Download The UBA2 Domain Functions as an Intrinsic Stabilization Signal that

Molecular Cell, Vol. 18, 225–235, April 15, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.03.015
The UBA2 Domain Functions as an Intrinsic
Stabilization Signal that Protects Rad23
from Proteasomal Degradation
Stijn Heessen,1,2 Maria G. Masucci,1
and Nico P. Dantuma1,3,*
1
Microbiology and Tumor Biology Center
Karolinska Institutet
Nobels väg 16
S-171 77 Stockholm
Sweden
2
Ludwig Institute for Cancer Research
Nobels väg 3
S-171 77 Stockholm
Sweden
3
Department of Cell and Molecular Biology
Karolinska Institutet
Nobels väg 3
S-171 77 Stockholm
Sweden
Summary
The proteasome-interacting protein Rad23 is a longlived protein. Interaction between Rad23 and the proteasome is required for Rad23’s functions in nucleotide excision repair and ubiquitin-dependent
degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting,
transferable stabilization signal that protects Rad23
from proteasomal degradation. Disruption of the UBA2
domain converts Rad23 into a short-lived protein that
is targeted for degradation through its N-terminal
ubiquitin-like domain. UBA2-dependent stabilization
is required for Rad23 function because a yeast strain
expressing a mutant Rad23 that lacks a functional
UBA2 domain shows increased sensitivity to UV light
and, in the absence of Rpn10, severe growth defects.
The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and
the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is
an evolutionarily conserved stabilization signal that
allows Rad23 to interact with the proteasome without
facing destruction.
Introduction
The Saccharomyces cerevisiae Rad23 protein is involved in regulation of nucleotide excision repair, proteolysis and cell cycle progression (Clarke et al., 2001;
Lambertson et al., 1999; Watkins et al., 1993). While the
precise mode of action of Rad23 is unknown, increasing evidence suggests that a close interplay between
Rad23 and the ubiquitin/proteasome system is essential for its diverse functions (Chen and Madura, 2002;
Russell et al., 1999; Schauber et al., 1998). The ubiquitin/proteasome system was originally identified as the
primary degradation machinery in the cytosol and nucleus of eukaryotic cells (Baumeister et al., 1998;
*Correspondence: [email protected]
Hershko and Ciechanover, 1998), but recent studies
have revealed additional nonproteolytic functions in
DNA repair and transcription (Ferdous et al., 2001; Gonzalez et al., 2002; Russell et al., 1999).
The presence of several characteristic structural domains highlights the close relationship of Rad23 with
the ubiquitin/proteasome system. Rad23 contains an
N-terminal ubiquitin-like (UbL) domain and two ubiquitin-associated (UBA) domains: an internal UBA1 domain and a C-terminal UBA2 domain (Buchberger,
2002). The UbL domain, which can be functionally replaced by ubiquitin (Watkins et al., 1993), mediates
the interaction between Rad23 and the proteasome
(Schauber et al., 1998). The UBA domain was originally
identified as a sequence motif present in proteins
linked to the ubiquitination system (Hofmann and
Bucher, 1996). It was later reported that, notwithstanding their low sequence homology, all UBA domains
share the ability to bind ubiquitin (Bertolaet et al.,
2001b; Chen et al., 2001; Wilkinson et al., 2001). Based
on its ability to simultaneously bind ubiquitinated proteins and the proteasome, it has been proposed that
Rad23 may function as a scaffold that facilitates interactions between substrates and the proteasome
(Chen and Madura, 2002; Kim et al., 2004). Recently,
direct biochemical evidence has been provided for a
role of Rad23 in targeting ubiquitinated substrates to
the proteasome (Elsasser et al., 2004; Saeki et al.,
2002a; Verma et al., 2004). Several studies revealed that
a concerted action of the ubiquitin/proteasome system
and Rad23 is important for nucleotide excision repair
(NER) although the precise mode of action of the proteasome in this event remains obscure (Russell et al.,
1999; Schauber et al., 1998). Interestingly, this DNA repair activity requires only the 19S regulatory particle of
the proteasome and is independent of the proteolytically active 20S core particle (Gillette et al., 2001).
Substrates of the ubiquitin/proteasome system are
targeted by degradation signals, which are small motifs
or domains that accommodate interaction with the
proteasome in a ubiquitin-dependent (Hershko and
Ciechanover, 1998) or, less common, a ubiquitin-independent manner (Chen et al., 2004; Murakami et al.,
1992). As a general rule, recruitment of proteins to the
proteasome results in their rapid inactivation by processive degradation. However, the interaction between
Rad23 and the proteasome does not lead to Rad23 destruction (Schauber et al., 1998; Watkins et al., 1993).
We have previously described a similar phenomenon
with a viral repetitive sequence that can be used for
cis-stabilization of proteasome substrates (Heessen et
al., 2002; Levitskaya et al., 1995; Sharipo et al., 1998).
Based on these findings, we postulated that cellular
proteins might contain similar protective “stabilization
signals” that spare them from proteolysis (Dantuma and
Masucci, 2002). Here, we show that the C-terminal UBA
domains of Rad23, Dsk2, Ddi1, and Ede1 function as
stabilization signals that can protect proteins from proteasomal degradation.
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Figure 1. Cis Stabilization of Proteasome Reporter Substrates by the UBA2 Domain
(A) Schematic drawing of the fusion of the N-end rule reporter substrate Ub-R-GFP and Rad23 fragments. The locations of the UbL, UBA1,
and UBA2 domains in Rad23 are indicated.
(B) Lysates of 10 OD600 units of log-phase yeast coexpressing Ub-R-GFP-Rad23⌬UbL and 3HAubiquitin were subjected to immunoprecipitation
with an anti-GFP antibody, followed by Western blotting with an anti-HA antibody. Yeast cells transformed with an empty vector and 3HAubiquitin were used as negative control. The molecular weight markers, ubiquitinated species, and the immunoglobulin heavy chain (HC) are indicated.
(C) Representative histograms of flow cytometric analysis of yeast expressing the Ub-M-GFP, Ub-R-GFP, or the Ub-R-GFP fusions shown in
(A). The mean fluorescence intensity of each sample is indicated in the upper-right corner (corrected for background fluorescence of vector
transformed yeast).
(D) Quantification of mean fluorescence intensities of yeast cells expressing Ub-M-GFP, Ub-R-GFP, or the Ub-R-GFP fusion proteins measured
by flow cytometry. Ub-M-GFP was standardized as 100%. Values are means and standard deviations of three independent experiments.
Asterisks indicate values that are significantly different from Ub-R-GFP (t test, p < 0.05).
(E) Representative histograms of flow cytometric analysis of yeast cells expressing UbG76V-GFP or UbG76V-GFP-Rad23⌬UbL.
UBA2 Is an Intrinsic Stabilization Signal
227
Figure 2. An Intact UBA2 Domain Protects from Proteasomal Degradation and Does Not Cause General Impairment of Proteasomal Degradation
(A) Western blot analysis of turnover of Ub-R-GFP, Ub-R-GFP-UBA1, and Ub-R-GFP-UBA2 levels in wild-type yeast and Ub-R-GFP in the
ubr1D strain at 0, 10, 20, and 40 min after promoter shutoff.
(B) Densitometric quantification of the Ub-R-GFP (closed circles) and Ub-R-GFP-UBA2 (open circles) signal in (A). The signal at t = 0 is
standardized as 100%.
(C) Mean fluorescence intensities of yeast expressing Ub-R-GFP-UBA1, Ub-R-GFP-UBA2, and Ub-R-GFP-UBA2L392A. Mean and standard
deviations of three independent experiments are shown. Asterisk indicates values that are significantly different from Ub-R-GFP-UBA1 (t test,
p < 0.01).
(D) Western blot analysis with specific GFP antibody of Ub-R-GFP and Ub-R-GFP-Rad23⌬UbL levels at 0, 10, 20, and 40 min after switching
off the GAL1 promoter in yeast expressing Ub-R-GFP alone or together with Ub-R-GFP-Rad23⌬UbL. Putative ubiquitinated species (asterisks)
and molecular weight markers are indicated.
Results
Cis Stabilization of Proteasome Reporter
Substrates by the UBA2 Domain
The remarkable stability of the proteasome-interacting
protein Rad23 prompted us to investigate whether this
protein may harbor domains that protect it from protea-
somal degradation. For this purpose, we analyzed
whether insertion of Rad23 fragments in green fluorescent protein (GFP)-based substrates of the ubiquitin/
proteasome system could inhibit their degradation (Figure 1A). These GFP-based substrates are targeted to
the proteasome through the presence of well-defined
degradation signals that deem the GFP for ubiquitin-
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228
Table 1. Yeast Strains Used in this Study
Strain
Genotype
Reference
DF5
MATa lys2-801 leu2-3, 112 ura3-52
his3-⌬200 trp1-1
DF5 ubr1D::LEU2
DF5 rad23D::kanMX-KlURA3
DF5 FLAG-RAD23
DF5 FLAG-rad23(L392A)
DF5 rpn10D::HIS3
DF5 rad23D::kanMX-KlURA3
rpn10D::HIS3
DF5 FLAG-RAD23 rpn10D::HIS3
DF5 FLAG-rad23(L392A) rpn10D::HIS3
Finley et al.
BBY47
SHY002
SHY003
SHY004
SHY012
SHY013
SHY014
SHY015
Bartel et al.
this study
this study
this study
this study
this study
this study
this study
dependent proteolysis (Dantuma et al., 2000b). Because it is well established that the UbL domain of
Rad23 mediates interaction with the proteasome, we
omitted the UbL domain to assure that only the degradation signal of the reporter substrate would target the
fusion to the proteasome.
Introduction of the Rad23⌬UbL did not interfere with
cleavage of the N-terminal ubiquitin of ubiquitin-R-GFP
(Ub-R-GFP; data not shown), which is required for activation of the N-end rule degradation signal in this reporter substrate (Varshavsky, 1996), nor did it affect
targeting of the Ub-R-GFP for ubiquitination because
we could readily detect ubiquitinated reporter substrates (Figure 1B). Insertion of the Rad23⌬UbL in the
Ub-R-GFP reporter resulted in a 14-fold increase in the
mean fluorescent intensity of the reporter-expressing
yeast to approximately 50% of the level observed with
the stable Ub-M-GFP that lacks a degradation signal
(Figures 1C and 1D). To identify the region in Rad23 that
was responsible for this effect, we produced a set of
C-terminal deletions of the Ub-R-GFP-Rad23⌬UbL fusion protein (Figure 1A). Removal of the C-terminal
UBA2 domain brought the fluorescence intensity almost back to the Ub-R-GFP level (Figures 1C and 1D).
Insertion of the UBA2 domain was sufficient to recapitulate the effect observed with Rad23⌬UbL, whereas the
related UBA1 domain alone had no appreciable effect
on the mean fluorescent intensity (Figures 1C and 1D).
To test whether the stabilizing effect of UBA2 was
dependent on the type of degradation signal, we analyzed the effect of the UBA2 domain on a reporter substrate carrying an ubiquitin fusion degradation (UFD)
signal, UbG76V-GFP. Of note, N-end rule and UFD substrates are targeted through distinct ubiquitination
pathways for degradation by the proteasome (Bartel et
al., 1990; Johnson et al., 1995; Koegl et al., 1999). We
found a very similar protective effect on the UFD reporter substrate, suggesting that the effect is largely independent of the type of degradation signal (Figure 1E).
An Intact UBA2 Domain Is Required for Protection
from Proteasomal Degradation
Promoter shutoff experiments demonstrated that the
increased steady-state levels of UBA2-containing substrates were due to delayed turnover of the reporter
while the UBA1 domain had no effect on degradation
(Figures 2A and 2B). The Ub-R-GFP reporter was stable
in the ubr1D yeast strain, which lacks the N-end rule
ubiquitin ligase (Table 1), confirming that the ubiquitin/
proteasome system is responsible for degradation of
this substrate in yeast (Bartel et al., 1990). Thus, the
UBA2 domain increases the steady-state levels of
Ub-R-GFP by delaying its proteasomal degradation.
Structural analysis of the UBA2 domains of the human homolog of Rad23 A (hHR23A) has revealed that a
conserved leucine corresponding with leucine 392 in
yeast Rad23 is important for the structural integrity of
the characteristic UBA fold (Mueller and Feigon, 2002).
Substitution of this single amino acid with an alanine
was sufficient to completely abrogate the protective effect, suggesting that a proper UBA fold is important for
the inhibitory activity (Figure 2C).
The UBA2 domain Does Not Cause General
Impairment of the Ubiquitin/Proteasome System
It has been reported that UBA domains can act as
general inhibitors of ubiquitination (Ortolan et al., 2000)
and deubiquitination (Hartmann-Petersen et al., 2003).
Thus, stabilization of the GFP reporter could be the
consequence of a general impairment of the ubiquitin/
proteasome system caused by the overexpressed
UBA2 domain. To probe into this possibility, we examined whether overexpression of the stable Ub-R-GFPRad23⌬UbL fusion protein led to a general accumulation
of proteasome substrates. Ub-R-GFP-Rad23⌬UbL and
the Ub-R-GFP or UbG76V-GFP substrates were coexpressed in yeast and their degradation was followed
after simultaneously switching off expression of both
proteins. Overexpression of Ub-R-GFP-Rad23⌬UbL did
not affect the turnover of Ub-R-GFP (Figure 2D) or
UbG76V-GFP (data not shown). Furthermore, while general impairment of the ubiquitin/proteasome system results in accumulation of ubiquitinated substrates and
induction of cell cycle arrest and apoptosis (Dantuma
et al., 2000b), overexpression of Ub-R-GFP-Rad23⌬UbL
did not increase the total pool of ubiquitin-conjugates
nor did it affect proliferation (data not shown). We conclude that insertion of the Rad23 UBA2 domain can selectively protect proteasome substrates carrying the
UBA2 domain from degradation without disturbing
overall proteasomal degradation.
The UBA2 Domain Functions as an Intrinsic
Stabilization Signal in Rad23
In order to explore whether the UBA2 domain plays a
protective role in the context of native Rad23, we took
advantage of the single L392A amino acid substitution
that abrogated the protective effect of the UBA2 domain. Introduction of this amino acid substitution converted Rad23 from a very stable into a short-lived protein with a half-life of approximately 10 min (Figures 3A
and 3B). Because mutations can result in rapid degradation due to misfolding, it was important to assess
whether the Rad23L293A was targeted for degradation
through a misfolded UBA2L392A domain or through an
endogenous targeting signal that is also functional in
the context of native Rad23. We argued that the UbL
domain could fulfill this role as it has been shown to
mediate the interaction of Rad23 with the proteasome
(Schauber et al., 1998). Deletion of the UbL domain resulted in full stabilization of the mutant Rad23L392A (Figures 3A and 3B), excluding the possibility that the
UBA2L392A domain targets the mutant Rad23 for degra-
UBA2 Is an Intrinsic Stabilization Signal
229
Figure 3. The UBA2 Domain Functions as an Intrinsic stabilization Signal in Rad23
(A) Turnover of FLAGRad23, FLAGRad23L392A, and FLAGRad23⌬UbL/L392A after promoter shutoff. Samples were collected at the indicated time
points after shutting off the GAL1 promoter and analyzed in anti-FLAG Western blot analysis.
(B) Densitometric quantification of Western blot shown in (A). FLAGRad23 (closed circles), FLAGRad23L392A (open circles), and FLAGRad23⌬UbL/
L392A
(squares). The signal at t = 0 for each of the constructs was standardized as 100%.
(C) Steady-state levels of FLAG-tagged Rad23 in the wild-type, rad23D, FLAGRad23, and FLAGRad23L392A yeast strains determined in an antiFLAG Western blot on 0.25 OD600 units. FLAGRad23 and FLAGRad23L392A are expressed from the endogenous Rad23 promoter.
(D) Turnover of FLAGRad23 and FLAGRad23L392A was determined by metabolic labeling with 35S-methionine/cysteine followed by chasing within
the presence of an excess cold methionine and cysteine for the indicated times. FLAGRad23 and FLAGRad23L392A were immunoprecipitated
with anti-FLAG antibodies. Asterisks indicate a nonspecific band.
dation. This shows that UbL domain is important for
targeting Rad23 to the proteasome (Schauber et al.,
1998) and, in the absence of the protective UBA2 domain, for induction of Rad23 proteolysis.
Three yeast strains were generated to gain insight in
the role and functional significance of the UBA2 domain
under physiological conditions: (1) a rad23D strain,
which lacks the Rad23 gene, (2) a FLAGRad23 strain,
and (3) a FLAGRad23L392A strain, which express FLAGtagged wild-type Rad23 and FLAG-tagged mutant
Rad23L392A, respectively, from the endogenous Rad23
promoter (Table 1). The FLAGRad23 could easily be detected in Western blot analysis, whereas the level of the
FLAG
Rad23L392A mutant was below the detection limit
(Figure 3C). Pulse-chase analysis revealed that this
striking difference in steady-state levels was due to accelerated degradation of the Rad23L392A mutant (Figure
3D). We conclude that the UBA2 domain is important
for physiological expression levels of Rad23 from its
endogenous promoter.
Rad23 Lacking a Protective UBA2 Domain
Is Functionally Compromised
It has been reported that the UBA1 and UBA2 domains
of Rad23 are dispensable for functional NER (Bertolaet
et al., 2001b). Consistent with this earlier report, we
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Figure 4. Rad23 Lacking a Protective UBA2 Domain Is Functionally Compromised
(A) UV sensitivity of the wild-type, rad23⌬, FLAGRad23, and FLAGRad23L392A yeast strains. Serial dilutions of yeast cultures were spotted on
YPD agar and were left untreated or exposed to 300 J/m2 UV light.
(B) UV sensitivity of the wild-type, rad23⌬, FLAGRad23, and FLAGRad23L392A yeast strains. Serial dilutions of yeast cultures were spotted
on YPD agar and were left untreated or exposed to one, two, or three exposures of 100 J/m2 UV light with 20 min intervals between
each exposure.
(C) Quantification of UV sensitivity by colony survival assay. Yeast was exposed to 3 × 100 J/m2 or left untreated. The numbers were
standardized to untreated controls. Mean and standards deviation of triplicate measurements are shown. Asterisks indicate that values are
significantly different from wild-type strain (t test, p < 0.02).
(D) Serial dilutions of the indicated yeast cultures were spotted on YPD agar and were grown at 30°C or 25°C.
found that yeast cells expressing the destabilized
FLAG
Rad23L392A were only slightly affected in their ability to survive a 300 J/m2 UV light exposure, whereas
rad23D cells were highly sensitive to this UV dose (Figure 4A). This suggests that the reduced Rad23 levels in
the FLAGRad23L392A strain are still sufficient for NER. A
more dramatic effect on cell survival was revealed
when the yeast was subjected to three subsequent UV
exposures of 100 J/m2 with 20 min intervals. The 1st UV
exposure had a strong inhibitory effect on the growth
of the rad23D strain, whereas the FLAGRad23 and
FLAG
Rad23L392A strains coped equally well (Figure 4B).
UBA2 Is an Intrinsic Stabilization Signal
231
of Rad23 as multiubiquitin chain receptor engaged in
the targeting of substrates to the proteasome, a notion
that was recently supported by detailed biochemical
studies (Verma et al., 2004). We investigated the significance of the protective UBA2 domain in relation to this
function of Rad23. Deletion of Rad23 and Rpn10 resulted indeed in slow growth, in particular at 25°C (Figure 4D). Whereas introduction of the FLAGRad23 restored the growth to wild-type levels, the destabilized
FLAG
Rad23L392A did not rescue the growth phenotype of
the rad23D/rpn10D double mutant. Thus, the presence
of a protective UBA2 domain is of critical significance
for Rad23 function.
Figure 5. C-terminal UBA Domains of Dsk2, Ddi1, and Ede1 Can
Protect from Proteasomal Degradation
(A) Schematic representation of the UbL and UBA domains in
Rad23, Ddi1, Dsk2, and Ede1.
(B) Mean fluorescence intensities of yeast expressing Ub-R-GFP,
Ub-R-GFP-UBA2(Rad23), Ub-R-GFP-UBA(Dsk2), Ub-R-GFP-UBA
(Ddi1), and Ub-R-GFP-UBA(Ede1). Mean and standard deviations
of three independent experiments are shown. Asterisks indicate
values that are significantly different from Ub-R-GFP: *(t test, p <
0.02), **(t test, p < 0.01).
(C) Western blot analysis with specific GFP antibody of Ub-R-GFP,
Ub-R-GFP-UBA(Dsk2),
Ub-R-GFP-UBA(Ddi1),
and
Ub-R-GFPUBA(Ede1) levels at 0, 10, 20, and 40 min after switching off the
GAL1 promoter.
However, a clear difference was observed in the survival of the FLAGRad23L392A strain after the second and
third UV exposures, with increased sensitivity to UV irradiation with each exposure (Figures 5B and 5C). Thus,
although the short-lived FLAGRad23L392A appeared to be
sufficient for UV resistance after a single UV exposure,
it was not capable of rescuing yeast from multiple
rounds of UV exposure.
Yeast strains lacking both Rad23 and the ubiquitin
binding proteasome subunit Rpn10 display impaired
degradation of proteasome substrates, accumulation
of ubiquitinated proteins, and growth retardation that is
most striking at low temperatures (Lambertson et al.,
1999). These findings are in line with a possible function
The C-Terminal UBA Domains of Dsk2, Ddi1,
and Ede1 Act as Stabilization Signals
Dsk2 and Ddi1 share with Rad23 the presence of an
N-terminal UbL domain and a C-terminal UBA domain
(Figure 5A). In addition, some proteins such as Ede1, a
protein engaged in membrane trafficking (Aguilar et al.,
2003), carry a C-terminal UBA domain but lack an UbL
domain (Figure 5A). In order to examine whether the
UBA domains of these proteins share the protective capacity of Rad23’s UBA2 domain, they were fused to the
C terminus of the Ub-R-GFP reporter. Flow cytometric
analysis clearly demonstrated a significant increase in
the steady-state fluorescence levels of the chimeric reporters (Figure 5B). Most striking was the effect of the
UBA domain of Dsk2, which even exceeded the effect
of the Rad23 UBA2 domain, while the Ddi1 and Ede1
had weaker effects. Analysis of the turnover of these
reporter constructs confirmed that the increase in the
steady-state levels was due to a delay in degradation
(Figure 5C). We conclude that the stabilizing effect of
the C-terminal UBA domain is not unique for Rad23 but
is a more general phenomenon shared with at least
three other UBA domains.
The Protective Effect of the UBA2 Domain
Is Evolutionarily Conserved
To examine whether the protective effect of the UBA2
domain has been conserved during evolution, we next
tested the activity of the UBA1 and UBA2 domains of
the human homolog hHR23A. We found that the UBA2
domains of Rad23 and hHR23A but not their UBA1
counterparts are equally capable of stabilizing the UbR-GFP reporter in yeast (Figure 6A). In order to assess
whether the protective effect also operates in mammalian cells, we used a previously described assay in
which human cervix carcinoma HeLa cells are transiently transfected with these fusions and the percentage of fluorescent cells is determined in the absence
or presence of a specific proteasome inhibitor (Dantuma et al., 2000a). Introduction of the UBA1 domain of
Rad23 or hHR23A in Ub-R-GFP did not affect the stability of this reporter in HeLa cells, whereas the UBA2
domains inhibited degradation (Figures 6B and 6C). We
conclude that the stabilizing effect of the UBA2 domain
in Rad23 is conserved during evolution.
Discussion
We have shown that the UBA2 domain of Rad23 act as
an intrinsic, cis-acting, transferable stabilization signal.
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Figure 6. Protective Effect of the UBA2 Domain Is Evolutionarily
Conserved
(A) Mean fluorescence intensities of yeast expressing Ub-M-GFP,
Ub-R-GFP, or Ub-R-GFP fused to the UBA1 or UBA2 domain of
yeast Rad23 and human hHR23A. Mean fluorescent intensity of UbM-GFP-expressing yeast was standardized as 100%. Means and
standard deviations of three independent experiments are shown.
Asterisks indicate values significantly different from Ub-R-GFP (t
test, p < 0.05).
(B) Dot plots of flow cytometric analysis of HeLa cells transiently
transfected with the indicated constructs. Cells were left untreated
or incubated with 10 ␮M of the proteasome inhibitor Z-L3-VS for 8
hr. The percentage of GFP-positive cells in each sample is indicated in the upper right corner. Values from one representative experiment out of three are shown.
(C) Quantitative analysis of flow cytometric analysis depicted in (B).
Relative fluorescence is expressed as the percentage of GFP positive cells in the absence of inhibitor divided by the percentage of
GFP positive cells in the presence of inhibitor. Means and standard
deviations from three independent experiments are shown. Asterisks indicate values significantly different from Ub-R-GFP (t test,
p < 0.05).
Thus, Rad23 harbors counteracting degradation and
stabilization signals, which enable the protein to interact with the proteasome without being degraded.
UBA2-dependent stabilization is required for Rad23
function in DNA repair and protein degradation.
Our data show that protein stabilization is a characteristic shared by at least four C-terminally positioned
UBA domains present in Rad23, Dsk2, Ddi1, and Ede1.
Rad23, Dsk2, and Ddi1 each contain an N-terminal proteasome-interacting UbL domain and a C-terminal UBA
domain (Jentsch and Pyrowolakis, 2000), whereas Ede1
lacks the UbL domain. Whether Ede1, which is the
yeast homolog of eps15 and involved in ubiquitindependent membrane trafficking (Aguilar et al., 2003),
interacts with the proteasome awaits clarification.
Rad23, Dsk2, Ddi1, and Ede1 are to our knowledge the
first examples of cellular proteins that harbor a specific
domain that enables proteins to resist degradation. It
remains to be seen how widespread this phenomenon
is, but there are several findings in the literature suggesting that other proteins may contain related and unrelated stabilization signals (Dantuma and Masucci,
2002). For instance, a truncated variant of the ataxin
1-interacting ubiquitin-like protein lacking the UBA domain was also found to be degraded by the ubiquitin/
proteasome system (Riley et al., 2004).
How do C-terminal UBA domains protect from degradation? There are indications that UBA domains do not
only bind ubiquitin but also UbL domains (Raasi and
Pickart, 2003; Wang et al., 2003). Based on these findings, it has been proposed that intramolecular interactions between the UbL and UBA domains may give
Rad23 a closed conformation that cannot bind to the
proteasome (Madura, 2002; Wang et al., 2003). Others
have proposed that UBA domains can inhibit elongation of polyubiquitin chains by capping conjugated
ubiquitin (Chen et al., 2001; Ortolan et al., 2000). Although either UbL binding or inhibition of polyubiquitination provides possible explanations for the stabilizing
effect of the C-terminal UBA domain, there are several
observations that do not support these models. First,
the ubiquitin binding activity of the UBA domains is not
sufficient for the protective effect since the very similar
UBA1 domains of Rad23 and hHR23A, that bind ubiquitin and block ubiquitination equally well as UBA2 domains (Bertolaet et al., 2001b; Chen et al., 2001; Rao
and Sastry, 2002), lacked this protective activity. Second, the presence of the UBA domain did not have any
striking effects on the handling of ubiquitin on the substrate: the N-terminal ubiquitin was efficiently cleaved
of the Ub-R-GFP to activate the N-end rule degradation
signal and the reporter was found to be polyubiquitinated. Third, Madura and colleagues previously reported that C-terminal tagging of Rad23 dramatically
affected its half-life and resulted in rapid proteasomal
degradation (Schauber et al., 1998). We have obtained
preliminary data suggesting that a C-terminal extension
disturbs the protective effect of the UBA2 domain (unpublished data). It seems unlikely that short motifs
lacking any secondary structure would interfere with
UBA interactions since both internal and C-terminal
UBA domains were found to bind ubiquitin in the
context of the native protein (Bertolaet et al., 2001b).
Fourth, the interaction between Rad23 and the protea-
UBA2 Is an Intrinsic Stabilization Signal
233
some has been studied in much detail (Elsasser et al.,
2002; Kim et al., 2004; Saeki et al., 2002b; Schauber et
al., 1998), leaving little doubt about the fact that Rad23
does interact with the proteasome in vivo.
A possibility that cannot be excluded is masking of
polyubiquitin chains by the UBA2 domain. Thus the
UBA2 domain could bind to polyubiquitin chains conjugated to Rad23, thereby modifying the interaction between Rad23 to the proteasome in such a way that it
does not promote degradation. It remains unclear
whether Rad23 itself is a target for ubiquitination, but
the fission yeast homolog Rph23 was shown to be
polyubiquitinated (Elder et al., 2002). In disfavor of this
model, it was recently shown that the UBA1 and UBA2
domains bind synthetic Lys48 tetraubiquitin chains
equally well (Raasi and Pickart, 2003), but more detailed analyses on the in vivo preferences of UBA domains for different polyubiquitin chains is required to
conclusively address this issue.
Our study shows that the structural integrity of the
UBA2 domain is an essential constraint for the protective effect. An attractive hypothesis that could explain
these results is that the C-terminal UBA2 domain hinders unfolding of proteasome-bound Rad23. Prakash
and coworkers have recently shown that degradation
signals require an unstructured initiation site for efficient unfolding and proteasomal degradation (Prakash
et al., 2004). Unfolding is a rate-limiting and crucial step
in the sequence of events that leads to degradation of
ubiquitinated substrates (Thrower et al., 2000), and in
vitro studies have shown that tightly folded C-terminal
domains can delay or block proteasomal degradation
(Navon and Goldberg, 2001). Disturbing the UBA2 fold
may enable the proteasome to unfold and degrade
Rad23. This would also explain the intriguing observation that simple C-terminal tagging can destabilize
Rad23 (Schauber et al., 1998) because extending the
UBA2 domain with unstructured epitope tags may provide the proteasome with a “handle” to unwind the UBA
domain. Notably, it has been hypothesized that processing domains, i.e., domains that interrupt proteasomal degradation of a precursor protein at a specific
point (Rape et al., 2001), may mediate protein-protein
interactions that terminate degradation by forming stable structures (Rape and Jentsch, 2002). UBA domains
can interact with each other (Bertolaet et al., 2001a),
and a similar mechanism may therefore apply for stabilization by UBA domains.
We have shown that yeast expressing destabilized
Rad23 can resist a high, single dose of UV exposure,
despite extremely low Rad23 levels. This observation is
consistent with earlier reports showing that deletion of
the UBA2 domain does not affect UV sensitivity after a
single UV exposure (Bertolaet et al., 2001b; Chen and
Madura, 2002). Apparently cells contain excess amounts
of Rad23. This may be related to the emerging role of
Rad23 in the targeting of substrates for ubiquitin/proteasome-dependent proteolysis (Verma et al., 2004).
Even more remarkable is the increased sensitivity of
yeast expressing Rad23L392A to repeated UV exposures. Conceivably, NER lesions may induce Rad23proteasome interactions and cause a further depletion
of the already low levels of Rad23 in this strain. Because UV exposure also causes proteotoxic stress and
can induce DNA damage-independent responses (Bryan
et al., 2004), it cannot be excluded that this unique phenotype is caused by a combination of reduced NER activity and reduced substrate targeting activity as a consequence of the low Rad23 levels.
Our study identifies C-terminal UBA domains as cellular stabilization signals and the primary element that
enables Rad23 and hHR23A to interact with the proteasome without facing destruction. An attractive hypothesis is that Rad23 by acquiring the UBA2 domain has
evolved from a proteasome substrate into a privileged
protected “substrate” that assists the ubiquitin/proteasome system as a dedicated proteasome scaffold in
DNA repair and substrate targeting.
Experimental Procedures
Yeast Strains and Plasmids
All strains used in this work are listed in Table 1 and are derivatives
of DF5. Correct integration of gene cassettes was confirmed by
whole-locus PCR analysis. SHY002 was constructed by replacing
the entire RAD23 open reading frame (ORF) by transforming DF5
with a PCR product encompassing the kanMX4-KlURA3 cassette
from pCORE (Storici et al., 2001). Ura+ G418R clones were isolated
and UV sensitivity was confirmed. To obtain SHY003, the SHY002
strain was transformed with a FLAG-RAD23 PCR fragment from
pYES3/CT-FLAG-RAD23 deleting the kanMX4-KlURA3 cassette,
giving rise to 5-fluorootic acid-resistant (5-FOAR) and G418S
clones. SHY004 was created similarly by transforming SHY002 with
a FLAG-RAD23L392A PCR product amplified from pYES3/CT-FLAGRAD23L392A. RPN10D strains SHY012-15 were generated by replacement of the complete RPN10 ORF with an HIS3 containing
cassette that was PCR amplified from pRS313.
The FLAGRad23 open reading frame and fragments were PCR amplified from pCS13-FLAGRad23 (gift from Dr. Madura, Robert Wood
Johnson Medical School, Piscataway, NJ) and subcloned under the
regulation of a GAL1 promoter in the yeast expression vector
pYES2 (Invitrogen, 2 ␮ URA3) or pYES3/CT (Invitrogen, 2 ␮ TRP1).
The UBA domains of Dsk2, Ddi1, and Ede1 were PCR amplified
from genomic yeast DNA. The hHR23A UBA1 (codons 159–200)
and UBA2 (codons 317–357) domains were PCR-amplified from total HEK293T cell cDNA.
Flow Cytometry
HeLa cells transiently transfected with lipofectamine were treated
8 hr with 10 ␮M of the proteasome inhibitor carboxybenzyl-leucylleucyl-leucine vinyl sulfone (Z-L3VS, gift from Dr. Ploegh, Harvard
Medical School, Boston, MA) and subjected to flow cytometric
analysis on a fluorescent activated cell sorter (Beckton & Dickinson).
Western Blot Analysis and Immunoprecipitations
Western blots of total protein extracts were probed with mouse
monoclonal anti-FLAG (M5, Sigma-Aldrich), anti-HA antibody (Boehringer-Mannheim), anti-HA ascites, and rabbit polyclonal anti-GFP antibody (Molecular Probes Europe). For immunoprecipitations, yeast
was lysed with acid-washed glass beads in HEPES lysis buffer (50
mM HEPES, 5mM EDTA, 1% TritonX-100, 150 mM NaCl), supplemented with 20mM NEM (Sigma-Aldrich) and 0.5 ␮M epoxomycin
(Affinity Research Products). For anti-FLAG immunoprecipitations,
sepharose beads with conjugated M2 anti-FLAG monoclonal antibody were used (M2-Sepharose, Sigma).
Turnover Analysis
For GAL1 promoter shutoff experiments, transcription and translation were arrested by adding glucose and cycloheximide (Sigma)
to final concentrations of 2% and 0.5 mg/ml, respectively. For endogenously expressed proteins, only cycloheximide was added to
the growth media (final concentration 100 ␮g/ml). Aliquots were taken
at the indicated time points, and total protein extracts were prepared by NaOH lysis and TCA precipitation. Samples were heated
Molecular Cell
234
10 min at 65°C and subjected to SDS-PAGE before Western blot
analysis with the appropriate antibodies. Pulse-chase analyses
were performed by metabolic labeling of 6 OD600 units of yeast
with 100 ␮C 35S-methionine/35S-cysteine (Redivue ProMix, Amersham) for 10 min and chasing in the presence of 10 mM cold methionine and 1 mM cold cysteine for the indicated time. Yeast was
washed, lysed with acid-washed glass beads, and the proteins of
interest were immunoprecipitated from the lysate.
UV Survival Assays
For drop assays, yeast cells were resuspended in sterile water and
serial 10-fold dilutions were spotted as 1.5 ␮l drops on YPD plates.
Plates were irradiated with a single dose of 300 J/m2 or a maximum
of 3× 100 J/m2 254 nm UV light using the Stratalinker UV crosslinker
(Stratagene). All plates were wrapped in aluminum foil and kept in
the dark both during the 20 min recovery intervals and after the
final UV exposure. Plates were photographed after 2 days of
growth at 30°C. For quantitative survival analyses, yeast cells were
diluted in a 24-well plate before serial exposures to 100 J/m2 UV
with 20 min intervals. After the final exposure, the yeast was evenly
spread out over YPD plates and the number of colonies was determined after growth at 30°C for 2 days in the dark.
Acknowledgments
We thank Per Ljungdahl for technical advise and support; Kiran
Madura, Hidde Ploegh, Alex Varshavsky, Sigurd Braun, and Stefan
Jentsch for generous gifts of reagents and advice; Marianne Jellne
for excellent technical support; Kristina Lindsten, Lisette Verhoef,
Victoria Menéndez-Benito, Florian Salomons, Claes Andréasson,
Steven Bergink, Wim Vermeulen, and Cecile Pickart for stimulating
discussions and useful comments. This work was supported by
grants awarded by the Swedish Research Council (N.P.D.), Swedish
Cancer Society (N.P.D. and M.G.M.), Swedish Foundation for Strategy Research (M.G.M.) and the Karolinska Institute. N.P.D. is supported by a fellowship from the Swedish Research Council. S.H.
has been supported in part by a grant from the Ludwig Institute for
Cancer Research.
Received: October 8, 2004
Revised: January 11, 2005
Accepted: March 21, 2005
Published: April 14, 2005
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