated the presence of novel proteins associated with rice RP. 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Identification of the rice RP subunit by ESI-Q-TOF mass spectrometry Prior to identification of the RP subunits by ESI-Q-TOF MS, we separated the subunits