The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants
ABSTRACT In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.Molecular and Cellular Biology 07/1998; 18(6):3149-62. · 5.37 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.Archives of Biochemistry and Biophysics 03/2000; 374(2):181-8. · 3.37 Impact Factor
- Plant physiology 12/1996; 112(3):871-8. · 6.56 Impact Factor
Molecular Biology of the Cell
Vol. 12, 383–392, February 2001
The Cellular Level of PR500, a Protein Complex
Related to the 19S Regulatory Particle of the
Proteasome, Is Regulated in Response to Stresses in
Zhaohua Peng,* Jeffrey M. Staub,*†Giovanna Serino,* Shing F. Kwok,*‡
Jasmina Kurepa,§Barry D. Bruce,? Richard D. Vierstra,§Ning Wei,* and
*Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven,
Connecticut 06520-8104;?Department of Biochemistry and Cellular and Molecular Biology, University
of Tennessee at Knoxville, Knoxville, Tennessee 37996; and§Cellular and Molecular Biology Program,
University of Wisconsin-Madison, Madison, Wisconsin 53706
Submitted September 21, 2000; Revised November 29, 2000; Accepted December 19, 2000
Monitoring Editor: Elliot Meyerowitz
In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory
subunit) was found in two distinct protein complexes of ?800 and 500 kDa, respectively. The large
complex likely represents the proteasome 19S regulator particle (RP) because it displays the
expected subunit composition and all characteristics. The small complex, designated PR500,
shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with
an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or
reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was
also diminished in response to potential protein-misfolding stresses caused by the heat shock and
canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization
pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher
plants to cope with the frequently encountered environmental stresses.
The 26S proteasome is a major proteolytic device for selec-
tive protein breakdown in eukaryotes (Hershko and Ciech-
anover, 1998). This targeted protein degradation is one of the
most important means utilized by all eukaryotes to control
cellular protein quality, transcription, signal transduction,
cell cycle progression, and metabolic activities.
The 26S proteasome is composed of a 20S catalytic core
and two 19S regulatory particles on both ends. The 20S core
particle (20S CP) is a hollow cylinder with interior proteo-
lytic sites. The 19S regulatory particle (19S RP) is composed
of six ATPases and at least 11 non-ATPase subunits in yeast
(Baumeister et al., 1998; Glickman et al., 1998b). ATP can
stimulate association of 19S RP with the 20S CP to form the
26S proteasome that is capable of degrading ubiquitin-
tagged proteins (Orino et al., 1991). The 19S RP is believed to
direct substrate recognition and processing (ubiquitin bind-
ing, unfolding, and deubiquitination) before substrates are
funneled into the 20S CP for destruction (Coux et al., 1996;
Baumeister et al., 1998). The 19S particle of Saccharomyces
cerevisiae can be divided into two subcomplexes, the base
and the lid (Glickman et al., 1998a). The base subcomplex
consists of all six ATPases and Rpn1 and Rpn2 (the two
largest non-ATPase subunits), and is in direct contact with
the 20S CP. The lid subcomplex contains eight non-ATPase
subunits, Rpn3, Rpn5–9, Rpn11, and Rpn12. It covers one or
both ends of the 26S proteasome cylinders by contacting the
base subcomplex. Rpn10 seems to play a role in connecting
the lid to the base, because the lid can be easily disassociated
from the 26S proteasome when Rpn10 is absent (Glickman et
al., 1998a). More recently, a lid-like complex was isolated as
a proteasome purification by-product of human red blood
cells (Braun et al., 1999; Henke et al., 1999). However, there is
no report until now to indicate that a lid-like subcomplex is
present in vivo under physiological conditions in any organism.
On the other hand, the COP9 signalosome, a highly con-
served cellular regulator (Wei and Deng, 1999), exhibits a
remarkable resemblance to the lid subcomplex of the pro-
Present addresses:†Monsanto Company, 700 Chesterfield Park-
way North, Mail Code BB3G, St. Louis, MO 63017;‡Ceres, Inc., 3007
Malibu Canyon Road, Malibu, CA 90265.
¶Corresponding author. E-mail address: email@example.com.
© 2001 by The American Society for Cell Biology383
teasome (Glickman et al., 1998a; Seeger et al., 1998; Wei et al.,
1998). The COP9 signalosome also consists of eight subunits,
and each of the subunits shares significant sequence simi-
larities with a particular lid subunit in a one-to-one correla-
tion. This suggests that the COP9 signalosome and the lid
have likely evolved from a common ancestor (Wei and
Deng, 1999). Recently, structures of purified human COP9
signalosome and lid subcomplex were compared by electron
microscopy imaging (Kapelari et al., 2000). The result indi-
cates that both complexes lack any symmetry in subunit
arrangement and exhibit a central groove, although they do
not appear to have identical structure. Although the lid is
conserved throughout eukaryotes (Glickman et al., 1998a),
COP9 signalosome is found from fission yeast to plants and
mammals but is apparently absent from S. cerevisiae (Deng et
The COP9 signalosome was initially defined as a light-
inactivable repressor of photomorphogenesis in Arabidopsis
(Wei et al., 1994; Chamovitz et al., 1996). Mutant seedlings
lacking the complex exhibit a constitutive photomorpho-
genic phenotype, constitutive activation of light-responsive
gene expression, and lethality after the seedling stage (Wei
and Deng, 1996, 1999). In mammals, subunits of the COP9
signalosome have been implicated to regulate multiple cel-
lular signaling pathways and cell cycle progression (Wei
and Deng, 1999). Most recently, a role of the COP9 signalo-
some in the proteasome-mediated degradation of a key pho-
tomorphogenic regulator HY5 has been documented (Oster-
lund et al., 2000). Together, available data points to a
functional relation between the COP9 signalosome and the
proteasome. However, there is no information available yet
regarding the nature of this relationship.
As an initial step in searching the possible relationship
between those two complexes, we characterized the in vivo
forms of the Rpn6 protein and its possible regulation by the
COP9 signalosome. Here we report the identification and
purification of am Rpn6-containing complex named PR500.
PR500 stably exists in plant cells under physiological condi-
tions and localizes to distinct nuclear foci in plant cell nuclei.
We showed that PR500 is absent or significantly reduced in
stress conditions in which abnormal proteins are expected to
accumulate and in the COP9 signalosome mutants of Arabi-
dopsis, suggesting a role of PR500 in cellular stress response
MATERIALS AND METHODS
Plant Materials and Arabidopsis Strains
The fus6–1, fus6-G236, fus6-T236, cop8-1, cop10-1, and fus11-U203
mutants have been described (Castle and Meinke, 1994; Wei et al.,
1994; Kwok et al., 1996; Karniol et al., 1999; Serino et al., 1999). The
det1-1 and det1-8 were described by Pepper et al. (1994). Seeds were
planted on agar plates containing growth medium (GM) and 1%
sucrose (Wei et al., 1994) and were cold treated at 4°C for 7–12 d
before being transferred to growth chambers. The fluence rate of
white light was 156 ?mol m?2s?1. Cauliflower heads used for gel
filtration and complex purification were purchased fresh from a
Heat Shock and Canavanine Treatments
Five-day-old Arabidopsis seedlings, grown continuously in white
light on GM plates with 1% sucrose, were transferred from a 22°C
growth chamber to a 42°C chamber for 3 h or the time period
indicated. For the canavanine treatment experiment, cold-treated
Arabidopsis seeds were germinated on GM medium for 3 d. Then,
the seedlings were transferred to GM medium supplemented with
0 or 4 mg/l canavanine and grown for 2 d more under continuous
Protein Extraction and Gel Filtration
Arabidopsis seedlings or cauliflower tissue were homogenized in
liquid nitrogen (Arabidopsis) or in cold buffer (cauliflower) contain-
ing 50 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 2 mM dithiothreitol
(DTT), 10% glycerol, protease inhibitor cocktail (Boehringer Mann-
heim, Indianapolis, IN) and 25 (cauliflower) or 50 (Arabidopsis) mM
Tris-HCl, pH 7.5, with phenylmethylsulfonyl fluoride and ?-mer-
captoethanol added to 5 mM, respectively, just before use. The
homogenate was centrifuged for 10 min, and the supernatant was
filtered through a 0.2-?m filter (Gelman Sciences, Ann Arbor, MI)
before loading onto a Superose 6 (HR 10/30) gel filtration column
(Pharmacia, Piscataway, NJ). The column was equilibrated with a
PBSM buffer (1.76 mm KH2PO4, 10 mm Na2HPO4, 136 mm NaCl, 2.6
mm KCl, 2 mm MgCl2, and 10% glycerol) or Tris buffer (50 mM Tris,
pH 7.5, 50 mM NaCl, 5 mM EDTA, 10 mM MgCl2, 10% glycerol, and
1 mM DTT). Proteins were eluted in the same buffer for column
equilibration at a flow rate of 0.2 ml/min. All manipulations were
carried out at 4°C. Fractions of 0.25 or 0.5 ml were collected starting
from the onset of the column void volume (7.5 ml) and concentrated
using StrataClean Resin (Stratagene, La Jolla, CA) as described
before (Kwok et al., 1998). Equal volumes of each fraction were
subjected to SDS-PAGE followed by immunoblot analysis. The mo-
lecular mass standards were described previously (Kwok et al.,
1999). When ATP was included, it was 10 mM in the homogeniza-
tion buffer and 2 mM in subsequent gel filtration buffers, or as
indicated in the text.
Immunoblot Analysis and Immunoprecipitation
Western blot and immunoprecipitation analyses were carried out as
previously described (Staub et al., 1996), except that Tween-20 was
not included in the PBS buffer during immunoprecipitation, and a
final concentration of 0.55 M NaCl was included in the PBS washing
buffer. Rabbit polyclonal antibodies to Rpn6 and Rpt5 were de-
scribed previously (Kwok et al., 1999). The polyclonal antibodies
against Arabidopsis Mbp1/Rpn10 and Rpt1 (subunits of 19S RP)
were reported before (van Nocker et al., 1996). The monoclonal
antibodies against 21D7/Rpn3 was described by Smith et al. (1997).
The antibodies against human Rpt2 (S4) were kindly provided by
Dr. Carlos Gorbea (University of Utah Medical School, Salt Lake
City, UT). The rabbit polyclonal antibodies against hsp70 were
made against purified wheat germ hsp70 protein (Crookes and
Olsen, 1998). The antibodies against Rpn5a will be described sepa-
rately (Kurepa and Vierstra, unpublished data).
Arabidopsis Protoplast Immunofluorescence
The procedure for protoplast preparation and immunofluorescence
was as described previously (Matsui et al., 1995; Kwok et al., 1998).
Fresh seedlings were picked and immediately chopped with razor
blades to small pieces. The pieces were added to the solution con-
taining cell-wall–digesting enzymes as described by Matsui et al.
(1995). Purified rabbit polyclonal antisera against Rpn6, Rpt5, or
COP9 signalosome subunits were used for staining the protoplasts
after affinity purification (Kwok et al., 1998). Secondary antibody
was a goat anti-rabbit antibody conjugated to fluorescein isothio-
cyanate (Sigma, St. Louis, MO) and was used at a dilution of 1:400.
The samples were double stained for nuclei with 1 mg/ml 4?,6-
Peng et al.
Molecular Biology of the Cell384
Purification of the Cauliflower 19S and PR500
The surface layer of cauliflower heads was shaved and homoge-
nized with a commercial blender in buffer A (25 mM Tris-HCl, pH
7.5, 20 mM NaCl, 6 mM MgCl2, 5 mM EDTA, 5 mM ?-mercapto-
ethanol, 2 mM DTT, and 10% glycerol) containing 2 mM phenyl-
methylsulfonyl fluoride and the protease inhibitor cocktail (Boehr-
inger Mannheim). Homogenates were filtered through eight layers
of cheesecloth and centrifuged for 30 min at 13,000 ? g at 4°C.
Ammonium sulfate fine powder was added slowly to the superna-
tant to bring the ammonium sulfate concentration to 30% saturation.
The mixture was incubated at 4°C for 1 h and centrifuged for 30 min
at 13,000 ? g. The supernatant was filtered through a 2-?m pore size
nylon filter and was applied to a 300 ml phenyl Sepharose (Phar-
macia) column. The column was initially equilibrated with buffer B
(25 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 10% glycerol, and 1 mM
DTT) containing ammonium sulfate to 30% saturation and 10 mM
NaCl. The column was then washed with (?3 volumes) buffer B
containing a 15% saturation of ammonium sulfate. The Rpn6-en-
riched fractions were eluted with buffer B supplemented with 10
mM NaCl. The eluted fraction was passed through a P-DG gel
desalting column (Bio-Rad, Hercules, CA) to completely remove
trace amounts of ammonium sulfate. The sample was loaded onto a
Q-Sepharose ion exchange column and was washed (?3 volume)
with buffer B containing 150 mM NaCl. The complexes were eluted
with 350 mM NaCl in buffer B. The eluted fraction was then ad-
justed to a NaCl concentration of 100 mM with buffer B before
separation on a heparin affinity column (Pharmacia). The heparin
column was washed with 4 volumes of the loading buffer (buffer B
with 100 mM NaCl), and the Rpn6-associated complexes were
eluted with 350 mM NaCl in buffer B. The Rpn6-containing fraction
was adjusted to a NaCl concentration of 200 mM and loaded onto a
1 ml Mono-Q column (Pharmacia). A linear 200–400 mM NaCl
gradient elution (50 ml total) was used to separate the PR500 com-
plex (eluted at 240 mM) from the 19S complex (eluted at 270 mM).
The eluted fractions (1 ml each) containing PR500 and 19S particles
were pooled separately, and each of the complexes was further
purified on a Superose 6 HR columns in the PBSM buffer (1.76 mM
KH2PO4, 10 mM Na2HPO4, 136 mM NaCl, 2.6 mM KCl, 2 mM
MgCl2and 10% glycerol). The proteins were collected in 0.5-ml
fractions, and each fraction was concentrated by Strataclean beads
(Stratagene). The proteins were eluted from the beads by mixing
with 2? SDS gel-loading buffer and boiling for 3 min, separated by
a 10% SDS-PAGE, and visualized by silver or Coomassie blue
staining. In large-scale purification, the peak fractions of PR500 from
the Superose 6 HR gel filtration column were pooled and applied
onto a 1-ml heparin column for a gradient elution after the NaCl
concentration was adjusted. The linear gradient from 100–300 mM
NaCl was carried out over 20 ml in buffer B plus 0.25% Nonidet P-40
Rpn6 Is Present in 19S RP as Well as a Smaller
We recently reported the molecular characterization of Rpn6
(AtS9) and Rpt5 (AtS6A), a non-ATPase and an ATPase
subunit of the Arabidopsis 19S RP (Kwok et al., 1999). To
further examine their functional forms, we tested whether
Rpn6 is also present in any form other than in the 19S RP.
Gel filtration chromatography was used to size fractionate
crude plant extracts prepared from Arabidopsis seedlings or
cauliflower florets. In both cases, Rpn6 was eluted in two
separate complexes with estimated molecular masses of
?800 and 500 kDa in all buffers tested (Figure 1A). In con-
trast, Rpt5 was eluted in only a single 800-kDa complex
cofractionating with Rpn6 and was not found in any other
major form in the absence of ATP. The 800-kDa complex size
is similar to the reported molecular size of the mammalian
19S RP (Chu-ping et al., 1994; Sawada et al., 1997). Consistent
with Rpn6 and Rpt5 being subunits of the 19S RP, Rpn6 and
Rpt5 not only cofractionated but also coimmunoprecipitated
each other from plant extract (Kwok et al., 1999). These
results led us to hypothesize that the 800-kDa Rpn6- and
Rpt5-containing complex is the Arabidopsis and cauliflower
One characteristic of the 19S particle is its ATP-dependent
association with the 20S CP to form the 26S proteasome.
Therefore, we tested whether Rpn6 and Rpt5 could be in-
corporated into larger complexes in the presence of ATP. As
shown in Figure 1B, both Rpn6 and Rpt5 shifted from the
800-kDa complex toward larger molecular mass complexes
when ATP was present in the extraction and the subsequent
gel filtration buffers. Thus, the 800-kDa complex is likely to
be the 19S RP, and the higher molecular mass complexes
that we observed in the presence of ATP are the 26S protea-
some with one or two 19S RP. In contrast, the 500-kDa
Rpn6-containing complex is not affected by ATP (Figure 1B).
We tentatively designate this 500-kDa complex as PR500 for
proteasome-related 500-kDa complex. To further confirm
that PR500 indeed exists in vivo, different buffer strengths
(25, 50, and 200 mM Tris), different ion strengths (6 or 10 mM
Mg2?,and 20, 100, or 200 mM NaCl), and different buffer pH
(6–8.5) were also used to extract proteins and elute proteins
from gel filtration column. PR500 peak was constantly ob-
served (unpublished results).
PR500 Contains a Subset of the 19S RP Subunits
To ascertain whether the 800-kDa complex is the 19S regu-
lator and to reveal the composition of PR500, we purified
both complexes to near homogeneity from cauliflower (see
MATERIALS AND METHODS). As shown in Figure 2A, the
two complexes could be separated from each other in a
Mono-Q column by a fine salt gradient elution. Peak frac-
tions containing PR500 (fractions 10–13) or 19S RP (fractions
plants. Cell extract from cauliflower head was fractionated in a
Superose 6 gel filtration column. Selected elution fractions (Fr.) of
0.25 ml each were analyzed by immunoblot for Rpn6 and Rpt5. The
elution positions of molecular mass markers are shown (in kDa)
above the gel blots. (A) Cell extraction and subsequent size fraction-
ation were conducted under standard condition (see MATERIALS
AND METHODS) in which no ATP was present. (B) ATP was
included in the extraction buffer, the column equilibration buffer,
and elution buffer in otherwise identical experimental conditions as
in A. Note that Rpn6 is eluted in a peak at ?500 kDa in the presence
and absence of ATP and that both Rpn6 and Rpt5 cofractionate in an
800-kDa peak in the absence of ATP and in fractions of larger
molecular mass in the presence of ATP.
Rpn6 is present in two distinct protein complexes in
PR500: a Proteasome Lid-Like Complex in Plants
Vol. 12, February 2001 385
16–22) from the Mono-Q column were pooled. Each of the
complexes was further purified by Superose 6 size fraction-
ation, which also ensured that the complexes of native sizes
The purified 800-kDa complex contained ?19 major pro-
tein bands (Figure 2B). Its protein profile resembled that of
19S RP purified from animal sources (Chu-ping et al., 1994).
Its identity as the 19S RP was confirmed by immunoblot
analysis using seven antibodies that recognize four non-
ATPase (Rpn3, Rpn6, Rpn10, and Rpn5a) and three ATPase
subunits of the 19S RP (Rpt1, Rpt2, and Rpt5). All of these
antibodies specifically reacted with proteins of the expected
sizes (Figure 2C).
Because the PR500 preparation following the gel filtration
column still contained a few minor protein bands that did
not comigrate with PR500, an additional purification step by
heparin gradient chromatography in the presence of 0.25%
Nonidet P-40 was carried out (see MATERIALS AND
METHODS). This resulted in nine distinct cofractionating
bands, ranging from 30–63 kDa (Figure 2B). A direct com-
parison of the protein profiles of 19S RP and PR500 com-
plexes (Figure 2B) indicated that essentially all proteins in
the PR500 complex might also be present in the 19S RP. The
only exception was the largest band (P1), which was a
doublet in PR500, whereas the corresponding protein of P1
in 19S RP preparation was not (Figure 2B). The nature of this
difference is not yet clear. The P1 doublet in PR500 could be
due to partial degradation, novel modification, or the pres-
ence of a new protein. Assuming all of the cofractionated
proteins were components of PR500, we further examined
the molecular identity of PR500 components by a Western
blot using available antibodies against seven 19S protea-
some subunits. Among the seven tested antibodies, three of
them (Rpn6, Rpn3, and Rpn5a) recognized a protein in the
purified PR500 (Figure 2C). The remaining four proteins,
one non-ATPase (Rpn10) and three ATPases (Rpt1, Rpt2,
and Rpt5), were not present in the PR500 complex. Because
the yeast lid subcomplex is composed of eight non-ATPase
subunits including Rpn3, Rpn5, and Rpn6 (Glickman et al.,
1998a), PR500 could potentially contain all subunits of the
lid subcomplex with room for one or two extra subunits.
An hsp70 Chaperone Protein May Associate with
the PR500 Complex
During the purification of the PR500 complex, we noted that
a 70-kDa protein cofractionated with PR500 even after the
Superose-6 gel filtration step but was lost in the final heparin
chromatography step. Because the hsp70 chaperones have
recently been shown to be required for the proteasome
function (Luders et al., 2000; Pirkakala et al., 2000), we tested
whether this 70-kDa protein is related to hsp70 chaperones.
Indeed, as shown in Figure 3A, this protein was recognized
by polyclonal antibodies against hsp70. To test whether this
hsp70 is physically associated with the PR500 complex, we
attempted to coimmunoprecipitate hsp70 with either Rpn6
or Rpt5. Whereas Rpn6 antibody could immunoprecipitate
hsp70, the Rpt5 antibody could not (Figure 3, B and C).
Therefore, we conclude that Arabidopsis hsp70 is specifically
associated with PR500 but not with the 19S RP.
The COP9 Signalosome Is Required for PR500
To investigate the possible role of COP9 signalosome in
regulating Rpn6-containing complexes, we examined the
effect of the COP9 signalosome mutations on the 19S RP and
PR500 accumulation. In Arabidopsis, FUS6/COP11, COP8,
and FUS11 encode the CSN1, CSN4, and CSN3 subunits of
the COP9 signalosome, respectively (Chamovitz et al., 1996;
Serino et al., 1999; Deng et al., 2000), and mutations in any of
these genes disrupt COP9 signalosome assembly. As shown
in Figure 4, a series of Rpn6 gel filtration analyses using
equal amounts of wild-type and mutant cell extracts was
carried out to examine the levels of the Rpn6-containing
complexes. In wild type, whereas Rpt5 was exclusively frac-
tionated with 19S RP, ?30–40% of Rpn6 was present in
PR500. Rpn6 was used as an indication of the PR500 abun-
from cauliflower. (A) PR500 was separated from the 19S RP in an
NaCl linear gradient on a Mono-Q column. Fractions (1 ml each)
taken from 216 mM (fraction 4) to 300 mM NaCl (fraction 25) were
analyzed for Rpn6 and Rpt5 by immunoblot. Fractions 10–13 were
pooled as PR500 and fractions 16–22 were pooled as 19S RP. These
two pooled samples were further purified by gel filtration as de-
tailed in MATERIALS AND METHODS. (B) Compositional com-
parison between the purified PR500 and 19S RP preparations. The
silver-stained SDS-PAGE lanes for each complex are shown, with
positions of molecular size markers indicated. The identities of the
seven subunits, three subunits shared between the two complexes
and the four subunits present in only 19S RP, were confirmed by
Western blot and are marked. Note that the corresponding protein
bands between 19S RP and PR500 are indicated by a line or arrow
(only the three with know identities). (C) Western blot analysis of
the two purified complexes. All seven antibodies against distinct
proteasome 19S RP subunits reacted with specific bands from the
purified 19S RP preparation as well as the total cell extracts, whereas
only three of them reacted with the purified PR500 preparation.
Purification and characterization of PR500 and 19S RP
Peng et al.
Molecular Biology of the Cell386
dance, and it is evident that the PR500 complex was either
significantly reduced (fus6-T236, 15%; fus6-G236, 8%; and
fus11-U203, 9%) or essentially absent (fus6-1 and cop8-1,
?5%) in the different COP9 signalosome mutants (Figure 4).
Furthermore, the level of PR500 accumulation correlates
with the severity of the molecular lesions as well as with the
severity of the phenotype (Figure 4; Peng, Staub, Serino,
Kwok, Kurepa, Bruce, Vierstra, Wei, and Deng, unpublished
results. In contrast, in det1-8, cop1-5, and cop10-1 mutants,
which affect photomorphogenesis to a similar degree as the
severe COP9 signalosome mutants but do not affect the level
or structural integrity of COP9 signalosome (Kwok et al.,
1998), their abundance of PR500 was ?30–40%, similar to
that of wild type (Figure 4; Peng, Staub, Serino, Kwok,
Kurepa, Bruce, Vierstra, Wei, and Deng, unpublished re-
sults). Furthermore, the presence of ATP in the extraction
and gel filtration buffers did not affect PR500 levels observed
in any mutant strains, although in these conditions a good
portion of the 19S were incorporated into higher molecular
mass complexes (Peng, Staub, Serino, Kwok, Kurepa, Bruce,
Vierstra, Wei, and Deng, unpublished results).
Under our experimental conditions, the distribution of
Rpt5 was unaffected by the various mutations examined. It
was present only in the fractions containing the 19S RP or
putative 26S proteasome in all the mutants described above
(Peng, Staub, Serino, Kwok, Kurepa, Bruce, Vierstra, Wei,
and Deng, unpublished results) as in wild-type Arabidopsis
seedlings (Figure 4, top). Therefore, we concluded that the
COP9 signalosome is essential for the accumulation of
PR500 but not for the accumulation of 19S RP and for the
assembly of the 26S proteasome.
Heat Shock and Canavanine Treatments Down-
Because the cop1-5, cop10-,1 and det1-8 mutants did not affect
PR500 level, it is unlikely that the defective photomorpho-
genic development in the COP9 signalosome mutants is
hsp70 cofractionation profile on Superose 6 HR gel filtration column
before the last step of purification (see MATERIALS AND METH-
ODS). The fractions from the gel filtration column were separated
by SDS-PAGE and immunoblot analyzed. (B and C) Coimmunopre-
cipitation with Rpn6 or Rpt5 antibodies, respectively. Proteins par-
tially purified by phenyl Sepharose high performance and Q-Sepha-
rose fast flow columns were used for the immunoprecipitation (IP)
experiments. The precipitants were subjected to Western blot anal-
ysis with antibodies against hsp70 and Rpn6 or Rpt5. PI IP, preim-
mune serum immunoprecipitation.
hsp70 is physically associated with PR500. (A) Rpn6 and
some is required for PR500 com-
plex accumulation in Arabidopsis.
Total cell extracts from light-
grown wild-type or mutant seed-
lings were fractionated on a Su-
fractions (0.5 ml per fraction)
were analyzed for Rpn6 or Rpt5
(top lane) by immunoblot. The
positions of 19S RP and PR500 are
indicated on top, and the molec-
ular mass standards are indicated
at the bottom. Gel filtration was
performed as in Figure 1 except
that the buffers were modified as
described in MATERIALS AND
METHODS. Seedlings of Arabi-
dopsis COP9 signalosome mutants
and wild-type (WT) are shown on
the right for phenotype compari-
son. The mutant and wild-type
seedlings were grown in constant
white light for 5 d. All images
were taken with the same magni-
The COP9 signalo-
PR500: a Proteasome Lid-Like Complex in Plants
Vol. 12, February 2001 387
responsible for the observed reduction of PR500 level. Be-
cause the COP9 signalosome is required for regulated pro-
tein degradation mediated by the 26S proteasome (Oster-
lund et al., 2000), it is possible that the defect in proteasome-
mediated protein degradation in the COP9 signalosome
mutants might be related to the observed PR500 level
change. To test the possibility, we examined PR500 levels
under conditions elevating the production of misfolded pro-
teins, thus increasing demand of the proteasome activity.
First, the arginine analogue canavanine was used to supple-
ment Arabidopsis GM. The incorporation of canavanine in-
stead of arginine into cellular proteins causes protein mis-
folding, thus generating a large amount of substrate for the
ubiquitin-proteasome pathway (Rosenthal et al., 1989;
Rosenthal, 1992). Furthermore, heat shock is well known to
cause misfolding of cellular proteins, thus increasing the
accumulation of substrates for the ubiquitin-proteasome
pathway (Alberts et al., 1994). As shown in Figure 5A, when
3-d-old seedlings were transferred to a plate supplemented
with 4 mg/l canavanine for 2 d, PR500 peak diminished
whether ATP was included in the buffers or not. Further-
more, when 5-d-old seedlings were heat shock treated for
3 h at 42°C, the PR 500 peak also diminished in the two
buffer systems tested (Figure 5A). In contrast, light- and
dark-grown seedlings had no detectable difference in PR500
abundance. These results suggested that cellular level of
PR500 was decreased under stresses where higher protea-
some activity is expected.
To evaluate whether PR500 was degraded under the stress
treatments, we examined the overall cellular levels of Rpn6
and Rpt5 in the controls and the stress-treated plants. Be-
cause PR500 contains a significant portion (30–40%) of the
total cellular Rpn6 protein, a net loss of PR500 would result
in a noticeable decrease of the Rpn6 relative to that of
tubulin, a control for loading, in the total protein Western
blot. As shown in Figure 5B, neither canavanine nor heat
shock treatments caused detectable changes in the level of
Rpn6 and Rpt5 (Figure 5B). Therefore, our result is consis-
tent with the notion that PR500 was not lost under these
treatments. Because there is no Rpn6 monomer before or
after the treatments, the Rpn6-containing PR500 likely
shifted to incorporate into the 26S proteasome or other com-
plexed forms. However, we cannot rule out a possibility that
the Rpn6 in PR500 was degraded, and at the same time
increased synthesis of Rpn6 was able to compensate this loss
PR500 Has a Nuclear Localization Pattern Distinct
from the COP9 Signalosome and the Proteasome
In wild-type Arabidopsis protoplasts, both Rpn6 and Rpt5
antibodies predominantly stained the nucleoplasm (Fig-
ure 6; Kwok et al., 1998, 1999). However, antibodies
against Rpn6 (Figure 6A), but not Rpt5 (Figure 6D), in-
tensely stained numerous discrete speckles (20–40 per
nucleus) within the nucleoplasm in all cell types exam-
ined (see Figure 6, A and D). Consistent staining patterns
were observed in 100 individual cells of each cell type.
Because Rpn6 is a component of both 19S RP and PR500
and it is present in these speckles, whereas Rpt5 is only in
the 19S RP and does not stain any speckle, it is possible
that the bright speckles represent the localization of
PR500. The uniform nuclear-enriched staining patterns of
Rpn6 and Rpt5 could represent the localization of 19S RP
alone or the 26S proteasome.
To substantiate this conclusion, we took advantage of the
fact that PR500 complex is significantly diminished in the
COP9 signalosome mutants but not in det1 and cop10 mu-
tants of Arabidopsis. Protoplasts were prepared from roots of
7-d-old mutant seedlings that were either completely lack-
ing PR500 (fus6-1 and fus12-R380)) or had normal PR500
accumulation (cop10-1 and det1-8). These protoplasts were
then probed with the Rpt5 and Rpn6 antibodies. As shown
in Figure 6, B and E, the Rpn6-associated speckles were not
detected in cells prepared from the COP9 signalosome-de-
ficient fus6-1 mutants. In contrast, the staining patterns of
accumulation. Gel filtrations were conducted as in Figure 4 and
selected fractions were analyzed. (A) Gel filtration profiles of Rpn6
from seedlings grown under indicated conditions. The positions of
PR500 and 19S RP are indicated on the top. Molecular size markers
are indicated at the bottom. “?ATP, protein extraction and gel
filtration were conducted in buffers without ATP. ?ATP, protein
extraction and gel filtration were conducted in buffers with 10 and
2 mM ATP each. (B) The protein levels of Rpn6 and Rpt5 in the
controls and stress-treated Arabidopsis seedlings. Lanes 1 and 3, the
protein extracts from the control and heat shock-treated (42°C for
3 h) seedlings. Lanes 2 and 4, the protein extracts from the seedlings
grown in the absence or presence of 4 mM canavanine. The tubulin
antibodies were used as a control for equal loading.
Effect of heat shock and canavanine treatments on PR500
Peng et al.
Molecular Biology of the Cell 388
Rpn6 in the mutants of cop10-1 (Figure 6C) and det1-8 (Peng,
Staub, Serino, Kwok, Kurepa, Bruce, Vierstra, Wei, and
Deng, unpublished results) were essentially the same as that
of wild-type seedlings. Therefore, the absence of the bright
speckles specifically correlates with the loss of PR500 in the
COP9 signalosome mutants and not in other cop/det/fus mu-
tants. A distinct localization in specific nuclear foci of PR500
also strongly supports the conclusion that PR500 complex
exists in vivo.
PR500 Is a Freely Existing 19S RP-Related Protein
The purified PR500 complex contains nine distinct proteins,
most of which appear to be components of the 19S RP
(Figure 2C). This complex is similar, but not identical, to the
recently reported proteasome lid subcomplex observed in S.
cerevisiae and human (Glickman et al., 1998a; Braun et al.,
some, PR500, and the COP9 signalosome in
Arabidopsis protoplasts. Protoplasts were pre-
pared from 7-d-old light-grown wild-type (A,
D, and F), mutant fus6-1 (B and E), and
cop10-1 (C) seedlings. The protoplasts (top of
each panel) were stained with purified Rpn6,
Rpt5, and CSN1 antibodies and decorated
with a fluorescein (FITC) secondary antibody.
The corresponding labeling of nuclei by 4?,6-
diamidino-2-phenylindole (DAPI) is shown
in the middle of each panel. A diagram of the
cell (c) and nucleus (n) outlines is shown at
the bottom of each panel. The fus6-1 mutant is
defective in the COP9 signalosome, whereas
the cop10-1 mutant accumulates a normal
level of the COP9 signalosome.
Subcellular localization of protea-
PR500: a Proteasome Lid-Like Complex in Plants
Vol. 12, February 2001389
1999; Henke et al., 1999). PR500 clearly shares common fea-
tures in its composition with the lid subcomplex. For exam-
ple, it contains at least three non-ATPase subunits (Rpn6,
Rpn5a, and Rpn3) that are also present in the lid subcom-
plex. Furthermore, PR500 does not contain Rpn10, the two
largest 19S RP subunits, Rpn1 and Rpn2, and the three
ATPase subunits, all of which are not part of the lid sub-
complex either. Thus, it is possible that PR500 contains the
entire lid subcomplex (eight subunits) plus one or two ad-
It is important to note that the PR500 complex is itself a
freely existing structure apart from the 19S particles in nor-
mal plant cells, whereas the yeast lid is a stable subcomplex
that can be detached in vitro only from the 26S proteasome
purified from the Rpn10 deletion strain. Although a lid-like
subcomplex can be isolated as a by-product during the
COP9 signalosome purification from human cells, its pres-
ence under physiological conditions has not been demon-
strated (Braun et al., 1999; Henke et al., 1999). Four major
lines of evidence support the conclusion that PR500 exists
under physiological conditions. First, PR500 was consis-
tently observed by gel filtration of crude cell extracts under
different buffer conditions with or without ATP. Second, the
PR500 complex appears to have a distinct nuclear localiza-
tion pattern. Third, the mutations of COP9 signalosome
subunits resulted in specific loss of PR500, as demonstrated
by both gel filtration and immunolocalization studies.
Fourth, both heat shock and canavanine treatments can spe-
cifically reduced PR500 levels.
A Possible Role of PR500 in Plant Stress Response
Our studies identified three conditions in which PR500 ac-
cumulation was diminished. PR500 was diminished in
COP9 signalosome mutants, in Arabidopsis seedlings grown
on the arginine analogue canavanine and subjected to heat
shock treatment. The later two treatments are known to
elevated accumulation of misfolded proteins that are sub-
strates for the 26S proteasome, thereby increasing the work-
load of the ubiquitin-proteasome pathway. Interestingly, the
COP9 signalosome was recently shown to be essential for
the proteasome-mediated degradation of a key transcription
factor (Osterlund et al., 2000). Furthermore, in the COP9
signalosome mutants, we recently observed that there is a
dramatic increase in the amount of cellular ubiquitinated
proteins (Peng and Deng, unpublished data). This potential
defect of the ubiquitin-proteasome pathway specific to the
COP9 signalosome mutants is likely sensed and interpreted
by Arabidopsis cells as an increase in demand for the ubiq-
uitin-proteasome pathway. Thus, in all three cases in which
we observed a decrease of PR500 levels share a common
link, a high demand for the proteasome activity. It is also
worth noting that, although our data support an indirect
role of the COP9 signalosome in regulating the abundance of
PR500, it does not exclude an additional and more direct role
of the COP9 signalosome in regulating PR500 function or
association with proteasome (as discussed below).
Because our result suggests that PR500 under those
stresses was not degraded, it is likely incorporated into 26S
or other forms of proteasome. It is interesting to speculate
about the biochemical nature of the association of PR500 to
the proteasome. As diagramed in Figure 7, one of several
alternative possibilities could occur. First and the simplest,
PR500 may be a cellular reservoir of 19S components for
either assembly or disassociation. For example, upon high
demand of the cellular proteasome activity, PR500 could be
rapidly mobilized to assemble into 19S RP and then the 26S
proteasome. In this case, any components present in PR500
but not in the final 19S RP will be discarded during the
assembly process. This hypothesis would be consistent with
an association with an hsp70 chaperone, which is known to
help multisubunit protein complex assembly and ordered
disassociation (Crookes and Olsen, 1998; Luders et al., 2000).
However, because the 26S proteasome is rather abundant in
most cells, it has only rather limited value in storing some
extra lid-like complex just for rapid 26S proteasome assem-
bly in response to the stresses. Second, PR500 could be used
as a special lid subcomplex. During stress situations, this
special lid subcomplex may be used to replace the regular
lid subcomplex in a portion of the proteasome pool. This
type of the proteasome with PR500 as lid may thus possess
special characteristics that may better suit the stress situa-
tion. However, no evidence is available at present to suggest
that PR500 is a distinct lid for 26S proteasome. Third, PR500
may physically associate with a normal and intact 26S pro-
teasome and modify its activity or specificity to better suit
Although our current data cannot differentiate among
these possibilities, there are precedents that the activity and
specificity of the proteasome can be modulated by addi-
tional regulatory proteins, including activators, inhibitors,
and modulators. For example, a heterotrimeric modulator,
containing two ATPase subunits (Rpt5/S6/Tbp1 and Rpt4/
namic relationships among PR500, 19S
regulatory particle, and other protea-
some structures. Under this model,
19S regulatory particle and PR500 are
both present in the cell, and cellular
stresses and other signals can modu-
late their equilibrium. Note that the
presence of ATP (?) will help assem-
bly of the 26S proteasome, whereas
absence of ATP (?) will cause the dis-
association of the 26S proteasome into
A model showing the dy-
20S and 19S components. Free subunits and subcomplexes apart from the 19S particle may also participate in other unknown cellular
activities. It is not clear whether the incorporation of PR500 into 19S or 26S proteasome would constitute a normal or distinct lid subcomplex,
which would have different functional implications. See DISCUSSION for more detail.
Peng et al.
Molecular Biology of the Cell390
S10b/Sug2) in common with the 19S RP and one novel
protein (p27), has been reported (DeMartino et al., 1996).
This modulator facilitates the ATP-dependent association of
the 19S and 20S particles. A 220-kDa activator containing
S10b/Rpt4 and S6? (TBP1/Rpt5) ATPases has also been iso-
lated (Hastings et al., 1999). Recently, a novel 530-kDa pro-
tein complex distinct from PR500 has been shown to be
associated with the 20S proteasome core (Tanaka et al., 2000).
Moreover, functionally distinct forms of 26S proteasome
have also been reported (Kawahara et al., 2000). PR500 could
act according to one of the above-proposed ways to enhance
the capacity of plant cells to deal with stress situations.
A proposed functional role of PR500 in plant stress re-
sponses could fit well with the immobile life style of higher
plants. Unlike animals, plants cannot escape and need to
endure and cope with the environmental stresses. For exam-
ple, direct exposure of a leaf to sunlight can cause a rise of
temperature of 4–5°C above ambient temperature when wa-
ter is not abundantly available (Taiz and Zeiger, 1991). Thus,
it is rather frequent that plant leaves encounter temperatures
above 40°C during the year. This stress is likely to increase
misfolding or denaturing of proteins. To survive, plants
would need to effectively deal with those situations. The
proteasome was demonstrated to be critical in dealing with
this type of stress. For example, several knock-out mutants
of the 19S RP subunits have been shown to be hypersensitive
to heat shock stress and canavanine treatments in both yeast
and plants (Finley et al., 1987; Lambertson et al., 1999; Takeu-
chi et al., 1999; Vierstra, 2000). Regardless of the specific
mechanism of PR500 action, it is not unreasonable to suggest
that this free pool of PR500 may be a unique feature of
higher plants evolved to cope with the frequent protein-
misfolding stresses and their immobile life style.
We are grateful to Maria W. Smith, Carlos Gorbea, Mina Kurepa,
and Steve van Nocker for kindly providing the monoclonal anti-
bodies against 21D7/Rpn3 (subunit 3 of 19S RP), polyclonal anti-
bodies against human Rpt2, Rpt1, and Rpn10, respectively. We are
also grateful to Magnus Holm and Claus Swechheimer for com-
ments and critical reading of this manuscript. This research is sup-
ported by a National Science Foundation grants (MCD-9513366 and
MCD-0077217) to X.-W.D., a grant from Council for Tobacco Re-
search, United States of America, to N.W., and an RDV-United
States Department of Agriculture-National Research Initiative Com-
petitive Grants Program grant (97-35301-4218) to R.D.V. X.-W.D. is
a National Science Foundation Presidential Faculty Fellow. Z.P. is,
in part, supported by a National Institutes of Health postdoctoral
fellowship. G.S is a recipient of an Institute Pasteur Fondazione
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