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

Identification of the 19S regulatory particle subunitsfrom the rice 26S proteasome Tadashi Shibahara, Hiroshi Kawasaki and Hisashi Hirano Yokohama City University, Kihara Institute for B

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 26S 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 six different ATPase subunits and,

at least, 11 non-ATPase subunits In this 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, OsRpt4, OsRpt5 and OsRpt6,

and five non-ATPase subunits, OsRpn7, OsRpn8, OsRpn10,

OsRpn11and 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, OsRpt4 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 The 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 were also identified Various types

of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome

Keywords: proteasomes; rice; gene duplication; purification; immunoaffinity chromatography

All organisms possess highly selective proteolytic systems

which are essential for cellular functions such as cell cycle

progression and apoptosis and also remove abnormal and

unnecessary intracellular proteins A major proteolytic

system of them in both cytoplasm and 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 19S

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 triple A-ATPases) and Rpn (RP non-ATPases), respectively [1] Eight of them, six Rpt subunits and two 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 covers the base and provides the binding or recognition specificity for ubiqui-tinated substrates [2] ATP hydrolysis in RP is indispensable

to assemble the 26S holocomplex, to recognize appropriate substrates, and 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 xylem 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 of the RP subsub-units in plants remains unclear

In this study, we identified the rice homologs of the yeast

RP subunit genes from the rice EST library, determined their nucleotide sequences, and found that their products assembled in an RP complex of rice

E X P E R I M E N T A L P R O C E D U R E S Identification of cDNAs

EST clones encoding the rice RP subunits were identified from the GenBank EST database byTBLASTNin theBLAST

program of NCBI network service, with 17 protein sequences of yeast (Saccharomyces cerevisiae) RP subunits

as queries The EST clones were provided by the DNA bank

Correspondence to H Kawasaki, Yokohama City University, Kihara

Institute for Biological Research/Graduate School of Integrated

Science, Maioka 641-12, Totsuka-ku, Yokohama 244-0813, Japan.

Fax: + 81 45 820 1901, Tel.: + 81 45 820 1904,

E-mail: kawasaki@yokohama-cu.ac.jp

Abbreviations: AAA, ATPases associated with various cellular

activities; CTAB, cetyltrimethylammonium bromide; EST, expressed

sequence tag; Q-TOF, quadrupole time-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); lysylendopeptidase

(EC 3.4.21.50).

Note: The novel nucleotide sequences reported here have been

sub-mitted to DDBJ with the accession numbers AB033535–AB033537,

AB037149–AB037155, AB070252–AB070262 and AB071016.

(Received 4 January 2002, accepted 17 January 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) at

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 EST 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 sequencer

(model 4000 L, LI-COR, Lincoln, NE, USA) The

nucleo-tide sequence information was analyzed by GENETYX

(Software Development, Tokyo, Japan)

Isolation and gel blot analysis of genomic DNA

Genomic DNA was extracted from mature leaves of 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 Biotech, 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 Module (Amersham Pharmacia

Biotech, Uppsala, Sweden) All hybridization reactions

were performed at 55 or 70°C with AlkPhos Direct

Labeling Module Chemiluminescent signals were generated

with CDP-Star (Amersham Pharmacia Biotech, Uppsala,

Sweden), and detected with Hyperfilm ECL (Amersham

Pharmacia Biotech, Uppsala, 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, and 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 mM potassium phosphate

buffer, pH 7.5, and the protein solution was concentrated to

1 mgÆmL)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 20S proteasome) Ig and Protein A beads were cross-linked with disuccinimidyl suberate (DSS) (Pierce, Rockford, IL, USA) Two milliliters (80 mgÆmL)1) of the anti-(rice 20S proteasome) serum were mixed with equal volume of binding/washing buffer containing 140 mM

NaCl, 8 mM Na2PO4, 2 mM potassium phosphate and

1 mMKCl, pH 7.4, and loaded onto HiTrap rProtein A FF

1 mL (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with binding/washing buffer The flow-through was completely washed with binding/washing buffer The DSS was dissolved in dimethylsulfoxide 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 then capped, and the column was kept at room temperature for 1 h The nonreacted DSS was eluted with quenching/washing buffer containing

25 mMTris and 0.15MNaCl, 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, and 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 containing

100 mMTris/HCl, pH 7.5, 20% (v/v) glycerol, 10 mMATP,

10 mMMgCl2, 10 mMEGTA, 14 mM2-mercaptoethanol and protease inhibitor cocktail tablets EDTA free (Roche Molecular Biochemicals, Mannheim, Germany), and grin-ded with an ice-cold mortar and a pestle The homogenate was then filtrated with a nylon mesh, and centrifuged at

12 000 g for 20 min The supernatant was centrifuged again,

at 40 000 g for 20 min The supernatant was ultracentri-fuged at 80 000 g for 1 h, and then at 370 000 g for 5 h The pellet was dissolved in a suitable volume of the extraction buffer and the insoluble material was removed by centri-fugation at 12 000 g for 20 min The 3-mL samples (300 mg

of protein) were loaded onto a HiTrap rProtein A FF column containing immobilized anti-(rice 20S proteasome)

Ig The column was equilibrated with the extraction buffer containing no protease inhibitor before sample loading The column was then washed with extraction buffer without protease inhibitor cocktail tablets EDTA free and the RPs were eluted with a RP elution buffer containing 100 mM

Tris/HCl, pH 7.5, 20% (v/v) glycerol, 10 mM EGTA,

14 mM2-mercaptoethanol and 1.0MNaCl 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)

Protein assay

The protein concentration of the sample was determined 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 mMTris/HCl buffer, pH 7.5, 500 mMNaCl and

0.05% Tween 20), the blots were incubated overnight with a

primary antibody (rabbit polyclonal anti-AtRpn6 Ig or

rabbit polyclonal anti-AtRpt5 Ig; Affinity Research

Prod-ucts, Mamhead Castle, UK) These primary antibodies were

then detected with alkaline phosphatase-conjugated goat

anti-(rabbit IgG) Ig (Vector Laboratories, 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 completely and then

rehydrated with digestion buffer containing 100 mMTris/

HCl, pH 9.0 and 1 ngỈlL)1 of lysylendopeptidase (Wako

Pure Chemical Industries, Osaka, Japan) The protein was

digested at 37°C for 18 h After digestion, 1 lL of acetic

acid was added to the buffer to stop the reaction The buffer

was collected in a tube and the 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 TipC18

(Millipore, Bedford, MA, USA), and then eluted with 1%

(v/v) formic acid/70% (v/v) acetonitrile The 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 were analyzed byMASSLYNXv3.4

software (Micromass, Manchester, UK) The MS spectra of

each subunit and MS/MS spectra of each peptide fragment

were processed by a maximum entropy data enhancement

program,MAXENT3, a component ofMASSLYNXv3.4 The

spectra of MS and MS/MS deconvoluted withMAXENT3

were used for the peptide mass fingerprinting and

amino-acid sequencing, respectively

R E S U L T S

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 of the yeast Rpt subunit genes, ScRpt1, ScRpt2 and ScRpt5, respectively Similarly, five EST clones, R3615, S13278, S13105, S4633 and E1287, contained the entire coding regions structurally similar to those of the yeast Rpn subunit genes, ScRpn7, ScRpn8, ScRpn10, ScRpn11 and ScRpn12, respectively However, the other 16 EST clones, E61121, C2890, E0641, C50126, R2695, E0746, R1494, E40363, E50789, E1935, C10189, C10401, S20324, C1087, C50129 and R1547, had parts of the coding regions structurally similar to those of ScRpt and ScRpn genes (Table 1) The 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, and ScRpn9, respectively, was complemented by the results of cDNA sequencing in the rice genome research project (Table 1)

On the other hand, in our study, the full length of homologs of ScRpt4 and ScRpt6 were cloned by 5¢ RACE using C50126 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 genes, 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, the 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 suffixes, ƠaÕ and ƠbÕ, e.g OsRpt1aand OsRpt1b as shown in Table 1

Rice genomic DNA was prepared from a plant developed 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 OsRpt6 in the EcoRV digests (Fig 1A) These results indicated that each gene has a single copy in the genome As OsRpt6has an EcoRV site in the cDNA sequence deter-mined, the probe of OsRpt6 must hybridize to two bands of the EcoRV digest (Fig 1A) OsRpt3 and OsRpt5b have no EcoRV site in the cDNA sequence, but the probes of each gene hybridized to two bands This probably means that OsRpt3and OsRpt5b have an intron containing one EcoRV site (Fig 1A) In hybridization under normal conditions (55°C), all the gene-specific probes, except OsRpt3, cross-hybridized weakly to the DNA fragments identical to those detected with the probes for another member of the 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,

several fragments were additionally detected in all three

digests with the OsRpt6-specific probe under normal

conditions (Fig 1A) This suggests that the rice genome

has another gene closely related to OsRpt, such as other

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 the 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 Da

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

ArabidopsisRpt5 and Rpn6 (Fig 3) In this experiment, two

bands that cross-reacted with the antibodies were detected

on the gel, suggesting that these bands contain OsRpt5 and

OsRpn6

The rice RP subunits were separated by SDS/PAGE, and

14 bands were detected on the gel (Fig 2B) The gel piece containing each band was removed and soaked in the lysylendopeptidase digestion buffer to digest into peptides The resultant peptides were collected and subjected to ESI-Q-TOF MS and MS/MS analyses In this analysis, the products of all the rice RP genes obtained from the EST library were found in the RP complex purified 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)

In the rice RP, the relative molecular masses of the 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 those of theor-etical ones The relative molecular masses of 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 ND, not determined.

Genea

Predicted peptide length (aa)/MW (kDa)/pI

Representative EST

Nucleotide length

of EST (bp)

Accession number

Full length cDNA clone

a Adapted from Finley et al [1] b EST accession number c previously reported Suzuka et al [19] d Not encoded full length.

The isoforms of OsRpt subunits ƠaÕ and ƠbÕ 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 identified 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) For 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 ionization 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 of 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 the

products of these genes by MS analysis

D I S C U S S I O N

In the present study, we identified 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 of the homologs have identical sequences to TBPOs-2 (OsRpt2a), TBPOs-1 (OsRpt5a) and OsS5a (OsRpn10), which have been reported as homologs of rice proteasome subunit genes found in other organisms [19,20] The rice RP subunits possess the amino-acid sequence motifs commonly found in RP subunits of the other eukaryotes For 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 PUbS2 (OsRpn10), and Cys box (OsRpn11) as reported

in other eukaryotes [22,23]

Duplicated genes of OsRpt1, OsRpt2, 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 for 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 blot analysis of genes encoding six OsRpt 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 were separated by agarose gel electrophoresis Each band marked by an arrow represents a genomic DNA fragment which corresponds to the gene-specific probe used in the panel (A) Hybridized under highly stringent condition (70 °C) (B) Hybridized under normal condition (55 °C).

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 RP complex from the rice bran was

purified by immunoaffinity chromatography with a

column immobilized antibody against the rice 20S

proteasome As RP attaches at the 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 specifically 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, we

identified the products of all rice homologs obtained

from the EST library, including both products of the

duplicated OsRpt genes 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 subunits, resulting

in the immuno-proteasome [26] Dahlmann et al reported

that there are five subtypes of the 20S proteasome in the

rat skeletal muscle, including immuno-proteasome These

subtypes exhibit different substrate specificities [27] In

rice, the different types of RP complex, each of which

contains only one of the products of duplicated OsRpt

genes, may have specific 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 Lane 1, molecular mass marker stained with CBB; lane 2, detected with anti-AtRpt5 Ig; lane 3, detected with 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 CBB Lane 1, molecular mass marker; lane 2, purified rice

20S proteasome (B) Purified rice RP resolved by SDS/PAGE

and stained with CBB Lane 1, molecular mass marker; lane 2, purified

rice RP.

Table 2 Protein identification of the rice 19S regulatory particle sub-units.

* Protein without sequence homology to RP subunits.

Table 3 Identification of the rice 19S regulatory particle subunits by ESI-Q-TOF MS myri, N-myristoylaton; ac, N-acetylation.

a

B and J were defined as oxidation of Met and acrylamidation of Cys, respectively.

Post-translational modifications of the proteasome, 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 Table 3) Rpt2 from

various organisms such as human, yeast, Arabidopsis,

Caenorhabditisand Drosophila, have the consensus motif

(M-G-X-X-X-S/T-X-X-X) for N-myristoylation at the

N-terminus [34] Although 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 myristoylated 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 regulate protein 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 study, we detected 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, a2 and a5 [37,38] We found differences between observed molecular masses and theoretical values in the rice

RP subunits OsRpn3 was identified 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 the carrot Rpn3 was 55 000 Da, but the observed mass was 45 000 Da [39]

It is likely that 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 interacts with the N-terminal region 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 similarity to RP subunits These

Fig 4 Determination of N-terminal

modifica-tion of OsRpt2 The MS/MS spectrum

deconvoluted with MaxEnt3 from doubly

charged ion at m/z 558.29 of OsRpt2 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 by ESI-Q-TOF B, oxidation of Met J, acrylamidation of Cys Substituted amino-acid residues between a and b are indicated in bold.

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 proteasome, probably related

to the functions specific to the rice proteasome

A C K N O W L E D G E M E N T

This work was supported in part by the Ministry of Agriculture,

Forestry and Fisheries of Japan (Rice Genome Project PR-1404).

R E F E R E N C E S

1 Finley, D., Tanaka, K., Mann, C., Feldmann, H., Hochstrasser,

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Fig 5 ESI-Q-TOF MS analysis of OsRpt2 (A) ESI-Q-TOF

spec-trum of OsRpt2 digested with lysylendopeptidase between m/z 772 and

800 Two pairs of peptide fragments with amino acid substitutions are

shown with * and #, respectively (B) The MS/MS spectrum of

deconvoluted with MaxEnt3 from doubly charged ion at m/z 795.92.

Major b-series ions are indicated (C) The MS/MS spectrum of

deconvoluted with MaxEnt3 from doubly charged ion at m/z 774.42.

Major b-series ions are indicated The identified amino-acid sequences

are shown above the figures.

human immunodeficiency virus-1 Tat binding protein and subunit

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