From the Department of Molecular Oncology, The Tokyo
Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613 and the § EPM Project Groups,
Osaka R&D Laboratories, Sumitomo Electric Industries Ltd., and New
Energy and Industrial Technology Development Organization, Taya-cho
1, Sakae-ku, Yokohama 244-8588, Japan
Received for publication, February 6, 2003
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ABSTRACT |
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The 26 S proteasome is an evolutionarily
conserved ATP-dependent protease complex that degrades
poly-ubiquitinated proteins and plays essential roles in a critical
part of cellular regulation. In vertebrates, the roles of the
proteasome have been widely studied by use of specific inhibitors, but
not genetically. Here, we generated a cell line
Z The 26 S proteasome with a molecular mass of ~2500 kDa consists
of the central 20 S protease (catalytic core) and two outer 19 S
regulatory particles (alias PA700), functioning as a protein-destroying machine responsible for energy-dependent proteolysis (1,
2). The 20 S proteasome is composed of two copies of 14 different subunits: 7 distinct To date, yeast proteasomal mutants and membrane-permeable inhibitors
have been used to determine in vivo functions of
proteasomes, which have created diverse arrays of evidence on the
biological importance of proteasomes such as the cell cycle, immune
response, signaling cascades, and protein quality control in various
eukaryotes (5, 6). Indeed, budding yeast mutants that lack some
peptidase activities have contributed greatly to our understanding of
the involvement of proteasomes in the degradation of many unstable key
proteins (7), but their application to higher organisms has not been
tested. Various substrate-related peptidyl compounds such as MG-132 and
Z-L3VS have been devised as potent inhibitors of
proteasomes (8, 9), but caution must be exercised in their use for
interpreting proteasome functions, because they inhibit not only
proteasomes but also other proteases. In contrast to these compounds,
new microbial metabolites, such as lactacystin and eponemycin, were
found to induce selective inhibition of proteasomes that do not affect
other proteases examined so far (10, 11). However, although these
metabolites bind to active threonine residues of proteasomes, the
possibility that they inhibit other as-yet-unidentified threonine
protease(s) cannot be ruled out completely. Therefore, genetic
approaches capable of manipulating proteasomal activities are still
required to determine the in vivo functions of proteasomes in higher organisms such as vertebrate cells.
For this purpose, we disrupted proteasome subunit Z (formally
designated Plasmid Constructs--
Partial chicken Z cDNA was obtained
from DT40 cells-derived mRNAs by reverse transcription-PCR method.
The primers (5'-GACACGAGGGCGACCGAAGGGATG-3' and
5'-GCGGCTGCTCAGGAAGTATCCATG-3') were synthesized based on expressed
sequence tag sequence (AJ397675). The full-length cDNA and genomic
DNA were obtained by screening chicken muscle cDNA and genomic DNA
libraries (Stratagene). The Z targeting vectors were designed by
replacing the DNA segment that encompasses exon 1 to exon 3, with
drug-resistant cassettes for blasticidin (Bsd), puromycin
(Puro), and phleomycin (Bleo). A hemagglutinin (HA) tag was fused to
the 3'-end of chicken Z cDNA coding regions by PCR amplifications.
HA-tagged chicken Z cDNA (Z-HA) was inserted into the pUHD10-3
vector at the EcoRI site and the SspI site
was replaced with the HindIII site by a HindIII
linker. To construct a tetracycline (tet)-regulatable Z expression
vector, tTA-dependent promoter flanked with HA-tagged
chicken Z cDNA were recovered from pUHD10-3-Z-HA by digestion with
HindIII and inserted into the HindIII site of
ptTA2-Neo vector (Clontech) that encode
tet-repressible tTA (tetR-VP16). The resulting plasmid
(ptTA2-Neo-tetZ-HA) expresses Z-HA protein under the control of the
tetR-VP16 (12).
Cell Culture and Transfection--
DT40 cells were cultured in
RPMI1640 medium containing 10% (v/v) fetal bovine serum, 5% (v/v)
chicken serum, 10 µM 2-mercaptoethanol, and antibiotics
(penicillin and streptomycin) at 39.5 °C under 5% CO2.
Cells were electroporated at 25 microfarads and 550 V (Bio-Rad) as
described previously (13). Stable transformants were selected with each
drug at the following concentrations: 2.0 mg/ml Geneticin (Sigma), 0.5 µg/ml puromycin (Sigma), 50 µg/ml blasticidin-S (Funakoshi), and
0.3 mg/ml phleomycin (Sigma). Cell viability was assayed by measuring
the metabolic activity using tetrazolium salt WST-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, Cell Counting Kit-8, Wako). After incubating the cells
with WST-8, the optical density was read as specified by the
manufacturer. Dox was used at a concentration of 2 µg/ml for the
indicated times.
Antibodies--
Anti-chicken Z polyclonal antibody was raised in
rabbits using a purified recombinant His-tagged Z protein expressed in
Escherichia coli BL21. Anti-Hsp70 (MBL), anti-Hsp40 (Stress
Gen), anti-actin (Chemicon), anti-Wee1 (Santa Cruz Biotechnology),
anti-poly-ubiquitin (MBL), horseradish peroxidase-conjugated
anti-rabbit and anti-mouse IgG antibodies (Amersham Biosciences) were purchased.
Western Blot Analysis--
Cells were lysed in 50 mM
Tris-HCl (pH 8.0) containing 0.1% TritonX-100 and protease inhibitor
mixture (Roche Molecular Biochemicals). Following a brief sonication,
the extracts were cleared by centrifugation and subjected to 10-20%
SDS-PAGE (14). After transfer onto polyvinylidene difluoride membranes
(Millipore), proteins were detected by specific antibodies with the ECL
method (Amersham Biosciences). Protein concentration was measured by
the method of Bradford with bovine serum albumin as a standard
(15).
Southern Blot Analysis--
Genomic DNAs were isolated by using
a DNeasy tissue kit (Qiagen). Genomic DNA (15 µg) was digested with
EcoRI, separated in a 0.7% (w/v) agarose gel, and
transferred onto a Hybond N+ nylon membrane (Amersham
Biosciences). The membrane was hybridized with 32P-labeled
probe (ClaI-EcoRI fragment indicated in Fig. 1A),
washed at high stringency, and then autoradiographed.
Northern Blot Analysis--
Total RNAs were isolated by using an
RNeasy Mini kit (Qiagen). Approximately 15 µg of total RNAs was
separated and transferred onto a Hybond N+ nylon membrane.
The membrane was hybridized with 32P-labeled full-length
chicken Z cDNA probe, washed at high stringency, and then autoradiographed.
Glycerol Gradient Fraction--
Cells were lysed in 25 mM Tris-HCl (pH 7.5) containing 1 mM
dithiothreitol with 2 mM ATP by sonication, and the lysates
were centrifuged at 15,000 × g for 30 min. The
supernatants were subjected to glycerol gradient centrifugation with
10-40% glycerol in the above buffer. After centrifugation at
83,000 × g for 22 h using a Beckman SW28 rotor,
the gradient was separated into 30 fractions of 1 ml each (16).
Assay of Peptidase Activity--
Hydrolysis of the synthetic
peptides, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methycoumarine
(Suc-LLVY-AMC), t-butyloxycarbonyl-Leu-Arg-Arg-AMC (Boc-LRR-AMC), and carbobenzoxy-Leu-Leu-Glu-AMC (Z-LLE-AMC) was measured under the presence or absence of 0.05% SDS as described previously (17). One unit of peptidase activity was defined as the
amount that degraded 1 nmol of a given fluorogenic peptide per minute.
Assay of [35S]ODC Degradation
Activity--
35S-Labeled ornithine decarboxylase (ODC)
was produced in vitro by translating rat ODC mRNA in
rabbit reticulocyte lysates with 35S-labeled Met and Cys
(PerkinElmer Life Sciences) and then immunopurified. The degradation of
ODC was assayed as described previously (17). In brief,
35S-labeled ODC (3000-4000 cpm) was incubated with
antizyme, ATP, and enzyme solution in buffer containing the ATP
regeneration system at 37 °C for 1 h. The reaction mixtures
were then precipitated with trichloroacetic acid, the radioactivity of
the trichloroacetic acid-soluble fraction was measured, and the
activity was expressed as a percentage of total ODC radioactivity added.
Flow Cytometric Analysis--
Cells were fixed in 70% ethanol
in phosphate-buffered saline at 4 °C. Fixed cells were washed in
phosphate-buffered saline, incubated with 0.25 mg/ml RNase A at
37 °C, and stained with 10 µg/ml propidium iodide at 4 °C. DNA
contents were measured by a flow cytometry and cell cycle profiles were
analyzed by the Expo ADC analysis program (Beckman Coulter).
Immunofluorescence and TUNEL Assay--
Cells were fixed in 1%
paraformaldehyde. For immunofluorescence analysis, anti-HA monoclonal
antibody (BAbCO) and Alexa Fluor 594 goat anti-mouse IgG
antibody (Molecular Probes) were used. Nuclei were
counterstained with TOTO3 (Molecular Probes). Apoptotic cells were
detected by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay
using an Apoptag kit (Intergen). The assay was performed according
to instructions provided by the manufacturer. Fluorescence images were
obtained using a confocal laser microscope (Zeiss and Bio-Rad).
Genetic Manipulation of Proteasome Function--
To examine the
cellular roles of proteasomes in vertebrates, we generated a cell line
that could genetically manipulate the level of the proteasome subunit
that confers a peptidase activity. Chicken B cell line DT40 is
advantageous for this purpose, because of its efficient rate of
homologous recombination (13). Full-length chicken Z cDNA was
obtained by screening a chicken muscle cDNA library using the
partial cDNA fragment obtained by reverse transcription-PCR. The
full-length chicken Z cDNA deduced a protein of 277 amino acids
(accession number AB098728), displaying 57.4% and 83.8% identities
with Saccharomyces cerevisiae and human, respectively, at
the amino acid level. To disrupt the proteasome Z gene
(cpsmb7), chicken Z genomic DNA was isolated from chicken
genomic DNA library, and the targeting vectors were constructed as
shown in Fig. 1A. The vectors
were designed to create a null allele by replacing the DNA segment that
encompasses exon 1 to exon 3, which encodes the first 85 amino acids,
including the essential catalytic site (threonine 44 in the exon 2),
with drug-resistant cassettes. DT40 cells that contain three functional
Z genes (cpsmb7) were successively transfected with each
targeting vector, and the homologously recombined clones were
identified by genomic Southern blot (Fig. 1B). Because the
null mutant was expected to be lethal, we transfected the tet-regulatable Z-HA expression vector (ptTA2-Neo-tetZ-HA vector), in
which the expression of Z protein could be shut off by Dox treatment,
and isolated their stable transformants after the first allele was
disrupted by Bsd construct. The second and third loci were disrupted by
Puro and Bleo constructs, respectively. Finally, we obtained the
Z
We then tested the effect of Dox treatment on Z-HA expression in
Z Depletion of Z Subunit Resulted in Loss of Proteasome
Activity--
Based on yeast studies, Pup1p, which corresponds to
Z/
To further confirm that these peptidase activities represent
proteasome-specific activities, Z
Western blot analysis revealed that the mature form of Z protein
migrated in the fractions 11-19 with 20-26 S proteasomes (Fig. 3,
lower panels). The precursor form of Z in wild-type cell extracts migrated at lighter fractions 7-11. These fractions
corresponded to 16 S pre-proteasomal particles as reported previously
(18). On the other hand, the precursor Z-HA in Dox-untreated
Z
The peptidase activities in fractions of the mature form of Z-HA were
high in untreated cells, whereas they were markedly low in Dox-treated
cells fractionated in the same manner (Fig. 3, upper
panels). Taken together, these results suggest that Z-HA is
functionally active and that proteasome activity is dependent on the
expression of Z-HA. Because not only trypsin-like activity but also
other peptidase activities were affected by loss of Z-HA, we concluded
that the integral functions of the proteasome are impaired probably due
to its inappropriate assembly and/or maintenance of the complex.
Accumulation of Poly-ubiquitinated Proteins Associated with Loss of
Z--
In the next experiment, we analyzed the levels of cellular
poly-ubiquitinated proteins after Z depletion. After wild-type DT40 and
Z Z Subunit Is Essential for Viability--
Because most of the
proteasome subunit is essential in yeast, we tested the effect of loss
of proteasome function on cell viability of Z Enhanced Expression of Hsp70 and Hsp40 in Proteasome-defective
Cells--
It is known that damaged proteins and/or misfolded proteins
are rapidly eliminated from the cells by the ubiquitin-proteasome system. Indeed, proteasome inhibitors induce accumulation of such abnormal proteins in the cells and trigger signals that up-regulate the
expression of certain molecular chaperones in the cells (22). Given
that the expression of proteasomes can be reduced in
Z The proteasome is a multifunctional protease complex and essential
for cell viability. We generated Z The proteasome is the major protease for poly-ubiquitinated proteins
and known to degrade many cell-cycle regulators during the cell cycle
progression. Most of the yeast proteasome subunit mutants, although not
all, exhibit cell-cycle arrest at the G2/M phase rather
than the G1/S phase (24). Our data also indicate that the
major function of the proteasome in cell-cycle regulation is required
at the G2/M phase rather than the G1/S phase.
The essential substrates to be degraded by the proteasome at
G2/M phase were not characterized in this study; however,
we observed accumulation of Wee1 kinase in Z-HA knockdown cells (Fig.
5C). The Wee1 kinase is known to phosphorylate
cyclin-dependent kinase 1 and thus plays a critical role in
the checkpoint mechanism by inhibiting cyclin-dependent
kinase 1 activity (21). Whether Wee1 kinase is one of the essential
substrates or simply accumulates due to cell-cycle arrest at
G2/M phase remains to be elucidated.
The ubiquitin-proteasome system has been implicated in quality control
of proteins in the cytosol and endoplasmic reticulum (ER). Accumulation
of unfolded proteins in the ER induces the unfolded protein response
(UPR), which (i) halts the translation of newly synthesized proteins,
(ii) enhances the expression of molecular chaperones, and (iii) back
translocates unfolded protein in the ER to the cytosol for proteasome
degradation (25). The last process is called ER-associated protein
degradation among the UPR reactions (26). In the present study, our
results showed that inhibition of the proteasome enhances the
expression of Hsp70 and Hsp40. The latter is known to collaborate with
the former for folding newly synthesized and damaged proteins (27). How the cells sense the level of the proteasome and induce these molecular chaperones is unknown at present. However, this process is most likely due to enhanced UPR, because failure of protein degradation results in accumulation of abnormal proteins. Furthermore, the expression of molecular chaperone might be further enhanced following failure of the ER-associated protein degradation pathway.
Ubiquitin and proteasome-dependent protein degradation play
essential roles in various biological events as mentioned in the introduction. In this regard, recent studies in the ubiquitin field
reveal novel functions for ubiquitin in various biological events such
as endocytosis, DNA repair, transcriptional regulation, and kinase
activation (28). Whether these events do or do not involve
proteasome-dependent degradation remain to be elucidated by
genetic means, because proteasome inhibitors are known to inhibit some
of the above biological events, such as endocytosis (29). Furthermore,
many de-ubiquitinating enzymes that counteract with ubiquitination are
vital in the cells (28). Thus, Z/
/
/Z-HA, in which the expression of the
catalytic subunit of the proteasome, Z (
2) could be manipulated.
This cell line expresses exogenous Z protein under the control of a
tetracycline-repressible promoter in a Z-nullizygous genetic
background. Treatment of these cells with doxycycline inhibited Z
expression and, hence, the function of the proteasome. The latter
resulted in accumulation of poly-ubiquitinated proteins and concomitant
induction of molecular chaperones Hsp70 and Hsp40. These results
suggest a synergistic role for the proteasome with these molecular
chaperones to eliminate misfolded or damaged proteins in
vivo. Furthermore, knockdown of the proteasome induced apoptotic
cell death following cell-cycle arrest at G2/M
phase. Our Z
/
/
/Z-HA cell line would be useful
for evaluating proteolytic processes catalyzed by the proteasome in
many biological events in vertebrate cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 7 distinct
type subunits. It is a barrel-like particle formed by the axial stacking of four rings made up
of two outer
-rings and inner
-rings, being associated in the
order of
. Three out of seven
-type subunits of each inner ring have catalytically active threonine residues at their N
termini, and these active sites reside in a chamber formed by the
centers of the abutting
-rings. The eukaryotic 20 S proteasome has
at least three different catalytic activities against synthetic peptide
substrates; i.e. a trypsin-like, chymotrypsin-like, and caspase-like (or peptidylglutamyl-hydrolyzing) activities, that contribute to the hydrolysis of multiple peptide bonds in a single polypeptide by a coordinated mechanism (1, 3, 4).
2) gene (cpsmb7) in chicken B cell line DT40
then established Z
/
/
/Z-HA cells that express a
tetracycline-repressible
HA1-tagged Z protein (Z-HA).
This construct could manipulate proteasome levels in vertebrate cells
by repressing the Z-HA by doxycycline (Dox) treatment. Using these
cells, we found that reduction of proteasomes caused not only
G2/M arrest during cell-cycle progression but also
induction of apoptosis. Moreover, our results surprisingly showed that
reduced proteasomes functions induced the expression of major molecular
chaperones Hsp70 and Hsp40, suggesting a potential link between
proteasome-mediated proteolysis and stress response for protein
homeostasis in the cell.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
/
/Z-HA clone that expressed Z-HA in a Z-nullizygous
genetic background. The genomic Southern blots of representative clones
are shown in Fig. 1B. Homologous recombination was
identified as appearance of new 5.2-, 12-, and 11.5-kb bands
corresponding to the targeted alleles generated by the Bsd, Puro, and
Bleo constructs, respectively.
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Fig. 1.
Conditional knockdown of proteasome Z subunit
in DT40 cells. A, schematic representation of part of Z
locus and targeting vectors. Wild-type (WT) and targeted
alleles (Z/Bsd, Z/Puro, and Z/Bleo)
are shown. B, genomic Southern blot analysis of wild-type
(+/+/+), two heterozygous mutants (+/+/ and +/
/
), and homozygous
mutant (
/
/
). Genomic DNAs were digested with EcoRI and
hybridized with the probe indicated in A. C,
expression of Z-HA mRNA upon Dox treatment.
Z
/
/
/Z-HA cells were Dox-treated for the indicated
times, and total RNAs were subjected to Northern blot analysis.
Expression of Z-HA (top) and ethidium bromide staining of
the total RNAs (bottom) are shown. D, expression
of Z-HA protein upon Dox treatment. Cell extracts (2 µg of protein),
obtained from cells treated as in C, were Western blotted
with anti-chicken Z antibody. The lower signal is the mature
form (solid arrowhead), whereas the upper signal
corresponds to the precursor form (open arrowhead).
/
/
/Z-HA cells. Dox treatment of
Z
/
/
/Z-HA cells reduced the mRNA and protein
expression of Z-HA to undetectable levels at 24 h after Dox
treatment (Fig. 1, C and D). Western blot with
anti-Z antibody detected both precursor and mature forms of Z-HA. The
latter form migrated below the size of the former (Fig. 1D).
It is known that the catalytic subunits are synthesized as precursor
forms and processed into the mature form during the assembly of the 20 S proteasome complex (18). The mature form co-sedimented with the 20 S
proteasome, whereas the precursor form was detected in the lighter
fraction when the cell extracts were fractionated by glycerol density
gradient centrifugation (see Fig. 3, lower panels),
suggesting that Z-HA is processed and incorporated into the 20 S proteasome.
2, is known to confer trypsin-like activity (19, 20). In the next step, we tested whether loss of Z results in specific loss of trypsin-like activity. After Dox treatment, cells were serially collected at the indicated times and cell fractions were prepared. The
cell fractions were first tested for their ability to degrade ODC, a
proteasome-specific substrate independent of ubiquitination. As shown
in Fig. 2A, ODC-degrading
activities gradually decreased upon Dox treatment. These effects were
not seen in Dox-untreated cells or Dox-treated wild-type DT40 cells.
Testing for the specificity of peptidase activities showed that not
only trypsin-like activity, but also other peptidase chymotrypsin-like
and caspase-like activities were reduced (Fig. 2,
B-D).
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Fig. 2.
Proteasome activities in Dox-treated
Z /
/
/Z-HA cells. Cell extracts (20 µg of
protein) were obtained from Dox-treated or untreated cells for
indicated times, and peptidase activities were measured against ODC
(A) and synthetic peptides; i.e. a
chymotrypsin-like (B, Suc-LLVY-AMC), a trypsin-like
(C, Boc-LRR-AMC), and a caspase-like (D,
Z-LLE-AMC) activities. Open circles, Dox-untreated
Z
/
/
/Z-HA cells; solid circles, Dox-treated
Z
/
/
/Z-HA cells; open triangles,
Dox-untreated wild-type cells; solid triangles, Dox-treated
wild-type cells. Note that a caspase-like activity was assayed in the
presence of 0.05% SDS, whereas the chymotrypsin-like and trypsin-like
activities were without SDS (for the reason, see Fig. 3, upper
panels, and text). Data represent the mean ± S.D. values of
four independent analyses.
/
/
/Z-HA cells were
treated with or without Dox for 30 h, and the cell extracts were
further fractionated by glycerol density gradient centrifugation and
subjected to peptidase assay and Western blot analysis. As shown in
Fig. 3 (upper panels), in
wild-type cells, active enzyme with chymotrypsin-like and trypsin-like
activities was sedimented with a sedimentation coefficient of ~26 S,
but low activity was found in slowly sedimenting fractions
corresponding to the sedimentation position of the purified 20 S
proteasome. Addition of 0.05% SDS, which is a potent artificial
activator of the latent 20 S proteasome, caused marked enhancement of
chymotrypsin-like activity in fractions sedimenting like the 20 S
proteasome, as reported previously (16). Note that no obvious
caspase-like activity was observed without SDS, but its strong activity
could be measured in the presence of 0.05% SDS. ODC-degrading activity as well as these three types of peptidase activities were high and
comparable in the Dox-untreated Z
/
/
/Z-HA cells at
wild-type levels, whereas they were greatly decreased in the
Dox-treated Z
/
/
/Z-HA cells (Fig. 3, upper
panels).
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Fig. 3.
Sedimentation velocity analysis of wild-type
and Z /
/
/Z-HA cell extracts. Samples (4 mg of
protein) of the wild-type (WT) and
Z
/
/
/Z-HA cells treated with or without Dox for
30 h were fractionated by glycerol density gradient centrifugation
(10-40% glycerol from fractions 1 to 30) as described under
"Experimental Procedures." After fractionation, aliquots (20 µl)
of individual fractions were used for an assay of three peptide
hydrolysis with (solid circles) or without (open
circles) 0.05% SDS. The degradation of 35S-ODC
(crosses) was also assayed. Elution positions of purified 20 and 26 S proteasomes are shown. Lower panel, Western blot
analysis. Proteins in 200 µl of each fraction were precipitated with
acetone, subjected to SDS-PAGE, and stained by Western blot analysis
using an anti-chicken Z antibody. Numbers correspond to
fraction numbers in the upper panels. The solid
and open arrowheads point to the mature and precursor forms
of Z, respectively, similar to Fig. 1D.
/
/
/Z-HA cell extracts was recovered in fractions
1-11. The precursor Z-HA observed in fractions 1-5 might be the free
form based on its exogenous overexpression. Nonetheless, most mature
Z-HA was recovered in the fractions containing sediments of 20-26 S
proteasomes, indicating that Z-HA is assembled into these proteasomes.
Of note, in the Z
/
/
/Z-HA cells with Dox, the mature
Z-HA was reduced, and precursor Z-HA was not observed in fractions
7-11. In addition, very low mature Z-HA was recovered near top
fractions 1-5, which might be generated by disassembly of the 20 S proteasome.
/
/
/Z-HA cells had been treated with Dox for 30 h, these cell extracts were analyzed by Western blotting using an
anti-poly-ubiquitin antibody. As shown in Fig.
4, poly-ubiquitinated proteins
considerably increased in crude extracts from the
Z
/
/
/Z-HA cells by Dox treatment, whereas the pattern
of poly-ubiquitinated proteins remained unchanged in wild-type cells,
irrespective of Dox treatment. These results suggested that the
dysfunction of proteasomes leads to accumulation of poly-ubiquitinated
proteins.
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Fig. 4.
Accumulation of poly-ubiquitinated proteins
in Z /
/
/Z-HA cells. The wild-type (WT)
and Z
/
/
/Z-HA cells were treated with (+) or without
(
) Dox for 30 h. Cell extracts (10 µg of protein) were
subjected to Western blot analysis using an anti-poly-ubiquitin
antibody.
/
/
/Z-HA
cells. For this purpose, Z
/
/
/Z-HA cells were treated
with Dox for the indicated times and their viability was measured (Fig.
5A). Dox-treated
Z
/
/
/Z-HA cells proliferated in the first 24 h
but began to decrease from 30 h of treatment. Flow cytometric
analysis of these cells showed their arrest at G2/M phase
(Fig. 5B). We next examined the expression of Wee1 kinase,
which plays a role in checkpoint mechanism at G2/M phase
(21). Considerable accumulation of Wee1 kinase was noted at 24 h
after Dox treatment (Fig. 5C). Furthermore, flow cytometry
revealed cell death at 30 h after Dox treatment (Fig.
5B). To examine whether this was due to apoptosis, we
performed TUNEL analysis (Fig. 5D). The apoptotic cells had
large nuclei and less Z-HA expression. Taken together, these results
indicated that loss of Z-HA and proteasomal activities result in
cell-cycle arrest at G2/M phase followed by cell death. It
is worth noting that residual Z-HA was present mainly in the cytoplasm
as a punctate-like structure and to a lesser extent in the nuclei of
Dox-treated cells, whereas it was uniformly present in the cytoplasm
and rather abundantly in the nuclei of untreated cells (Fig.
5D).
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Fig. 5.
The Z subunit is essential for cell
viability. A, cell survival after Dox treatment.
Z /
/
/Z-HA cells (2 × 104cells) were
cultured in the presence (solid circles) or absence
(open circles) of Dox for indicated times (0, 6, 24, 30, and
48 h), and their viabilities were measured. Data represent the
mean ± S.D. values of four independent analyses. B,
cell cycle analysis. Cells were treated as in A and
subjected to flow cytometric analyses. Left panel,
Dox-untreated Z
/
/
/Z-HA cells; right panel,
Dox-treated Z
/
/
/Z-HA cells. Displayed data show
typical patterns, and essentially similar results were obtained in at
least three independent experiments. C, increment of Wee1 in
Z-depleted cells. The crude extracts (20 µg of protein), prepared
from wild-type (WT) (upper panels) and
Z
/
/
/Z-HA (lower panels) cells that had
been cultured with or without Dox for 24 h were immunoblotted with
anti-Wee1 antibody. D, immunofluorescence analysis. Cells
were treated as in A for the indicated times (0, 30, and
48 h) and mounted onto slides, fixed, and stained. DNA stain
(TOTO-3, blue), the expression of Z-HA (
HA,
red), and apoptotic signals (TUNEL, green), in
the same cells are shown together with the merged images
(Merge).
/
/
/Z-HA cells, we next examined whether Z depletion
could up-regulate the expressions of major molecular chaperones, Hsp40
and Hsp70 in Z
/
/
/Z-HA cells. As shown in Fig.
6 (lower panels), Hsp40 and
Hsp70 were consistently up-regulated by Z depletion in
Z
/
/
/Z-HA cells. In contrast, these effects were not
observed in Dox-treated wild-type DT40 cells (Fig. 6, upper
panels). These results suggested that these molecular chaperones
are up-regulated upon loss of proteasome function.
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Fig. 6.
Enhanced expression of molecular chaperones
Hsp70 and Hsp40 in Dox-treated Z /
/
/Z-HA cells.
Wild-type (WT) (upper panels) and
Z
/
/
/Z-HA (lower panels) cells were treated
with or without Dox for 30 h. Cell extracts (10 µg of protein)
were prepared and used for immunoblotting with antibodies against Z,
Hsp40, Hsp70, and actin. The solid arrowheads indicate the
respective proteins, whereas open arrowheads show the
precursor form of Z.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
/
/Z-HA cell line
in which the expression of proteasome subunit Z could be manipulated by
Dox. This cell line expressed Dox-repressible Z protein, tagged with
C-terminal HA peptide. The C-terminal HA-tag did not interfere with the
wild-type function of Z, because (i) Z-HA complemented the lethality of
Z null-phenotype (Fig. 5A) and (ii) Z-HA was processed in
mature form and incorporated into the proteasome complex (Figs.
1D and 3). Depletion of Z-HA resulted in inhibition of three
types of proteasomal peptidase activities and ODC-degrading activity
(Figs. 2 and 3). Furthermore, considerable accumulation of
poly-ubiquitinated cellular proteins was observed in vivo
(Fig. 4). This is consistent with the fact that cellular poly-ubiquitinated proteins accumulate in the yeast proteasomal temperature-sensitive mutants under restrictive temperature (23). Taken
together, these results suggest that the Z subunit is essential for the
integrity of proteasome.
/
/
/Z-HA cells could
be used as a tool for examining the function of proteasomes in various
cellular events that, at least, involve ubiquitination.
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ACKNOWLEDGEMENTS |
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We thank Y. Yamaguchi-Iwai for providing the parental DT40 cell line, the drug-resistance cassettes, and the pUHD10-3 vector and Y. Murakami for providing ODC and antizyme. We also thank K. Iwatsuki, T. Yasuda, and all members of the Tanaka laboratory for the advice and technical assistance.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB098728.
¶ To whom correspondence should be addressed. Tel./Fax: 81-3-3823-2237; E-mail: tchiba@rinshoken.or.jp.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M301331200
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ABBREVIATIONS |
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The abbreviations used are: HA, hemagglutinin; Bleo, phleomycin; Boc-LRR-AMC, t-butyloxycarbonyl-Leu-Arg-Arg-AMC; Bsd, blasticidin; Dox, doxycycline; ODC, ornithine decarboxylase; Puro, puromycin; Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methycoumarine; tTA, tetracycline-controlled transactivator; Z-LLE-AMC, carbobenzoxy-Leu-Leu-Glu-AMC; TdT, terminal deoxytransferase; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; TUNEL, TdT-mediated dUTP-biotin nick end-labeling; ER, endoplasmic reticulum; UPR, unfolded protein response.
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