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Primary structural features of the 20S proteasome subunits of rice (Oryza sativa)
Gene 250 (2000) 61–66 www.elsevier.com/locate/gene
Primary structural features of the 20S proteasome subunits of rice (Oryza sativa) Hidenori Sassa 1, So Oguchi 1, Takeshi Inoue, Hisashi Hirano * Kihara Institute for Biological Research & Graduate School of Integrated Science, Yokohama City University, Maioka 641-12, Totsuka-ku, Yokohama 244-0813, Japan Received 25 January 2000; received in revised form 4 April 2000; accepted 20 April 2000 Received by T. Sekiya
Abstract The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins, and it also has other diverse biological functions. Its structure comprises 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and two inner rings form an a7b7b7a7 structure, and each subunit, a and b, exists as seven different types, thus giving 14 kinds of subunits. In this study, we report the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all of the subunits from monocots. Amino acid sequence homology within the rice family (a-type: 28.9–42.1%; b-type: 17.2–31.9%) were lower than those between rice subunits and corresponding orthologs from Arabidopsis and yeast (a-type: 49.2–94.5%; b-type: 34.8–87.7%). Structural features observed in eukaryotic proteasome subunits, i.e., a- or b-type signature at the N-termini, Thr active sites in b1, b2 and b5 subunits, and nuclear localization signal-like sequences in some a-type subunits, were shown to be conserved in rice. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Oryza sativa; 20S proteasome subunits
1. Introduction The proteasome is a multicatalytic proteinase complex that is involved in an ATP/ubiquitin-dependent proteolytic pathway. In eukaryotes, there are two types of complexes, with sedimentation coefficients of 20S (20S proteasome) and 26S (26S proteasome). The 26S proteasome consists of two 19/22S regulatory complexes and the 20S proteasome as the catalytic core. The 20S proteasome subunits can be classified, on the basis of sequence similarities, into two families, a and b. Eukaryotic proteasomes contain 14 different subunits, seven a-type subunits and seven b-type subunits. The subunits are arranged into four seven-membered rings, with the a-type subunits forming the two outer rings and the b-type subunits, the two inner rings. Collectively, they form a barrel-shaped catalytic 20S complex (Coux Abbreviations: PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends. * Corresponding author. Tel.: +81-45-820-1904; fax: +81-45-820-1901. E-mail address: [email protected] (Hisashi Hirano) 1 These authors contributed equally to this work.
et al., 1996; Baumeister et al., 1998). In yeast and mammals, proteasomes have been shown to be involved in not only degradation of misfolded or truncated proteins, but also diverse biological functions including cell-cycle progression (Coux et al., 1996). However, in plants, limited findings are available for not only the biological roles of the proteasome (Genschik et al., 1998; Woffenden et al., 1998; Girod et al., 1999), but also the primary structural features of the subunits of the 20S unit (Ito et al., 1997; Parmentier et al., 1997; Umeda et al., 1997; Fu et al., 1998). The complete amino acid sequences of all of the 20S proteasome subunits, seven a- and seven b-types, were recently reported for Arabidopsis thaliana (Parmentier et al., 1997; Fu et al., 1998), but not yet for other plant species. We have analyzed a crude preparation of 20S proteasome of rice (Oryza sativa L. cv. Nipponbare) by twodimensional gel electrophoresis followed by protein microsequencing, and identified seven a- and seven b-type subunits on the 2D-gel with their partial amino acid sequences (Ito, K., Nakazawa, M., Sassa, H., Hirano, H., manuscript in preparation). In this study, we report the primary structural features of the seven
0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 19 0 - 6
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a-type and seven b-type subunits of the 20S proteasome of rice, representing the first set for all of the subunits from monocots.
GAG CC-3∞), and b6 (b6R1: 5∞-CCA TGG CAG GGC AGC TCA TC-3∞, b6R2: 5∞-TTG TAG TAC AGG GTA TTG GAG AG-3∞). Amplified RACE products were cloned in pT7Blue-T vector (Novagen).
2. Materials and methods
2.3. Sequence analysis
2.1. Identification of rice cDNAs encoding 20S proteasome subunits
EST clones and RACE clones were sequenced for both strands by using a DNA sequencer (model 4000; Li-Cor, Lincoln, NE ). The deduced amino acid sequences were aligned by Clustal X ( Thompson et al., 1997). Phylogenetic trees of a- and b-subunit families were generated by the neighbor-joining method (Saitou and Nei, 1987). Pairwise comparisons of sequence homology were conducted using the Genetyx-Mac ver. 10 computer program (Software Development, Tokyo, Japan).
We have previously analyzed crude preparation of rice 20S proteasome by two-dimensional gel electrophoresis followed by amino acid sequence analysis, and determined partial amino acid sequences of seven a-type and seven b-type subunits (Ito et al., unpublished data). By using the partial amino acid sequences of the rice 20S proteasome subunits, we searched for rice EST clones encoding 20S proteasome subunits using the BLAST program (Altschul et al., 1990). Deduced amino acid sequences of recently published 20S proteasome subunits of Arabidopsis (Parmentier et al., 1997; Fu et al. 1998) were also used as queries to search the rice EST clones. Identified EST clones from a japonica rice cultivar Nipponbare (Sasaki et al., 1994) were provided from a DNA bank at the Ministry of Agriculture, Forestry, and Fisheries of Japan (http://bank.dna. affrc.go.jp/). 2.2. RNA extraction and RACE (rapid amplification of cDNA ends) Seeds of a japonica rice (cv. Nipponbare) were surface-sterilized and hydroponically grown in 1/4 strength Murashige–Skoog medium (Murashige and Skoog, 1962). Total RNA was extracted from the roots of 1 week old seedlings by the hot-phenol method (DeVries et al., 1988). First-strand cDNA for 3∞RACE was synthesized from the total RNA by using a (dT ) adapter 17 primer essentially as described before (Sassa and Hirano, 1998). 3∞RACE was conducted by using an adapter primer (5∞- CGA CGT TGT AAA ACG ACG GCC AGT -3∞) and gene-specific primers for respective subunit genes: b3 (b3F: 5∞- ATG TCG ATC TTC GAG TAC AAT GG-3∞), b4 (b4F: 5∞- GCA GTT CAC CGA GTT CAT CC-3∞), and b6 (b6F1: 5∞-ACG AGA ACA ACG GCG GGA C-3∞). 5∞RACE was performed by the method of Hug and Klobeck (1996) or by using CapSite cDNA (Nippon Gene, Toyama, Japan) derived from shoots of Nipponbare. Gene-specific primers used for 5∞RACE were as follows: a1 (a1RiOg: 5∞-TGT CCA AAG AAG TGA CCT GC-3∞; a1RoOg: 5∞-TAA GAC CGG CGC TAG TTG CC-3∞), b3 (b3RiOg: 5∞-ACA GCT TGT GCC TGA ACA CCA GG-3∞; b3RoOg: 5∞-ACA GGC TGG CAG AAG TAT GGT CC-3∞), b4 (b4RiOg: 5∞-ATA GTA CGG ATT CTT CCG CAG GG-3∞; b4RoOg: 5∞-AGC GAG AGA CAG AAG TAG
3. Results and discussion Based on the partial amino acid sequences of the rice 20S proteasome subunits (Ito et al., unpublished results) and recently published cDNA sequences of Arabidopsis (Parmentier et al., 1997; Fu et al., 1998), DNA databases were searched by the BLAST program, and candidate EST clones for 10 subunits of a japonica rice cultivar ‘Nipponbare’ (Sasaki et al., 1994) were identified; a1 (Accession No. D49098), a2 (Accession No. D40076), a3 (Accession No. D41015), a4 (Accession No. C74810), a5 (Accession No. D24985), a7 (Accession No. D40644), b1 (Accession No. D41150), b2 (Accession No. D15408), b5 (Accession No. D48392) and b7 (Accession No. D15785). The cDNA sequence of the a6 subunit of japonica rice (cv. Yamahousi) was recently reported ( Umeda et al., 1997). The 10 EST clones were provided from the DNA bank at the Ministry of Agriculture, Forestry and Fisheries of Japan, completely sequenced, and found to encode the fulllength amino acid sequences of the respective 20S proteasome subunits, except for the a1 subunit (D49098). The EST clone (D49098) encoded the rice a1 subunit, but was not the full-length cDNA as it was found to lack a part of the sequence encoding the N-terminal region. In order to obtain the lacking part, we conducted 5∞RACE by using RNA derived from young root of Nipponbare. For the b4 subunit, an identified EST clone (Accession No. D24758) was not available. For b3 and b6 subunits, corresponding EST clones could not be identified from japonica rice, but from indica rice (b3, Accession No. AA752242) or maize (b6, Accession No. T18278). Primers were designed from the three EST sequences, and used for 3∞ or 5∞RACE to obtain b3, b4, and b6 cDNA sequences. Finally, primary structures of all the subunits of the 20S proteasome of japonica rice were determined by complete sequencing of the EST clones
H. Sassa et al. / Gene 250 (2000) 61–66
and RACE products. Designations of the 20S proteasome subunits of rice were based on those of Arabidopsis (Fu et al., 1998), i.e., OsPAA1 represents proteasome a1-1 subunit of Oryza sativa. The Accession Nos of the cDNAs encoding the rice 20S proteasome subunits are OsPAA1 (AB026558), OsPAB1 (AB026559), OsPAC1 (AB026560), OsPAD1 (AB032061), OsPAE1 (AB026561), OsPAG1 (AB026562), OsPBA1 (AB026563), OsPBB1 (AB026564), OsPBC1 (AB026565), OsPBD1 (AB026566), OsPBE1 (AB026567), OsPBF1 (AB014058) and OsPBG1 (AB026568). OsPAF1 (D49098) had previously been cloned and designated as the C2 subunit of rice ( Umeda et al., 1997). Based on comparisons of the derived amino acid sequences from rice, Arabidopsis and yeast, phylogenetic trees for a- and b-families of 20S proteasome subunits were generated ( Fig. 1). The phylogenetic trees showed that each rice gene was closely related to each of the seven a and seven b subfamily genes in Arabidopsis and yeast and formed seven subfamily-specific groups together with corresponding Arabidopsis and yeast genes in the trees. All rice a and b subunits were more homologous to those of corresponding subfamilies of other organisms, Arabidopsis and yeast, than to other subunits of rice, as observed in Arabidopsis genes ( Table 1; Parmentier et al., 1997; Fu et al., 1998). These findings suggest that the rice genes for 20S proteasome subunits are the orthologs of each of Arabidopsis and yeast. An alignment of the deduced amino acid sequences of the a-subunit family of rice and Arabidopsis is shown in Fig. 2. Pairwise comparisons of sequence homology of a-type subunits are shown in Table 1. All of the seven a-type subunits of rice were found to have the a-type signature at their N-termini. The amino acid sequence homology within the rice family (28.9–42.1%) was lower than that between rice subunits and corresponding orthologs from Arabidopsis and yeast (49.2–94.5%; Fig. 1 and Table 1). Some a-type subunits of eukaryotes and Thermoplasma have been shown to bear putative nuclear localization signals (NLS ) and are assumed to be involved in the nuclear translocation of proteasomes ( Tanaka et al., 1990; Nederlof et al., 1995). The probable NLS sequence ( X–X–K–K(R)–X–K(R); Roberts 1989) was found in the a1 subunit of rice ( K–K–M–K ). Parmentier et al. (1997) and Fu et al. (1998) suggested similar NLS-like sequences of clusters of basic amino acids in Arabidopsis a-type subunits, a2 (R–K–S–R–K ) and a1 ( K–K–V–P–D–K ), respectively. The a1 and a2 subunits of rice were also found to have the putative NLS sequences at the corresponding position. These putative NLS sequences in the a-type subunits are possibly involved in the nuclear import of the proteasome in rice. An alignment of the amino acid sequences of b-type subunits of rice is also presented in Fig. 2. The b-type
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Fig. 1. Phylogenetic trees of the 20S proteasome subunits from rice (Oryza sativa), Arabidopsis, and yeast (S. cerevisiae). (A) a subunits; (B) b subunits. The phylogenetic trees of amino acid sequences were generated by using ClustalX ( Thompson et al., 1997). The distance along the horizontal axis separating two sequences is proportional to the divergence between the sequences. The amino acid sequence homology between Arabidopsis or yeast subunits and the corresponding rice subunits is indicated in parentheses on the right.
signature motif was conserved in all of the seven subunits in rice. The amino acid sequence homology within the rice family (17.2–31.9%) was lower than that between rice subunits and corresponding orthologs from Arabidopsis and yeast (34.8–87.7%; Fig. 1 and Table 1). The b-type subunits participate in the proteolytic activity of proteasomes. In eukaryotes, three b-type subunits (b1, b2, and b5) were shown to be autocatalytically processed to remove their propeptides, and their resultant N-terminal Thr residues to form the catalytic site of the 20S proteasome (Baumeister et al. 1998). The
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Fig. 2. Amino acid sequence alignments of the 20S proteasome subunits of rice. (A) a subunits; (B) b subunits. The proteasomal a-type and b-type signatures were determined by using the ‘PROSITE’ database (http://www.expasy.ch/tools/scnpsit1.html ) and are boxed. Putative nuclear localization signals (NLS) are in reverse type. Potential N-glycosylation sites are enclosed by circles. The propeptide sequences of b-type subunits were identified by comparison between the results of N-terminal sequencing (Ito et al., unpublished results) and sequences deduced from cDNA, and are indicated by lower-case letters.
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H. Sassa et al. / Gene 250 (2000) 61–66 Table 1 Amino acid sequence homology (percentage identity) of the 20S proteasome subunits from rice a subunits
OsPAB1
OsPAC1
OsPAD1
OsPAE1
OsPAF1
OsPAG1
OsPAA1 OsPAB1 OsPAC1 OsPAD1 OsPAE1 OsPAF1
33.8
35.0 37.7
36.6 39.3 40.6
32.6 33.8 42.1 39.2
32.9 32.1 30.9 32.8 38.3
28.9 30.3 34.9 33.7 36.3 40.4
b subunits
OsPBB1
OPsPBC1
OPsPBD1
OsPBE1
OsPBF1
OsPBG1
OsPBA1 OsPBB1 OsPBC1 OsPBD1 OsPBE1 OsPBF1
31.9
22.2 17.2
23.8 19.0 20.3
30.9 30.8 23.4 23.1
27.2 24.0 31.4 25.7 26.5
23.8 20.5 19.3 23.3 23.6 19.6
N-terminal amino acid sequences of rice b1, b2, and b5 subunits (Ito et al., unpublished results) and their comparison to the amino acid sequences deduced from the cDNAs showed that these three catalytic subunits of rice are cleaved between a Gly/Thr pair to expose the Thr active sites in the same way as in the proteasome of yeast (Baumeister et al., 1998). The result of the present study on the rice proteasome supports the prediction of cleavage sites for the three active b1, b2, and b5 subunits of Arabidopsis based on sequence alignment (Fu et al., 1998) and provides experimental evidence that the active b-type subunits of plants are processed in the same way as those of other eukaryotes. Our protein sequence analysis of the rice 20S proteasome subunits showed that the b7 subunit had a free N-terminal, as did the b1, b2, and b5 subunits but that the N-terminals of b3, b4, and b6 subunits were blocked (Ito et al., unpublished results). Comparison of the b7 N-terminal sequence determined by protein sequencing and that deduced from the cDNA showed that the b7 proprotein is cleaved between R-29 and T-30 to release the propeptide. This evidence for the cleavage site of rice b7 propeptide supports the previous prediction for the cleavage site of the b7 subunit of Arabidopsis (Fu et al., 1998). Fu et al. (1998) predicted that the cleavage site of the Arabidopsis b6 subunit is between Glu-4 and His-5, based on the finding that the yeast b6 subunit is cleaved between Asp/His. In rice, however, the cleavage site of the b6 subunit could not be predicted because of the low sequence similarity between the b6 subunit of Arabidopsis and that of rice around the probable cleavage site, and because of the N-terminal block of the rice b6 subunit (Ito et al., unpublished results). Putative N-glycosylation sites were found in a2, a3, a4, a6, b2, b3 and b4 subunits of rice ( Fig. 2). In Arabidopsis, a2, a4, a6, a7, b1, b3 and b4 subunits have been reported to contain putative N-glycosylation sites (Parmentier et al., 1997). The sequences and locations
of the putative glycosylation motifs were conserved between rice and Arabidopsis for a2, a4, and b3 subunits. One of the two glycosylation sites found in the Arabidopsis b4 subunit was also conserved in the rice b4 subunit. This study represents the first set of the 20S proteasome subunit genes, seven a-types and seven b-types, in monocots, and serves genetic tools for the functional study of the 20S proteasome in rice. Based on extensive search of the large collection of EST clones, the majority of the 20S proteasome subunits of Arabidopsis were shown to be encoded by at least two genes ( Fu et al., 1998). This finding is in contrast to the molecular organization of the 20S proteasome subunit genes in yeast, where all the subunits are encoded by a single gene, and most of them are essential for yeast viability (Coux et al., 1996). Although the reason why the 20S proteasome subunit genes of Arabidopsis are duplicated is not yet clear, members of each a-type and b-type family encode proteins that are capable of imparting distinct proteolytic and/or biological functions to the 20S complex ( Fu et al., 1998). A further search of the growing collection of rice EST clones will be needed to determine whether 20S proteasome subunit genes are also duplicated in rice and to assess how they are implicated in the functions of plant proteasomes.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Baumeister, W., Walz, J., Zu¨hl, F., Seemu¨ller, E., 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380. Coux, O., Tanaka, K., Goldberg, A.L., 1996. Structure and functions of the 20S proteasomes. Annu. Rev. Biochem. 65, 801–847. DeVries, S., Hoge, H., Bisseling, T., 1988. Isolation of total and polysomal RNA from plant tissues. In: Glevin, S.B., Schilperoot, R.A.,
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Verma, D.P.S. ( Eds.), Plant Molecular Biology Manual. Kluwer Academic, Dordrecht, pp. 1–13. Fu, H., Doelling, J.H., Arendt, C.S., Hochstrasser, M., Vierstra, R.D., 1998. Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana. Genetics 149, 677–692. Genschik, P., Criqui, M.C., Parmentier, Y., Derevier, A., Fleck, J., 1998. Cell cycle-dependent proteolysis in plants: identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132. Plant Cell 10, 2063–2075. Girod, P.-A., Fu, H., Zryd, J.P., Vierstra, R.D., 1999. Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens. Plant Cell 11, 1457–1471. Hug, A.M., Klobeck, H.G., 1996. An improved 5∞ RACE technique facilitates the detection of multiple transcript initiation sites. Technical Tips Online, No. 40024. http://tto.biomednet.com. Ito, N., Tomizawa, K., Matsui, M., Kendrick, R.E., Sato, T., Nakagawa, H., 1997. Characterization of 26S proteasome a- and b-type and ATPase subunits from spinach and their expression during early stages of seedling development. Plant Mol. Biol. 34, 307–316. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Nederlof, P.M., Wang, H.R., Baumeister, W., 1995. Nuclear localization signals of human and Thermoplasma proteasomal a subunits are functional in vitro. Proc. Natl. Acad. Sci. USA 92, 12060–12064.
Parmentier, Y., Bouchez, D., Fleck, J., Genschik, P., 1997. The 20S proteasome gene family in Arabidopsis thaliana. FEBS Lett. 416, 281–285. Roberts, B., 1989. Nuclear location signal-mediated protein transport. Biochim. Biophys. Acta 1008, 263–280. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sasaki, T., et al., 1994. Toward cataloguing all rice genes: large-scale sequencing of randomly chosen rice cDNA from a callus cDNA library. Plant J. 6, 615–624. Sassa, H., Hirano, H., 1998. Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR 5-like protein in Japanese pear (Pyrus serotina Rehd.). Planta 205, 514–521. Tanaka, K., Yoshimura, T., Tamura, T., Fujiwara, T., Kumatori, A., Ichihara, A., 1990. Possible mechanism of nuclear translocation of proteasomes. FEBS Lett. 271, 41–46. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Umeda, M., Fujii, N., Manabe, Y., Uchimiya, H., 1997. Molecular and biochemical characterization of a proteasome subunit from rice and carrot cells. Mol. Gen. Genet. 255, 19–27. Woffenden, B.J., Freeman, T.B., Beers, E.P., 1998. Proteasome inhibitors prevent tracheary element differentiation in zinnia mesophyll cell cultures. Plant Physiol. 118, 419–430.
Primary structural features of the 20S proteasome subunits of rice (Oryza sativa) Hidenori Sassa 1, So Oguchi 1, Takeshi Inoue, Hisashi Hirano * Kihara Institute for Biological Research & Graduate School of Integrated Science, Yokohama City University, Maioka 641-12, Totsuka-ku, Yokohama 244-0813, Japan Received 25 January 2000; received in revised form 4 April 2000; accepted 20 April 2000 Received by T. Sekiya
Abstract The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins, and it also has other diverse biological functions. Its structure comprises 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and two inner rings form an a7b7b7a7 structure, and each subunit, a and b, exists as seven different types, thus giving 14 kinds of subunits. In this study, we report the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all of the subunits from monocots. Amino acid sequence homology within the rice family (a-type: 28.9–42.1%; b-type: 17.2–31.9%) were lower than those between rice subunits and corresponding orthologs from Arabidopsis and yeast (a-type: 49.2–94.5%; b-type: 34.8–87.7%). Structural features observed in eukaryotic proteasome subunits, i.e., a- or b-type signature at the N-termini, Thr active sites in b1, b2 and b5 subunits, and nuclear localization signal-like sequences in some a-type subunits, were shown to be conserved in rice. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Oryza sativa; 20S proteasome subunits
1. Introduction The proteasome is a multicatalytic proteinase complex that is involved in an ATP/ubiquitin-dependent proteolytic pathway. In eukaryotes, there are two types of complexes, with sedimentation coefficients of 20S (20S proteasome) and 26S (26S proteasome). The 26S proteasome consists of two 19/22S regulatory complexes and the 20S proteasome as the catalytic core. The 20S proteasome subunits can be classified, on the basis of sequence similarities, into two families, a and b. Eukaryotic proteasomes contain 14 different subunits, seven a-type subunits and seven b-type subunits. The subunits are arranged into four seven-membered rings, with the a-type subunits forming the two outer rings and the b-type subunits, the two inner rings. Collectively, they form a barrel-shaped catalytic 20S complex (Coux Abbreviations: PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends. * Corresponding author. Tel.: +81-45-820-1904; fax: +81-45-820-1901. E-mail address: [email protected] (Hisashi Hirano) 1 These authors contributed equally to this work.
et al., 1996; Baumeister et al., 1998). In yeast and mammals, proteasomes have been shown to be involved in not only degradation of misfolded or truncated proteins, but also diverse biological functions including cell-cycle progression (Coux et al., 1996). However, in plants, limited findings are available for not only the biological roles of the proteasome (Genschik et al., 1998; Woffenden et al., 1998; Girod et al., 1999), but also the primary structural features of the subunits of the 20S unit (Ito et al., 1997; Parmentier et al., 1997; Umeda et al., 1997; Fu et al., 1998). The complete amino acid sequences of all of the 20S proteasome subunits, seven a- and seven b-types, were recently reported for Arabidopsis thaliana (Parmentier et al., 1997; Fu et al., 1998), but not yet for other plant species. We have analyzed a crude preparation of 20S proteasome of rice (Oryza sativa L. cv. Nipponbare) by twodimensional gel electrophoresis followed by protein microsequencing, and identified seven a- and seven b-type subunits on the 2D-gel with their partial amino acid sequences (Ito, K., Nakazawa, M., Sassa, H., Hirano, H., manuscript in preparation). In this study, we report the primary structural features of the seven
0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 19 0 - 6
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H. Sassa et al. / Gene 250 (2000) 61–66
a-type and seven b-type subunits of the 20S proteasome of rice, representing the first set for all of the subunits from monocots.
GAG CC-3∞), and b6 (b6R1: 5∞-CCA TGG CAG GGC AGC TCA TC-3∞, b6R2: 5∞-TTG TAG TAC AGG GTA TTG GAG AG-3∞). Amplified RACE products were cloned in pT7Blue-T vector (Novagen).
2. Materials and methods
2.3. Sequence analysis
2.1. Identification of rice cDNAs encoding 20S proteasome subunits
EST clones and RACE clones were sequenced for both strands by using a DNA sequencer (model 4000; Li-Cor, Lincoln, NE ). The deduced amino acid sequences were aligned by Clustal X ( Thompson et al., 1997). Phylogenetic trees of a- and b-subunit families were generated by the neighbor-joining method (Saitou and Nei, 1987). Pairwise comparisons of sequence homology were conducted using the Genetyx-Mac ver. 10 computer program (Software Development, Tokyo, Japan).
We have previously analyzed crude preparation of rice 20S proteasome by two-dimensional gel electrophoresis followed by amino acid sequence analysis, and determined partial amino acid sequences of seven a-type and seven b-type subunits (Ito et al., unpublished data). By using the partial amino acid sequences of the rice 20S proteasome subunits, we searched for rice EST clones encoding 20S proteasome subunits using the BLAST program (Altschul et al., 1990). Deduced amino acid sequences of recently published 20S proteasome subunits of Arabidopsis (Parmentier et al., 1997; Fu et al. 1998) were also used as queries to search the rice EST clones. Identified EST clones from a japonica rice cultivar Nipponbare (Sasaki et al., 1994) were provided from a DNA bank at the Ministry of Agriculture, Forestry, and Fisheries of Japan (http://bank.dna. affrc.go.jp/). 2.2. RNA extraction and RACE (rapid amplification of cDNA ends) Seeds of a japonica rice (cv. Nipponbare) were surface-sterilized and hydroponically grown in 1/4 strength Murashige–Skoog medium (Murashige and Skoog, 1962). Total RNA was extracted from the roots of 1 week old seedlings by the hot-phenol method (DeVries et al., 1988). First-strand cDNA for 3∞RACE was synthesized from the total RNA by using a (dT ) adapter 17 primer essentially as described before (Sassa and Hirano, 1998). 3∞RACE was conducted by using an adapter primer (5∞- CGA CGT TGT AAA ACG ACG GCC AGT -3∞) and gene-specific primers for respective subunit genes: b3 (b3F: 5∞- ATG TCG ATC TTC GAG TAC AAT GG-3∞), b4 (b4F: 5∞- GCA GTT CAC CGA GTT CAT CC-3∞), and b6 (b6F1: 5∞-ACG AGA ACA ACG GCG GGA C-3∞). 5∞RACE was performed by the method of Hug and Klobeck (1996) or by using CapSite cDNA (Nippon Gene, Toyama, Japan) derived from shoots of Nipponbare. Gene-specific primers used for 5∞RACE were as follows: a1 (a1RiOg: 5∞-TGT CCA AAG AAG TGA CCT GC-3∞; a1RoOg: 5∞-TAA GAC CGG CGC TAG TTG CC-3∞), b3 (b3RiOg: 5∞-ACA GCT TGT GCC TGA ACA CCA GG-3∞; b3RoOg: 5∞-ACA GGC TGG CAG AAG TAT GGT CC-3∞), b4 (b4RiOg: 5∞-ATA GTA CGG ATT CTT CCG CAG GG-3∞; b4RoOg: 5∞-AGC GAG AGA CAG AAG TAG
3. Results and discussion Based on the partial amino acid sequences of the rice 20S proteasome subunits (Ito et al., unpublished results) and recently published cDNA sequences of Arabidopsis (Parmentier et al., 1997; Fu et al., 1998), DNA databases were searched by the BLAST program, and candidate EST clones for 10 subunits of a japonica rice cultivar ‘Nipponbare’ (Sasaki et al., 1994) were identified; a1 (Accession No. D49098), a2 (Accession No. D40076), a3 (Accession No. D41015), a4 (Accession No. C74810), a5 (Accession No. D24985), a7 (Accession No. D40644), b1 (Accession No. D41150), b2 (Accession No. D15408), b5 (Accession No. D48392) and b7 (Accession No. D15785). The cDNA sequence of the a6 subunit of japonica rice (cv. Yamahousi) was recently reported ( Umeda et al., 1997). The 10 EST clones were provided from the DNA bank at the Ministry of Agriculture, Forestry and Fisheries of Japan, completely sequenced, and found to encode the fulllength amino acid sequences of the respective 20S proteasome subunits, except for the a1 subunit (D49098). The EST clone (D49098) encoded the rice a1 subunit, but was not the full-length cDNA as it was found to lack a part of the sequence encoding the N-terminal region. In order to obtain the lacking part, we conducted 5∞RACE by using RNA derived from young root of Nipponbare. For the b4 subunit, an identified EST clone (Accession No. D24758) was not available. For b3 and b6 subunits, corresponding EST clones could not be identified from japonica rice, but from indica rice (b3, Accession No. AA752242) or maize (b6, Accession No. T18278). Primers were designed from the three EST sequences, and used for 3∞ or 5∞RACE to obtain b3, b4, and b6 cDNA sequences. Finally, primary structures of all the subunits of the 20S proteasome of japonica rice were determined by complete sequencing of the EST clones
H. Sassa et al. / Gene 250 (2000) 61–66
and RACE products. Designations of the 20S proteasome subunits of rice were based on those of Arabidopsis (Fu et al., 1998), i.e., OsPAA1 represents proteasome a1-1 subunit of Oryza sativa. The Accession Nos of the cDNAs encoding the rice 20S proteasome subunits are OsPAA1 (AB026558), OsPAB1 (AB026559), OsPAC1 (AB026560), OsPAD1 (AB032061), OsPAE1 (AB026561), OsPAG1 (AB026562), OsPBA1 (AB026563), OsPBB1 (AB026564), OsPBC1 (AB026565), OsPBD1 (AB026566), OsPBE1 (AB026567), OsPBF1 (AB014058) and OsPBG1 (AB026568). OsPAF1 (D49098) had previously been cloned and designated as the C2 subunit of rice ( Umeda et al., 1997). Based on comparisons of the derived amino acid sequences from rice, Arabidopsis and yeast, phylogenetic trees for a- and b-families of 20S proteasome subunits were generated ( Fig. 1). The phylogenetic trees showed that each rice gene was closely related to each of the seven a and seven b subfamily genes in Arabidopsis and yeast and formed seven subfamily-specific groups together with corresponding Arabidopsis and yeast genes in the trees. All rice a and b subunits were more homologous to those of corresponding subfamilies of other organisms, Arabidopsis and yeast, than to other subunits of rice, as observed in Arabidopsis genes ( Table 1; Parmentier et al., 1997; Fu et al., 1998). These findings suggest that the rice genes for 20S proteasome subunits are the orthologs of each of Arabidopsis and yeast. An alignment of the deduced amino acid sequences of the a-subunit family of rice and Arabidopsis is shown in Fig. 2. Pairwise comparisons of sequence homology of a-type subunits are shown in Table 1. All of the seven a-type subunits of rice were found to have the a-type signature at their N-termini. The amino acid sequence homology within the rice family (28.9–42.1%) was lower than that between rice subunits and corresponding orthologs from Arabidopsis and yeast (49.2–94.5%; Fig. 1 and Table 1). Some a-type subunits of eukaryotes and Thermoplasma have been shown to bear putative nuclear localization signals (NLS ) and are assumed to be involved in the nuclear translocation of proteasomes ( Tanaka et al., 1990; Nederlof et al., 1995). The probable NLS sequence ( X–X–K–K(R)–X–K(R); Roberts 1989) was found in the a1 subunit of rice ( K–K–M–K ). Parmentier et al. (1997) and Fu et al. (1998) suggested similar NLS-like sequences of clusters of basic amino acids in Arabidopsis a-type subunits, a2 (R–K–S–R–K ) and a1 ( K–K–V–P–D–K ), respectively. The a1 and a2 subunits of rice were also found to have the putative NLS sequences at the corresponding position. These putative NLS sequences in the a-type subunits are possibly involved in the nuclear import of the proteasome in rice. An alignment of the amino acid sequences of b-type subunits of rice is also presented in Fig. 2. The b-type
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Fig. 1. Phylogenetic trees of the 20S proteasome subunits from rice (Oryza sativa), Arabidopsis, and yeast (S. cerevisiae). (A) a subunits; (B) b subunits. The phylogenetic trees of amino acid sequences were generated by using ClustalX ( Thompson et al., 1997). The distance along the horizontal axis separating two sequences is proportional to the divergence between the sequences. The amino acid sequence homology between Arabidopsis or yeast subunits and the corresponding rice subunits is indicated in parentheses on the right.
signature motif was conserved in all of the seven subunits in rice. The amino acid sequence homology within the rice family (17.2–31.9%) was lower than that between rice subunits and corresponding orthologs from Arabidopsis and yeast (34.8–87.7%; Fig. 1 and Table 1). The b-type subunits participate in the proteolytic activity of proteasomes. In eukaryotes, three b-type subunits (b1, b2, and b5) were shown to be autocatalytically processed to remove their propeptides, and their resultant N-terminal Thr residues to form the catalytic site of the 20S proteasome (Baumeister et al. 1998). The
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Fig. 2. Amino acid sequence alignments of the 20S proteasome subunits of rice. (A) a subunits; (B) b subunits. The proteasomal a-type and b-type signatures were determined by using the ‘PROSITE’ database (http://www.expasy.ch/tools/scnpsit1.html ) and are boxed. Putative nuclear localization signals (NLS) are in reverse type. Potential N-glycosylation sites are enclosed by circles. The propeptide sequences of b-type subunits were identified by comparison between the results of N-terminal sequencing (Ito et al., unpublished results) and sequences deduced from cDNA, and are indicated by lower-case letters.
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H. Sassa et al. / Gene 250 (2000) 61–66 Table 1 Amino acid sequence homology (percentage identity) of the 20S proteasome subunits from rice a subunits
OsPAB1
OsPAC1
OsPAD1
OsPAE1
OsPAF1
OsPAG1
OsPAA1 OsPAB1 OsPAC1 OsPAD1 OsPAE1 OsPAF1
33.8
35.0 37.7
36.6 39.3 40.6
32.6 33.8 42.1 39.2
32.9 32.1 30.9 32.8 38.3
28.9 30.3 34.9 33.7 36.3 40.4
b subunits
OsPBB1
OPsPBC1
OPsPBD1
OsPBE1
OsPBF1
OsPBG1
OsPBA1 OsPBB1 OsPBC1 OsPBD1 OsPBE1 OsPBF1
31.9
22.2 17.2
23.8 19.0 20.3
30.9 30.8 23.4 23.1
27.2 24.0 31.4 25.7 26.5
23.8 20.5 19.3 23.3 23.6 19.6
N-terminal amino acid sequences of rice b1, b2, and b5 subunits (Ito et al., unpublished results) and their comparison to the amino acid sequences deduced from the cDNAs showed that these three catalytic subunits of rice are cleaved between a Gly/Thr pair to expose the Thr active sites in the same way as in the proteasome of yeast (Baumeister et al., 1998). The result of the present study on the rice proteasome supports the prediction of cleavage sites for the three active b1, b2, and b5 subunits of Arabidopsis based on sequence alignment (Fu et al., 1998) and provides experimental evidence that the active b-type subunits of plants are processed in the same way as those of other eukaryotes. Our protein sequence analysis of the rice 20S proteasome subunits showed that the b7 subunit had a free N-terminal, as did the b1, b2, and b5 subunits but that the N-terminals of b3, b4, and b6 subunits were blocked (Ito et al., unpublished results). Comparison of the b7 N-terminal sequence determined by protein sequencing and that deduced from the cDNA showed that the b7 proprotein is cleaved between R-29 and T-30 to release the propeptide. This evidence for the cleavage site of rice b7 propeptide supports the previous prediction for the cleavage site of the b7 subunit of Arabidopsis (Fu et al., 1998). Fu et al. (1998) predicted that the cleavage site of the Arabidopsis b6 subunit is between Glu-4 and His-5, based on the finding that the yeast b6 subunit is cleaved between Asp/His. In rice, however, the cleavage site of the b6 subunit could not be predicted because of the low sequence similarity between the b6 subunit of Arabidopsis and that of rice around the probable cleavage site, and because of the N-terminal block of the rice b6 subunit (Ito et al., unpublished results). Putative N-glycosylation sites were found in a2, a3, a4, a6, b2, b3 and b4 subunits of rice ( Fig. 2). In Arabidopsis, a2, a4, a6, a7, b1, b3 and b4 subunits have been reported to contain putative N-glycosylation sites (Parmentier et al., 1997). The sequences and locations
of the putative glycosylation motifs were conserved between rice and Arabidopsis for a2, a4, and b3 subunits. One of the two glycosylation sites found in the Arabidopsis b4 subunit was also conserved in the rice b4 subunit. This study represents the first set of the 20S proteasome subunit genes, seven a-types and seven b-types, in monocots, and serves genetic tools for the functional study of the 20S proteasome in rice. Based on extensive search of the large collection of EST clones, the majority of the 20S proteasome subunits of Arabidopsis were shown to be encoded by at least two genes ( Fu et al., 1998). This finding is in contrast to the molecular organization of the 20S proteasome subunit genes in yeast, where all the subunits are encoded by a single gene, and most of them are essential for yeast viability (Coux et al., 1996). Although the reason why the 20S proteasome subunit genes of Arabidopsis are duplicated is not yet clear, members of each a-type and b-type family encode proteins that are capable of imparting distinct proteolytic and/or biological functions to the 20S complex ( Fu et al., 1998). A further search of the growing collection of rice EST clones will be needed to determine whether 20S proteasome subunit genes are also duplicated in rice and to assess how they are implicated in the functions of plant proteasomes.
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