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INTRODUCTION |
The ubiquitin-proteasome system is responsible for the degradation
of many of the short-lived proteins in eukaryotic cells. The pathway
targets proteins for degradation by the proteasome via covalent tagging
of the substrate protein with a polyubiquitin chain. This is
accomplished in three sequential steps. Ubiquitin, a 76-amino acid
protein, is initially activated by
E1,1 the ubiquitin-activating
enzyme. Activated ubiquitin is then transferred to a
ubiquitin-conjugating enzyme (E2), which generally shuttles ubiquitin
to a ubiquitin ligase (E3). The E3 is bound to the targeted substrate
and catalyzes the covalent attachment of ubiquitin to the substrate.
Once the first ubiquitin molecule is transferred to the substrate, a
polyubiquitin chain is generated via a series of isopeptide linkages
between a lysine residue of the attached ubiquitin and the
carboxyl-terminal glycine of the next ubiquitin molecule to be added.
The multi-ubiquitinated substrate protein is then degraded by the 26 S
proteasome in an ATP-dependent reaction (1).
This highly selective proteolytic system is essential to the regulation
of a wide range of nuclear proteins including cell cycle regulators,
transcription factors, tumor suppressors, and oncoproteins (2-4). All
of the components of the ubiquitin-proteasome system are found in the
cytoplasm, but the role of the nucleus in the degradation of nuclear
proteins is not well understood although many of the components of the
ubiquitin-proteasome system can also be found within the nucleus.
Among these components, ubiquitin is found in the nucleus (5). The
117-kDa isoform of the ubiquitin-activating enzyme (E1a) is found in
the nucleus in a cell cycle-dependent manner (6-8). E2
forms that are found in the nucleus include RAD6, an E2 involved in DNA
repair (9) and cdc34 (9-12), an E2 associated with the SCF-type
ubiquitin ligase complex (13, 14). Moreover, proteasomal subunits have
been immunolocalized to both the cytoplasm and the nucleus throughout
the cell cycle (15-18).
These observations suggest that a functional ubiquitin-proteasome
system may be operative within the nucleus. A functional nuclear
ubiquitin-proteasome system may provide additional levels of regulation
of intracellular proteolysis via the cellular targeting of a substrate
as well as via compartment-specific activities of its components.
Studies examining the degradation of transforming growth
factor-
-activated Smad2 (19) and the dioxin receptor, a member of
the basic helix-loop-helix/PER-ARNT-Sim family of transcription factors
(20), show that nuclear localization occurs prior to the ultimate
ubiquitin-dependent degradation of these substrates.
However, other studies have determined that nuclear export is required
for the ubiquitin-dependent degradation of p27kip1 (21), cyclin D1 (22), p53 (23), and
I
B
(24) as well as the aryl hydrocarbon receptor (AHR) (25).
These results, which couple nuclear export with degradation of the
substrate, suggest that export to the cytoplasmic ubiquitin-proteasome
machinery may represent a common route for the degradation of nuclear substrates.
However, ubiquitin-dependent degradation of nuclear
substrates in the cytoplasm envisions no role for the proteasome or the ubiquitin activating and conjugating enzymes that are localized to the
nucleus. This raises questions about the role of the
ubiquitin-proteasome components found in the nucleus and whether the
nucleus will support ubiquitin-dependent degradation. To
determine if the nucleus and its complement of components are
sufficient to support ubiquitin-dependent degradation, we
have developed a cell-free system based on highly purified HeLa
nucleoplasm. We have evaluated both model substrates and MyoD as
a physiological substrate using this system. In addition, we have
extended these studies in vivo and taken advantage of the
nuclear export inhibitor, leptomycin B (26).
MyoD is a nuclear basic helix-loop-helix transcription factor that is
pivotal in skeletal muscle differentiation (27). In addition, MyoD can
act as a cell cycle inhibitor during G1 (28, 29). Although
the cellular locus for the degradation of MyoD has not been
established, in vivo (10) and in vitro (30, 31), studies including those using proteasomal inhibitors have shown that
MyoD is a substrate of the ubiquitin-proteasome system. The present
results now demonstrate that highly purified nucleoplasm will degrade
MyoD in a ubiquitin-proteasome-dependent manner and that
in vivo, nuclear export is not required for the degradation of MyoD, indicating that MyoD is degraded by a nuclear
ubiquitin-proteasome system.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HeLa cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (100 units/ml
penicillin G and 100 µg/ml streptomycin) (Life Technologies, Inc.).
The cells were maintained at 37 °C and 5% CO2 in a
humidified chamber and were harvested at 50-75% confluence. For large
scale culture, HeLa cells were maintained in suspension in Dulbecco's
modified Eagle's medium with 5% fetal calf serum and were harvested
at 0.5 × 106 cells/ml. Large scale cultures were
obtained from the Tissue Culture Support Center at the Washington
University School of Medicine.
Transient transfections with MyoD were performed using LipofectAMINE
according to the manufacturer's instructions (Life Technologies, Inc.).
Preparation of Extracts--
Rabbit reticulocyte lysate was
prepared as described previously (32). Nucleoplasm was obtained from
HeLa cells via a procedure that is a modification of those of Dignam
et al. (33) and Blobel and Potter (34). The preparation was
monitored microscopically at each step. The HeLa cell pellet was rinsed
twice in ice-cold phosphate-buffered saline and once in a hypotonic
buffer (10 mM Tris-Cl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT). The pellet resuspended in the hypotonic buffer was then homogenized for two or three strokes with a "B" type Dounce pestle to ensure a single cell suspension. Thereafter the cells were swollen via a 45-min incubation on ice. All buffer volumes were 25 ml/g pellet (25 ml/liter
suspension culture). The swollen cells were then dounced for 35 stokes
with an "A" type pestle, and the resulting crude nuclei were
pelleted at 600 × g for 10 min. The pelleted nuclei were resuspended in 0.25 M sucrose in hypotonic buffer and
incubated on ice for 10 min. Thereafter, 2.3 M sucrose in
hypotonic buffer was added to yield a 1.62 M sucrose
solution. The nuclei suspension was layered over a step gradient of
1.80 M and 2.30 M sucrose and centrifuged at
28,000 rpm for 1 h in a SW 28 rotor (Beckman). The pellet of
nuclei was resuspended in hypotonic buffer and rinsed once by
centrifugation in the same buffer. The washed nuclei were then
incubated in the hypotonic buffer without MgCl2 on ice for 15 min. Following sonication, the resulting nucleoplasm was collected after centrifugation at 10,000 rpm for 10 min in an Eppendorf microcentrifuge at 4 °C and stored at
70 °C after rapid
freezing in liquid N2. When MyoD was used as the substrate,
the sonicated nuclei were incubated with 2.5 units of DNase I/mg of
protein at room temperature for 20 min before the final centrifugation step. This step was necessary for removal of DNA, which is inhibitory in the ubiquitin-dependent degradation of MyoD (data not
shown). Control experiments showed no effect on substrate degradation when DNase I was added to reticulocyte lysate. A typical preparation of
nucleoplasm (from 4 liters of HeLa cell suspension culture) contained
4-6 mg/ml protein in a total volume of 1-1.5 ml. Protein concentrations were determined by the Bradford method (35) using bovine
serum albumin as the standard.
Characterization of HeLa Nucleoplasm--
Nuclei were evaluated
for cytoplasmic protein contamination using both morphological and
biochemical criteria. Nuclei were visualized under phase contrast at
each step of the preparation and were considered substantially free of
cytoplasmic remnants if the nuclear envelope appeared smooth and the
nuclei did not clump. The washed nuclei were assayed for lactate
dehydrogenase activity as a biochemical marker of cytoplasmic
contamination (8). Total activity was determined using nuclei volume
based on total number of nuclei × 0.3 pl/nuclei (36). HeLa
homogenate was obtained from an aliquot of the disrupted HeLa cells
prior to centrifugation. HeLa cytosol was the supernatant obtained from 100,000 × g centrifugation of the homogenate (8).
Nucleoplasmic and cytoplasmic fractions were also evaluated by 12.5%
SDS-PAGE and protein staining with Coomassie Brilliant Blue. In
addition, nuclear protein contamination of the cytoplasm was assessed
by Western analysis of histones using a histone pan antibody (Roche Molecular Biochemicals, 25 mg/ml, catalog no. 1 492 519).
Preparation of MyoD Substrate--
Wild-type MyoD was cloned
into the bacterial expression vector pT7-7 as described previously
(31). BL21(DE3) pLysS Escherichia coli cells were used for
expression of MyoD. Expression was induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside at an absorbance of 0.6 at 600 nm, and the cells were harvested after a 5-h induction at
30 °C. The cells were lysed by sonication in 20 mM
HEPES, pH 7.9, 0.2 M KCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, and 2 mM PMSF, and
nucleic acids were removed by precipitation with 0.3%
polyethyleneimine. MyoD was precipitated from the resulting supernatant
using 0.6 M ammonium sulfate as described (37).
Degradation Assays--
Degradation assays were performed
according to the method of Hershko et al. (38). Briefly,
reactions were carried out in a final volume of 12.5 µl and contained
either crude reticulocyte lysate (3 µl, ~550 µg of protein) or
HeLa nucleoplasm (30 µg o protein) as the source of
ubiquitin-proteasomal system components. The reaction mixtures
contained 50 mM Tris-Cl, pH 7.6, 5 mM
MgCl2, 2 mM DTT along with an exogenous protein
substrate (MyoD, 400 ng/reaction) supplemented with either an
ATP-depleting system (0.5 µg of hexokinase and 10 mM
2-deoxyglucose) or ATP-regenerating system (0.5 mM ATP, 10 mM phosphocreatine, and 0.5 µg of phosphocreatine kinase). The ATP-regenerating system maintains the ATP level at ~1
mM. Degradation reactions were incubated at 37 °C for
2 h and were terminated by boiling after the addition of an equal
volume of 2× Laemmli sample buffer (39). The reaction products were resolved by 10% SDS-PAGE and electroblotted onto nitrocellulose. The
immunoblots were incubated with a monoclonal anti-MyoD antibody (1:250
dilution, Novacastra, NCLMyoD1) and, after incubation with a
secondary horseradish peroxidase-conjugated antibody, were detected by
chemiluminescence (Amersham Pharmacia Biotech). Statistical analysis of
the Western blot data was performed by Student's t test
after the data was quantified using Un-Scan-It gel version 5.1 software
(Silk Scientific, Inc.).
For the inhibition studies, MG132
(N-carbobenzoxyl-Leu-Leu-Leucinal) (Peptide Institute, Inc.)
was diluted to 10 mM in Me2SO and was added to
the reactions containing the ATP-regenerating system at 20 µM. A reaction containing an equal volume of the solvent
Me2SO was run as a control. Methylated ubiquitin was
prepared as described previously (40). Methylated ubiquitin and the
ubiquitin K48R mutant (Boston Biochem) were each prepared as a 25 mg/ml stock in H2O and were added to the degradation reaction
containing the ATP-regenerating system at 5, 10, or 15 µg/12.5 µl
of reaction volume. ATP
S (Sigma) was prepared as a 0.1 M
stock in 10 mM Tris-Cl, and the pH was adjusted to 7.0.
Conjugation of MyoD in Nuclear Extract--
The MyoD cDNA
was translated in the presence of [35S]methionine using a
transcription-translation coupled reticulocyte lysate system (Promega).
Prior to its addition to the conjugation assay, the labeled
substrate-containing mixture was treated with 9 mM N-ethylmaleimide (NEM; Sigma) for 10 min at room temperature
to inactivate components of the ubiquitin system. The NEM was
neutralized with excess (9 mM) NEM.
Conjugation assays were performed in a reconstituted cell-free system
as described (31). Briefly, the reaction mixture contained, in a final
volume of 12.5 µl: 40 µg of nucleoplasmic proteins, or 40 µg of
BSA as indicated, 5 µg of ubiquitin, and ~25,000 cpm of in
vitro translated and NEM-treated 35S-MyoD. Reactions
were performed in the presence of the proteasome inhibitor ATP
S (4 mM) (41) and the isopeptidase inhibitor, ubiquitin aldehyde
(0.5 µg) (42). Conjugation assays were incubated at 37 °C for
1 h. Reactions were terminated by the addition of sample buffer
and resolved by SDS-PAGE (10%). MyoD was visualized by PhosphorImager
(Molecular Dynamics/Fuji).
Stability of MyoD in Vivo--
At 24 h following
transfection, protein synthesis was inhibited by incubation with
cycloheximide (final concentration of 100 µg/ml) (43) along with
either MG132 (10 µM) or leptomycin B (10 nM).
MG132 was prepared as a 10 mM stock solution in
Me2SO, and leptomycin B was prepared as a 10 µM solution in ethanol. Control experiments were carried
out with either Me2SO or ethanol alone. At the indicated
time points, the cells were lysed in PBS with 0.5% Igepal, 1 mM EDTA, 1 mM DTT, and 2 mM PMSF.
The lysed cells were sonicated, and cellular debris was removed by
centrifugation at 13,000 rpm × 1 min in an Eppendorf
microcentrifuge. Equal volumes of the supernatant at each time point
were analyzed by 10% SDS-PAGE followed by Western blotting as
previously described for the degradation assays. The relative amounts
of MyoD were analyzed using Un-Scan-It gel version 5.1 software, and
the half-life was determined based on three separate experiments.
Pulse-chase analysis of MyoD stability was carried out in HeLa cells at
24 h after transfection. Cells were transfected with pC1neoMyoD or
the pC1neo vector as a control. The cells were pretreated for 2 h
with 10 nM leptomycin B (LMB) or an equal volume of ethanol for the (
) LMB condition prior to metabolic labeling with
[35S]cysteine (200 µCi/ml) (Amersham Pharmacia Biotech)
for 1 h in media devoid of exogenous cysteine. The cells were
washed and chased in media containing unlabeled cysteine with or
without 10 nM LMB and harvested after 3 h. Cell
lysates were prepared by resuspension of cells in lysis buffer
(phosphate-buffered saline, 0.5% Igepal, 1 mM DTT, 1 mM EDTA, 2 mM PMSF) followed by sonication. Cellular debris was removed by centrifugation, and the resulting supernatant was pre-cleared overnight at 4 °C with protein
A-Sepharose (RepliGen). Immunoprecipitations were performed by
incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa
Cruz, sc-760) followed by incubation with protein A-Sepharose.
The beads were extensively washed, and the immunoprecipitates were
analyzed by 10% SDS-PAGE followed by autoradiography.
Control experiments were carried out to confirm LMB's biological
activity. Herein, HeLa cells growing on coverslips were transiently transfected with GFP-I
B
(44). Twenty-four hours after
transfection, the cells were treated for 1 h with 10 nM LMB and localization of the GFP-I
B
fusion protein
was determined by indirect immunofluorescence (45) with anti-GFP
antibody (1:500 dilution, Chemicon, AB16901). The resulting images were
photographed (magnification, ×40) using an Olympus BX 60 microscope.
Immunofluorescent Localization of MyoD--
HeLa cells were
transfected with pClneo myoD. 24 h later cells were incubated with
MG132 or LMB for 3 h as described above. Thereafter, localization
of MyoD was determined by indirect immunofluorescence with a polyclonal
anti-MyoD antibody (1:500 dilution, Santa Cruz, sc-760), and the
resulting images were photographed (magnification, ×40) using an
Olympus BX60 microscope.
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RESULTS |
Isolation of Purified Nuclei and Preparation of
Nucleoplasm--
In an effort to examine the role of the nucleus in
the ubiquitin-proteasome-dependent degradation of
short-lived nuclear proteins, we established a cell-free system that
supports the ubiquitin-dependent degradation of exogenous
protein substrates. This cell-free system is dependent upon highly
purified nucleoplasm obtained from HeLa nuclei that were isolated by
sucrose density centrifugation. The nuclei are essentially free of
cytoplasmic contamination by both microscopic examination and lactate
dehydrogenase analyses (Fig. 1,
A and C). A typical result for the lactate
dehydrogenase analysis yielded 1329 units/min of activity for the
cytoplasm and 0.17 units/min of activity for the nuclei, for a relative
contamination of 0.013%. In addition, SDS-PAGE and Western analysis
using histones as a marker indicate histones are the major protein
species present in the nucleoplasm with essentially no contamination
(undetectable by Western blot scanning) of the post-nuclear supernatant
(Fig. 1B).

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Fig. 1.
Purification of HeLa nucleoplasm.
A, nuclei were isolated from HeLa cells following disruption
in a hypotonic solution followed by sucrose density gradient
centrifugation. The purified nuclei were subjected to sonication and
incubated with DNase I prior to centrifugation to obtain the
nucleoplasm. B, the resulting nucleoplasm (N)
along with the HeLa cytoplasm (C) (50 µg of protein/lane)
was analyzed by 12.5% SDS-PAGE followed by Coomassie staining and
Western blotting for histones. C, the purified nuclei ( ),
whole cell homogenate ( ), cytoplasm ( ), and cytosol ( ) were
assayed for lactate dehydrogenase activity using equivalent amounts of
protein. Total activity was calculated as described under
"Experimental Procedures."
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ATP-dependent Degradation of MyoD in HeLa
Nucleoplasm--
With this system, we first examined
ATP-dependent degradation of lysozyme as a model protein
substrate of the ubiquitin-proteasome system (38). HeLa nucleoplasm
served as the source of ubiquitin-proteasome components. All assays
were also performed using rabbit reticulocyte lysate in control
reactions. After establishing that our cell-free system supports the
ATP-dependent degradation of lysozyme (data not shown), we
next determined if a physiological substrate of the
ubiquitin-proteasomal system could be degraded by the nucleoplasm. As
seen in Fig. 2B, MyoD is
degraded by HeLa nucleoplasm in an ATP-dependent manner.
The results from five similar experiments were quantified using
Un-Scan-It gel version 5.1 software and are reported here as total
pixels corrected for background pixels. The results seen in the lower
portions of Fig. 2B show the ATP-dependent degradation of MyoD in nucleoplasm. The difference between the degree
of degradation for the ATP-containing and ATP-depleted reactions is
significant at p < 0.001. This is also the case for MyoD degradation in the reticulocyte lysate (Fig. 2A). In
addition, we observed a second immunoreactive band of slightly slower
mobility in the (+) ATP reaction for the nucleoplasm (Fig.
2B). This band was consistently present in reactions with
nucleoplasm, was not observed with the reticulocyte lysate, and may
represent the phosphorylated form of MyoD described in Song et
al. (10).

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Fig. 2.
MyoD is degraded in an
ATP-dependent manner by HeLa nucleoplasm. MyoD (400 ng) was added to the reaction mixtures, and degradation was assayed by
Western blotting with anti-MyoD as described under "Experimental
Procedures." The reaction mixtures contained an ATP-regenerating
(+ATP) or ATP-depleting (to,
ATP) system and were incubated for 2 h at 37 °C
except for the to reaction, which was kept at
4 °C during the incubation period. The results of five independent
assays were quantified. Asterisk (*) indicates significance
(< 0.001).
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Nucleoplasm Degrades MyoD in a Ubiquitin- and
Proteasome-dependent Manner--
To determine if the
degradation of MyoD that occurs in nucleoplasm is
proteasome-dependent, we examined the effect of ATP
S and
MG132. As seen in Fig. 3, MyoD is not
degraded in the presence of ATP
S alone. A >5-fold molar excess of
ATP
S is associated with a decrease in the ATP-dependent
degradation of MyoD in nucleoplasm as well as in reticulocyte lysate
(Fig. 3, ATP
S).

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Fig. 3.
Degradation of MyoD in HeLa nucleoplasm is
dependent on the 26 S proteasome. Degradation reactions were
carried out in the presence of either 5 mM ATP S or 20 µM MG132. The ATP S was added alone or in addition to
the ATP-regenerating system (ATP reg). MG132 dissolved in
Me2SO was added to reactions containing the
ATP-regenerating system. Me2SO alone was run as a
control.
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The decrease in degradation in the presence of ATP
S is consistent
with proteasome-mediated degradation since the ATPase activity at the
19 S cap structure of the 26 S proteasome is the
ATP-dependent step in the ubiquitin-proteasome pathway
requiring hydrolysis of the
-phosphate (46). To confirm involvement
of the proteasome in nucleoplasm-supported MyoD degradation, we
examined the effect of the proteasomal inhibitor, MG132. As seen in
Fig. 3, MG132 inhibited MyoD degradation in both HeLa nucleoplasm and
reticulocyte lysate. These observations indicate that the nuclear
proteasome is functional in the recognition and processing of a nuclear substrate.
To verify that the nucleoplasm-supported degradation of MyoD is
ubiquitin dependent, we examined the specificity of the
ATP-dependent degradation by using inhibitors directed at
the ubiquitin-substrate conjugation reactions. Proteins are tagged for
recognition by the 26 S proteasome via the conjugation of a
polyubiquitin chain to the (1). This polyubiquitin chain is generated
by isopeptide linkages between the carboxyl terminus of each ubiquitin
molecule with lysine 48 of the preceding ubiquitin. This process is
competitively inhibited by methylated ubiquitin (40) as well as the
ubiquitin K48R mutant (47). Thus, methylated ubiquitin (MeUb) and
ubiquitin K48R (Ub K48R) were added in increasing amounts to the
degradation reaction in an effort to overcome the effect of endogenous
ubiquitin. As seen in Fig. 4, the
ATP-dependent degradation of MyoD is competitively inhibited by MeUb and by UbK48R in either HeLa nucleoplasm or reticulocyte lysate. This competitive inhibition can be overcome by the
addition of excess wild-type ubiquitin in either case. These results
indicate that the ubiquitin-proteasome pathway is responsible for the
ATP-dependent degradation of MyoD in HeLa nucleoplasm.

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Fig. 4.
Degradation of myoD in HeLa nucleoplasm is
dependent on ubiquitin. Degradation reactions were carried out in
the presence of the polyubiquitin chain terminators MeUb and ubiquitin
K48R. Each inhibitor was added in increasing amounts (5, 10, and 15 µg/12.5-µl reaction) to reactions containing the ATP-regenerating
system (ATP reg). Wild-type ubiquitin (20 µg) was added in
2-fold excess over MeUb or Ub K48R (10 µg) in additional reactions.
Both the to and (ATP ) reactions contained the
ATP-depleting system. The to reactions were kept
at 4 °C during the 2-h incubation. Reticulocyte lysate or HeLa
nucleoplasm served as the cellular fraction.
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Ubiquitin Conjugation of MyoD in Nucleoplasm--
Ubiquitin
conjugation assays were performed with isolated nucleoplasm as a direct
measurement of the ability of the nuclear ubiquitin system components
to support conjugation (Fig. 5).
35S-Labeled MyoD is found in higher molecular mass forms
typical of ubiquitin conjugate ladders. The MyoD-ubiquitin conjugate
ladders occur only in the presence of nuclear extract and are increased by addition of the proteasome inhibitor, ATP
S (41) and ubiquitin aldehyde, an inhibitor of ubiquitin isopeptidases (42). In addition, high molecular mass ubiquitin conjugates accompany nucleoplasm-mediated myoD degradation (Fig. 6). These results
support our conclusion that the nuclear ubiquitin-proteasome system is
responsible for the degradation of MyoD.

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Fig. 5.
HeLa nucleoplasm supports the generation of
myoD-ubiquitin conjugates. 35S-Labeled myoD was
prepared in a coupled transcription-translation system as described
under "Experimental Procedures." The reaction was thereafter
inactivated with NEM/DTT. Conjugation assays were performed in the
absence or presence of HeLa nucleoplasm (NE), and in the
absence or presence of the proteasome inhibitors ATP S plus ubiquitin
aldehyde (ATP S), as described in the text.
Lanes 1 and 4 were incubated at
4 °C. High molecular mass myoD-ubiquitin conjugates resolved on
SDS-PAGE are noted (conj). MyoD is indicated by the
arrowhead.
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Fig. 6.
High molecular mass ubiquitin conjugates
accompany HeLa nucleoplasm-mediated myoD degradation. Degradation
reactions containing 100 ng of myoD and supported by either
reticulocyte lysate (left) or nucleoplasm (right)
were carried out in the presence of ATP and/or MG132, plus ubiquitin
aldehyde (Ubal) and ATP S as described under
"Experimental Procedures." Each reaction mix was analyzed via
SDS-PAGE and immunoblot with antibody to myoD. High molecular mass
myoD-ubiquitin conjugates are noted (conj.). MyoD is noted
by the arrowhead. Note that in the left
lane (+ATP) the myoD band is weak as it is
degraded efficiently.
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MyoD Is Degraded in Vivo in the Presence of Leptomycin B--
To
determine if the nucleus supports the degradation of MyoD in
vivo, we examined the degradation of MyoD in HeLa cells in the
presence of either MG132 or leptomycin B. LMB inhibits the CRM-1-dependent nuclear export pathway through a covalent
interaction at Cys-529 of CRM-1 (26) and has been used to determine if
nuclear export is required in the degradation of nuclear proteins. At 24-48 h after transfection, MyoD is localized to the nucleus under control conditions as well as during incubation with MG132 and/or leptomycin B (Fig. 7). As seen in Fig.
8, following inhibition of protein
synthesis in HeLa cells, the half-life of MyoD in control cells is
~1.3 h, i.e. there is an 80% decrease in MyoD levels during the 3-h "chase" period. In the presence of MG132, there is
only a 7% decrease in the level of MyoD (t1/2 = 28.4 h). This is consistent with previous reports of the
proteasome-dependent degradation of MyoD (10, 30). In
contrast, MyoD is not significantly stabilized in the presence of
leptomycin B. The 63% decrease in the level of MyoD
(t1/2 = 2.1 h.) (Fig. 8) in the presence of
leptomycin B is comparable to that seen in the control cells. This
result is confirmed in biosynthetic radiolabeling pulse-chase
experiments (data not shown).

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Fig. 7.
Immunofluorescent localization of MyoD.
HeLa cells were transfected with pClneo MyoD. 24 h later cells
were incubated with MG132 or LMB for 2 h as described above.
Thereafter, localization of MyoD was determined by indirect
immunofluorescence with a polyclonal anti-MyoD antibody (Santa Cruz)
and the resulting images were photographed (magnification, ×40) using
an Olympus BX60 microscope. A = control,
B = MG132 (20 µM), C = LMB (10 µM).
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Fig. 8.
MyoD is degraded in vivo in
the presence of leptomycin B. A, HeLa cells were
transiently transfected with MyoD and 24 h later were incubated
with cycloheximide (100 µg/ml) for the indicated times. The relative
amount of MyoD at each time point was determined by 10% SDS-PAGE,
followed by Western blotting with anti-MyoD as described under
"Experimental Procedures." The reactions were carried out in the
presence of MG132 (20 µM) or leptomycin B (10 nM) as indicated. B, the relative amounts of
MyoD were quantified from three independent experiments and the
half-lives determined as described under "Experimental
Procedures."
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To ensure that leptomycin B was active in inhibition of nuclear export,
we carried out a series of experiments using HeLa cells. Recent studies
have shown that I
B
shuttles between the cytoplasm and nucleus
(44, 48) and is degraded in the cytoplasm (24). These studies have also
shown that nuclear export and the subsequent degradation of I
B
alone or as a fusion protein with GFP is inhibited by LMB in HeLa cells
(24, 44). We therefore transfected HeLa cells with GFP-I
B
and
followed its fate with immunofluorescence in the presence or absence of
LMB (Fig. 9). Our results demonstrate
absence of GFP-I
B
from the nucleus under control conditions and
accumulation of GFP-I
B
in the nucleus in the presence of LMB,
confirming the LMB is biologically active.

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Fig. 9.
Leptomycin B inhibits the nuclear export of
GFP-I B . HeLa
cells were transiently transfected with GFP-I B . 24 h later
the cells were incubated for 1 h in the absence (A) or
presence (B) of 10 nM leptomycin B. GFP-I B
was localized by indirect immunofluorescence as described under
"Experimental Procedures."
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DISCUSSION |
The ubiquitin-proteasome system is essential for the turnover of
many short-lived regulatory proteins that are active within the
nucleus. The selective degradation of these nuclear proteins is
required for cellular processes such as cell cycle progression and
transcriptional regulation. Cell cycle progression is marked by the
ubiquitin-dependent degradation of nuclear substrates at the G1/S transition and during mitosis. The targeted
nuclear proteins include cell cycle kinases and their regulators as
well as proteins required for DNA replication (49) and repair (9, 50).
In addition, ubiquitin-dependent proteolysis of proteins
that regulate sister chromatid adhesion serves as a trigger for exit
from mitosis (51). Degradation of transcription factors and tumor
suppressors by the ubiquitin-proteasome pathway is well established (1) and recently described examples include ligand-activated transcription factors such as the aryl hydrocarbon receptor (20, 24, 25) and Smad2
(19).
Studies examining the degradation of p53 (23, 52),
p27kip1 (21), cyclin D1 (22), I
B
(24), and
the aryl hydrocarbon receptor (25) have demonstrated that nuclear
export is required for the turnover of a subset of nuclear proteins.
For several of these proteins, ubiquitin-dependent
degradation of the substrate appears to be linked to the nuclear export
of associated "molecular chaperones."
The Mdm2 oncoprotein targets p53 for degradation (53, 54) and
inhibiting the nuclear export of Mdm2 results in stabilization of p53
in the nucleus (23). Jab1-mediated degradation of p27 also requires
nuclear export. In this instance, inhibition of nuclear export blocks
the ability of Jab1 to shuttle the phosphorylated form of p27 to the
cytoplasm (21). Degradation of the AHR, which exists in the nucleus as
an AHR-hsp90 complex, is inhibited by blocking nuclear export or by
mutating the putative nuclear export sequence of AHR (25). From these
examples, a model of nuclear protein degradation emerges in which the
substrates are shuttled to the cytoplasmic ubiquitin-proteasome pathway
after being tagged for degradation by a post-translation modification
such as phosphorylation or via association with a molecular chaperone.
Furthermore, studies with p27 (21), AHR (25), and Smad2 (19) indicate
that nuclear proteasomal components may act in a modulating capacity in
the export of nuclear proteins. The possibility that nuclear
ubiquitin-proteasomal system may function to degrade the selected
substrates is strengthened by several observations. Most, if not all,
of the components of the ubiquitin-proteasome system necessary for the
degradation of a substrate are found in the nucleus, including
ubiquitin (5) and the 117-kDa isoform of the ubiquitin-activating
enzyme, E1 (8). Several of the ubiquitin conjugation enzymes (E2) have been shown to be localized primarily to the nucleus. Cdc34 (11, 55), an
E2 associated with the SCF-type E3 complexes (13, 14), is of particular
interest. Expression of dominant-negative deletions of Cdc34 result in
the stabilization of p27kip1 in cell extracts
(56) and of MyoD in vivo (10). The SCF-type E3-ubiquitin
ligases have been shown to be responsible for the destruction of a
large number of proteins including Sic1p (13, 14) and E2F-1 (57) and
function as a multisubunit complex (58). The Cdc53/Cul-1 subunit of
SCFSKP2 is localized to the nucleus when expressed with the
p45SKP2 F-box subunit of the E3 (11). These subunits can be
immunoprecipitated in a complex containing a third subunit of the E3
complex, p19SKP1 as well as Cdc34, suggesting that the
active ubiquitin ligase complex can be formed within the nucleus. In
addition, all of the components of the 26 S proteasome are found in the
nucleus (16-18, 59, 60). Finally, studies using a folding mutant of the influenza virus nucleoprotein that is localized to the
promyelocytic leukemia oncogenic domains in the nucleus indicate that
the promyelocytic leukemia oncogenic domains are the site of
concentration, and perhaps generation of polyubiquitin conjugates.
Proteasomal subunits are also colocalized therein. Indirect evidence
suggests that the ubiquitin-proteasome-dependent
degradation of the misfolded substrate occurs within the nucleus (61).
A functional nuclear ubiquitin-proteasome system may provide additional
levels of regulation of intracellular proteolysis via the cellular
targeting of a substrate as well as via compartment-specific activities
of its components.
In the present study, we developed a cell-free system based on HeLa
nucleoplasm in order to examine the ability of isolated nucleoplasm to
support ubiquitin-dependent degradation. This cell-free system allows for the direct examination of any specific nuclear component of the ubiquitin-proteasome pathway required for the turnover
of nuclear substrates. It was essential to ensure that the nucleoplasm
is not contaminated with cytoplasmic ubiquitin-proteasome components.
We assessed cross-contamination of the cytoplasm and nucleoplasm with
protein markers; lactate dehydrogenase activity (cytoplasmic marker)
and histone content (nuclear marker). Using this approach, we were able
to obtain nucleoplasm that is essentially free of cytoplasmic
contamination (i.e. < 0.02%).
We have established that HeLa nucleoplasm contains all the components
of the ubiquitin-proteasome pathway necessary for degradation of a
physiological nuclear substrate, MyoD. Recent studies have investigated
the ubiquitin-dependent degradation of MyoD using both
in vivo (10, 62) and in vitro approaches (30,
31). In vitro studies (31) using rabbit reticulocyte lysate
have shown that MyoD is degraded by a mechanism involving attachment of
ubiquitin at the N-terminal residue. In vivo studies
indicate that phosphorylation at serine 200 is required for the
proteasome-mediated degradation of MyoD (10, 62). We demonstrate herein
that degradation of MyoD in isolated nucleoplasm involves the 26 S
proteasome as shown by the stabilization of MyoD in the presence of
ATP
S and MG132. We confirmed that the ATP-dependent
degradation of MyoD in nucleoplasm is ubiquitin-dependent
by using the polyubiquitin-conjugation inhibitors methylated ubiquitin
and ubiquitin K48R. Inhibition by each of these ubiquitin variants can
be overcome by the addition of excess wild-type ubiquitin, indicating
that ubiquitin activation and conjugation are required for the
nucleoplasm-dependent degradation of MyoD. Moreover, in the
nucleoplasm, high molecular mass MyoD-ubiquitin conjugates are formed
in the presence of ATP
S, ubiquitin aldehyde, and MG132. Taken
together, our results demonstrate that the nuclear components of the
ubiquitin-proteasome system are able to function in the recognition and
degradation of a nuclear protein.
However, these results do not directly address the question of the
cellular locus of MyoD degradation. To confirm that MyoD is degraded in
the nucleus in vivo, we examined the stability of MyoD in
the presence of leptomycin B. Biosynthetic radiolabeling pulse-chase
experiments demonstrate that MyoD is degraded in the presence or
absence of leptomycin B. Furthermore, experiments in which protein
synthesis is inhibited with cycloheximide show that although the
addition of MG132 prolongs the half-life of MyoD from 1.3 to greater
than 20 h, the half-life of MyoD is relatively unchanged (1.3-2.1
h) in the presence of leptomycin B (Fig. 8). These results indicate
that nuclear export is not required for the degradation of MyoD.
Although the specific role(s) and importance of nuclear protein
degradation are not known at present, it is clear that the ubiquitin-mediated degradation of various proteins is regulated by
compartmentalized localization within the cell. Herein, the nucleus
appears to be a dynamic organelle in terms of protein degradation. As
discussed above, many of the components of the ubiquitin-proteasome
system are localized to the nucleus, some in a temporally controlled
manner. For example, although most of the subunits of the SCF complex
are localized to both cytoplasm and nucleus, several F-box proteins
(e.g. Cdc4 and Met30 in yeast; Ref. 63) localize
specifically to the nucleus. This suggests that the subcellular
compartmentalization of F-box proteins may be responsible for spatially
regulated SCF-mediated degradation (58). Individual substrates of the
ubiquitin-proteasome system including several nuclear proteins
(e.g. p53) are, however, degraded within the cytoplasm
following chaperone-mediated export from the nucleus (see above). The
precise signals that target an individual substrate for site-specific
ubiquitin-proteasome mediated destruction remain largely unclear. Since
submission of the present study, Blondel et al. (64) has
shown that the yeast cyclin-dependent kinase inhibitor Far1
is ubiquitinated and degraded in the nucleus via the nuclear
SCFCdc4 complex. Thus, these results, together with the
present results, demonstrate that a functional ubiquitin-proteasome
system exists within the nucleus and allows for the regulated
proteolysis of selected nuclear proteins. The signals that govern the
targeting to the nucleus as well as those processes that expose the
determinants for ubiquitin-protein ligase (E3) recognition and
polyubiquitination are yet to be fully defined. It is possible that
turnover of MyoD, the focus of the present study, is regulated
differently from other nuclear proteins given the unique requirement
for ubiquitination at the N-terminal amino group rather than an
internal lysine (31). This will be the subject of future studies.