Báo cáo Y học: Identification of the 19S regulatory particle subunits from the rice 26S proteasome potx

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:
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
( 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. It is possible that the rice cells have
compositional variation in the p roteasome, p robably related
to the functions specific to the rice proteasome.
ACKNOWLEDGEMENT
This work was supported in part by the Ministry of Agriculture,
Forestry and Fisheries of Japan (Rice Genome Project PR-1404).
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