Two Nuclear Localization Signals in USP1 Mediate
Nuclear Import of the USP1/UAF1 Compl ex
Iraia Garcia-Santisteban
1
, Kerman Zorroza
2
, Jose Antonio Rodriguez
1
*
1 Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain, 2 BioEF, Basque Foundation for Health
Innovation and Research, Bilbao, Spain
Abstract
The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and
the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by
its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes
cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has
been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear
import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show
that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these
results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in
which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import
mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed
therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction
with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a
high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.
Citation: Garcia-Santisteban I, Zorroza K, Rodriguez JA (2012) Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex. PLoS
ONE 7(6): e38570. doi:10.1 371/journal.pone.0038570
Editor: Sergey Korolev, Saint Louis University, United States of America
Received March 1, 2012; Accepted May 7, 2012; Published June 6, 2012
Copyright: ß 2012 Garcia-Santisteban et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Basque Country Government Department of Industry (grant number ETORTEK BioGUNE2010 to JAR), the Spanish
Government MICINN (Ministerio de Ciencia e Innovacio
´
n) (grant number BFU2009-13245 to JAR), the University of the Basque Country (UFI11/20), and a
fellowship from the Department of Education of the Basque Country Government (to IG-S). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: josean.rodriguez@ehu.es
Introduction
Ubiquitination is an important posttranslational modification
that modulates the stability, localization and activity of many
cellular proteins [1]. Protein ubiquitination is a dynamic and
reversible process, and the removal of ubiquitin moieties from
ubiquitinated substrates is catalyzed by a group of functionally
related enzymes termed deubiquitinases (DUBs) [2,3]. Nearly
100 DUBs are encoded by the human genome [4]. By
deubiquitinating a variety of substrate proteins, human DUBs
have been found to be involved in important cellular processes,
such as cell cycle control [5], apoptosis [6], or the response to
DNA damage [7], which are frequently deregulated in tumor cells.
Ubiquitin-specific protease 1 (USP1) is a DUB that plays several
roles related to the DNA damage response. On one hand, USP1
contributes to regulate the Fanconi anaemia pathway through
deubiquitination of FANCD2 [8,9]. On the other hand, USP1
counteracts the monoubiquitination of PCNA and thus, prevents
the recruitment of low fidelity DNA polymerases in the absence of
damage [10]. Furthermore, USP1 is involved in the repair of
double-strand DNA breaks through homologous recombination
[11], and knockout of the USP1 gene in mice results in genomic
instability [12]. Besides these DNA damage-related functions, a
recent study has shown that USP1 deubiquitinates and stabilizes
differentiation-inhibiting proteins of the ID (inhibitors of DNA
binding) family, and thus contributes to preserve the undifferen-
tiated state of osteosercoma cells [13].
All these data indicate that USP1 function is primarily related to
nuclear processes. Consistent with these findings, USP1 has been
shown to localize to the nucleus [8,14]. However, although the
amino acid sequence of USP1 bears several sequences that have
been predicted to be putative nuclear localization signals (NLSs),
no functional evaluation of the sequence motifs that mediate USP1
nuclear import has previously been carried out.
A precise regulation of DUB activity is critical to maintain the
functional integrity of the processes controlled by these enzymes.
Several regulatory mechanisms of USP1 activity have been
described, including autocleavage at a diglycine motif [10], and
interaction with the WD40 repeat-containing protein UAF1
(USP1-associated factor 1), also called p80 or WDR48 [15].
UAF1 has been shown to interact directly with USP1, forming a
complex that contains stoichiometric amounts of both proteins
[15]. UAF1 binding stabilizes USP1 and dramatically increases its
catalytic activity [15]. Given the close functional relationship
between USP1 and UAF1, their subcellular localization would also
be expected to be closely related. Intriguingly, and in marked
contrast to USP1, ectopically expressed UAF1 has been reported
to be predominantly localized in the cytoplasm [16,17]. However,
the mechanisms that may modulate the nucleocytoplasmic
PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38570
localization of the USP1/UAF1 complex have not yet been
studied.
Human DUBs are becoming increasingly recognized as
potential therapeutic targets in the treatment of cancer
[3,18,19]. The role of USP1 in processes that are frequently
deregulated in tumor cells, suggests that this enzyme may
represent a particularly promising target for anticancer therapy.
In fact, it has been shown that USP1 gene ablation in chicken cells
increases sensitivity to DNA cross-linkers [9]. Further supporting
this view, a recent report has shown that inhibiting the
deubiquitinase activity of the USP1/UAF1 complex sensitizes
non-small cell lung cancer cells to DNA damaging agents [20].
The crucial role that UAF1 binding plays as regulator of USP1
deubiquitinating activity raises the possibility that interfering with
the formation of the USP1/UAF1 complex may constitute a useful
therapeutic strategy. A detailed characterization of the USP1/
UAF1 interaction motifs is a necessary first step in order to explore
this possibility. In this regard, previous studies have shown that a
relatively large UAF1 segment, encompassing its WD repeats,
mediates interaction with USP1 [15]. On the other hand, the
USP1 sequences that mediate its binding to UAF1 have not yet
been mapped.
Here, we identify two sequence motifs that mediate USP1
nuclear import. These motifs are one bipartite NLS, and a second
monopartite NLS in the amino-terminal half of the protein. Our
findings indicate that these NLSs mediate the nuclear localization
of the USP1/UAF1 complex, supporting a model in which USP1
and UAF1 form a complex in the cytoplasm that subsequently
relocates to the nucleus through active nuclear import mediated by
USP1 NLSs. Moreover, we have mapped the UAF1-binding
domain to a highly conserved 100 amino acid motif in the middle
of USP1 (residues 420–520).
In addition to providing further insight into the biological
consequences of the USP1/UAF1 interaction, the results present-
ed here have practical implications for the development of the
USP1/UAF1 as an anticancer target. On one hand, the UAF1-
interacting region of USP1 identified here might potentially be
targeted to disrupt the USP1/UAF1 interaction with therapeutic
purposes. On the other hand, our findings provide the basis for a
relocation assay that can be easily implemented in a high
throughput setting to search for drugs that may dissociate the
USP1/UAF1 complex and thus interfere with the activity of this
complex in tumor cells.
Materials and Methods
Plasmids, Cloning Procedures, and Site-directed
Mutagenesis
Mammalian expression plasmids encoding GFP-USP1 and
Xpress-UAF1 were kindly provided by Dr. Rene Bernards and Dr.
Jae Jung, respectively. USP1 cDNA was used as template in PCR
reactions using high fidelity Pfu UltraII fusion HS DNA
polymerase (Stratagene) to generate DNA fragments encoding
the USP1 deletion mutants 1–672, 1–520, 1–500, 1–350, 1–285
and 1–269. These DNA fragments were cloned into pEYFP-C1
(Clontech) using KpnI and BamHI restriction sites. On the other
hand, a double-stranded DNA sequence encoding USP1 NLS1
(KGNGKRKSDTEFGNMKKKVKLS) was generated by an-
nealing two complementary oligonucleotides, and cloned up-
stream of GFP in the pEGFP-N1 plasmid using the HindIII and
BamHI restriction sites. Subsequently, DNA fragments encoding
USP1 deletion mutants 420–785, 450–785 and 420–520 were
generated by PCR and cloned between the NLS1 and GFP-
encoding sequences using the BamHI and PinAI restriction sites.
The interstitial deletion mutant USP1 Del(420–520) was created
using a two-step PCR procedure. Briefly, two separate DNA
fragments, encoding USP1 sequences 1–419 and 521–785 with
short overlapping sequences, were generated in a first PCR
reaction. These fragments were purified, combined, and re-
amplified to produce a final PCR product encoding USP1-
Del(420–520), which was cloned into pEYFP-C1 using KpnI and
BamHI restriction sites.
USP1 mutations were introduced using QuickChange Light-
ning Site-Directed Mutagenesis Kit (Stratagene).
The NES-GFP plasmid used in the nuclear import assay was
derived from the Rev(1.4)-GFP nuclear export assay plasmid,
kindly provided by Dr. Beric Henderson. We have previously
described the generation of a collection of Rev(1.4)-GFP-based
constructs that encode different DUB nuclear export sequences
(NESs) cloned between the Rev(1.4) and the GFP moieties [14].
One of these plasmids, termed Rev(1.4)-NET22-GFP, contains a
relatively strong NES (LKSLHQLLEVLLALLDKDV) identified
in USP24, which was classified as 5+ according to the export assay
activity score [21]. The NES-GFP plasmid was created by
removing the Rev(1.4)-encoding sequence from Rev(1.4)-
NET22-GFP. Subsequently, DNA sequences encoding each of
USP1 candidate NLSs were inserted using HindIII and BamHI
restriction sites. The SV40 NLS (PKKKRKV) and a mutant
version of this sequence (PAAARAV) were similarly cloned as
positive and negative controls, respectively.
All the new constructs generated were confirmed by DNA
sequencing. The sequences of the oligonucleotides used in cloning
and mutagenesis procedures are available upon request.
Cell Culture, Transfection and Leptomycin B Treatment
Human embryonic kidney cells 293T, obtained from ATCC,
were grown in Dulbecco’s modified Eagle’s medium (Invitrogen),
supplemented with 10% fetal bovine serum (Invitrogen), 100 U/
ml penicillin and 100
mg/ml streptomycin (Invitrogen). Twenty
four hours before transfection cells were seeded onto sterile glass
coverslips in 12-well trays. Transfections were carried out with X-
tremeGENE 9 transfection reagent (Roche Diagnostics) following
manufacturer’s protocol.
Leptomycin B (Apollo Scientific) was added to the culture
medium 24 hours after transfection to a final concentration of
6 ng/ml for 3 hours.
Immunofluorescence and Confocal Microsc opy Analysis
Cells expressing proteins tagged with GFP or YFP were fixed
with 3.7% formaldehyde in phosphate-buffered saline (PBS) for
30 min, incubated with Hoechst 33285 (Sigma) to visualize the
nuclei, washed with PBS, and mounted onto microscope slides
using Vectashield (Vector). Endogenous USP1 and Xpress-tagged
UAF1 were stained with anti-USP1 polyclonal antibody (Abcam
ab27868, 1:200) and anti-Xpress monoclonal antibody (Invitro-
gen, 1:300), respectively, using a previously described immuno-
staining procedure [22].
Slides were examined and images acquired using an Olympus
Fluoview FV500 confocal microscope. Sequential acquisition of
each fluorochrome was performed in order to avoid overlapping of
fluorescent emission spectra.
Immunoblot Analysis and Co-immunoprecipitation
For immunoblot analysis, protein samples were resolved in a
10% SDS-PAGE gel and transferred to a nitrocellulose mem-
brane. Membranes were blocked with 5% non-fat dry milk diluted
in TTBS for 1 hour, probed with either anti-GFP (Chromotek,
1:1000) or anti-Xpress (Invitrogen, 1:5000) antibodies for 1 hour
Nuclear Import of USP1/UAF1 Complex by UPS1 NLSs
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at RT, and subsequently incubated with the corresponding
horseradish peroxidase-conjugated secondary antibody (Amer-
sham, 1:3000). Finally, immunoblots were developed with ECL
chemiluminiscence reagent (Thermo Scientific). Anti-GFP immu-
noprecipitation was carried out using the GFP-Trap_M reagent
(Chromotek), following manufacturer’s directions. Immunoprecip-
itated proteins were analysed by immunoblot as described above.
Bioinformatics analysis
Prediction of potential NLS sequences in USP1 was carried out
using PSORT (http://psort.hgc.jp/) [23] and cNLS mapper
(http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.
cgi) [24].
Multiple sequence alignment of USP1 amino acid sequences
was carried out with ClustalW2 [25].
Results and Discussion
Three Candidate Nuclear Localization Signals (cNLSs) in
Human USP1 Amino Acid Sequence
Human USP1 is a 785 amino acid protein which, consistent
with previous reports [8] localizes to the cell nucleus when
expressed as a GFP-tagged protein in 293T cells (Figure 1A, left
panels). In addition, immunofluorescence analysis with an anti-
USP1 antibody revealed a predominanlty nuclear staining in 293T
cells (Figure 1A, right panels) and HeLa cells (data not shown). We
have recently reported the presence of an NES in the amino acid
sequence of human USP1 [14] but, given the steady state nuclear
localization of the protein, any potential nuclear export mediated
by the NES appears to be effectively counteracted by rapid nuclear
import. However, the USP1 sequences that mediate its import into
Figure 1. Three candidate nuclear localization signals in human USP1. A. onfocal microscopy images showing nuclear localization of GFP-
(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs
(cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the
previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo
nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES.
Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to
show the nuclei (DNA panels).
doi:10.1371/journal.pone.0038570.g001
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the nucleus have not been functionally mapped. Using two
different web-based NLS prediction programs, PSORT [23] and
cNLS mapper [24], we examined USP1 primary amino acid
sequence searching for motifs that may constitute nuclear
Figure 2. Identification of two NLSs that mediate USP1 nuclear import. A. Schematic representation of USP1 deletion mutants. The positions
of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C.
Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear,
except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1
using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of
the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic
residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization
of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to
show the nuclei (DNA panels).
doi:10.1371/journal.pone.0038570.g002
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localization signals (NLSs). Three sequences containing short
stretches of basic residues were identified by the programs as
candidate NLSs (cNLSs) in human USP1 (Figure 1B).
These cNLS were tested using an in vivo nuclear import assay in
293T cells. In this assay, we evaluate the ability of these sequences
to induce the nuclear relocation of a cytoplasmic chimeric protein
(NES-GFP) that contains the green fluorescent protein fused to the
previously identified nuclear export sequence NET22 (Figure 1C)
[14]. NET22 is a relatively strong NES, with an export assay score
of 5+ over a maximum score of 9+ [21]. As positive and negative
controls, the SV40 large T antigen NLS (PKKKRKV) and a
mutant version of this NLS (PAAARAV) were used, respectively.
As shown in Figure 1C, the three USP1 motifs tested were able to
induce the nuclear relocation of NES-GFP, although to a different
extent. The (14–37) fragment behaved as a very weak NLS, being
able to only partially overcome NES-mediated export, and leading
to an even distribution of NES-GFP in the nucleus and cytoplasm.
In contrast, the (266–287) fragment induced a near-complete
nuclear relocation of the NES-GFP protein, similar to the
relocation afforded by the SV40 NLS. Finally, a clear, but less-
marked relocation, was induced by the (298–321) fragment.
Similar results were obtained when the assay was carried out in
HeLa cells (data not shown). These results, therefore, identify three
USP1 amino acid motifs, having different degrees of nuclear
import activity, that might contribute to the nuclear localization of
this protein.
Two NLSs are Necessary and Sufficient for USP1 Nuclear
Import
In order to assess the contribution of each cNLS to USP1
nuclear localization, we generated a series of USP1 deletion
mutants fused to YFP (Figure 2A). The expression and size of each
USP1 fragment was confirmed by immunoblot (Figure 2B), and
their nucleocytoplasmic localization was evaluated in transfected
293T cells (Figure 2C). Like the full-length protein, the fragment
(1–350), lacking the carboxy-terminal half of USP1, but bearing
the three cNLSs, was nuclear. The fragment (1–285), lacking
cNLS[298–321], also localized to the nucleus. In contrast, the
Figure 3. USP1 NLSs mediate nuclear import of the USP1/UAF1 complex. A. Confocal images showing that Xpress-UAF1 localizes to the
cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing
Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1
NLS1/2m
. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1
wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1
NLS1/2m
. Cells were counterstained with
Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP,
GFP-USP1 wild type (WT) or GFP-USP1
NLS1/2m
. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC)
or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating
the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.
doi:10.1371/journal.pone.0038570.g003
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Figure 4. Mapping the UAF1-binding site in USP1. A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site.
The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (2)
nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different
USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-
Nuclear Import of USP1/UAF1 Complex by UPS1 NLSs
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fragment (1–269), which lacks both cNLS[266–287] and
cNLS[298–321], localized exclusively to the cytoplasm. These
results indicate that cNLS[14–37] is not sufficient to maintain
nuclear localization of USP1.
Since the cytoplasmic 1–269 fragment contains the previously
identified NES [14], we hypothesized that active CRM1-
dependent export might contribute to its nuclear exclusion. To
test this possibility, cells were treated with the specific CRM1
inhibitor leptomycin B (LMB) [26]. Indeed, inhibition of CRM1
resulted in a partial relocation of the fragment (1–269) to the
nucleus (Figure 2D), suggesting that CRM1-mediated nuclear
export contributes to the marked cytoplasmic localization of this
USP1 fragment. The prominent nuclear localization of full-length
USP1, however, suggests that either CRM1 access to USP1 NES is
somehow hindered, or any eventual nuclear export of the protein
is efficiently counteracted by rapid re-import into the nucleus.
Further studies should clarify this issue. Altogether, our data
strongly suggested that, in the physiological context of the full-
length protein, the critical nucleocytoplasmic localization motifs of
USP1 were cNLS[266–287] (hereafter called NLS1) and
cNLS[298–321] (hereafter called NLS2). NLS1 is a classical
bipartite nuclear import signal, bearing two runs of basic amino
acids separated by a short linker, whereas NLS2 is a monopartite
signal with a single run of basic residues [27].
In order to further delineate the role of each NLS in the context
of full-length USP1, we used site-directed mutagenesis to introduce
alanine substitutions of basic residues in either or both NLSs
(Figure 2E). GFP-USP1
NLS1m
was still a predominantly nuclear
protein, but a fluorescent signal was also evident in the cytoplasm
of some transfected cells, indicating that mutation of NLS1
reduces, but does not abrogate, USP1 nuclear import. GFP-
USP1
NLS2m
, on the other hand, was exclusively located in the
nucleus, like wild type USP1. Strikingly, simultaneous mutation of
both NLSs completely abrogated GFP-USP1 nuclear import. Of
note, GFP-USP1
NLS1/2m
did not relocalize to the nucleus upon
LMB treatment (not shown). These results suggest that both NLS1
and NLS2 contribute to USP1 import, although NLS1 appears to
play a more critical role. In summary, we have identified the two
nuclear localization signals that mediate USP1 nuclear import.
Interestingly, a proteomic study has identified importin alpha 5
(KPNA1) as a USP1-interacting protein [28]. It seems, thus,
reasonable to propose that the NLSs identified here may mediate
the interaction of USP1 with this nuclear import receptor.
USP1 NLSs Mediate Nuclear Import of the USP1/UAF1
Complex
As previously reported [16,17], Xpress-tagged UAF1 localized
to the cytoplasm of 293T cells (Figure 3A). LMB treatment did not
induce its nuclear relocation, suggesting that CRM1-mediated
nuclear export is not necessary to maintain the cytoplasmic
localization of UAF1.
We hypothesized that the USP1 NLSs could mediate the
nuclear import of the USP1/UAF1 complex. To test this
possibility, we co-expressed Xpress-UAF1 with either GFP-USP1
wild type or with GFP-USP1
NLS1/2m
. As a control, Xpress-UAF1
was co-expressed with GFP. As shown in Figures 3B and 3C,
Xpress-UAF1 was cytoplasmic when co-expressed with GFP, but
readily relocalized to the nucleus when co-expressed with GFP-
USP1. The import-deficient mutant GFP-USP1
NLS1/2m
was
unable to relocate Xpress-UAF1 to the nucleus. These results
indicate that the two USP1 NLSs identified here mediate the
import of the USP1/UAF1 complex into the nucleus. The
cytoplasmic localization of Xpress-UAF1 in the absence of
ectopically expressed USP1 suggests that endogenous levels of
USP1 in 293T cells are not sufficient to relocalize overexpressed
UAF1 to the nucleus.
These findings parallel the previous observation that the E1
helicase encoded by some strains of human papillomavirus (HPV),
can bind and relocate UAF1 to the nucleus [17]. HPV E1-
mediated nuclear relocation of UAF1 also depends on E1 NLS.
Thus, UAF1 subcellular localization appears to rely on the
transport signals of its different interacting partners. In this regard,
it has been reported that, besides USP1, UAF1 also binds and
regulates the activity of two other DUBs, namely USP12 and
USP46 [29]. Thus, three different DUB/UAF1 complexes have
been detected in human cells: USP1/UAF1, USP12/UAF1 and
USP46/UAF1 [29]. The localization and function of USP12 and
USP46 is still poorly characterized, but it has recently been shown
that these enzymes deubiquitinate two nuclear proteins, namely
histones H2A and H2B, in Xenopus [30]. It is tempting to speculate
that nuclear relocation of the USP12/UAF1 and USP46/UAF1
complexes could be mediated by, as yet unidentified, NLSs in the
DUBs.
Our results are consistent with a model in which USP1 and
UAF1 form a complex in the cytoplasm that is subsequently
USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and
(420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with
Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown
in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap,
showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T
cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the
panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the
(420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay.
The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence
encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence
VERIV, which resembles a UAF1-interacting motif in HPV E1 protein [17], is boxed.
doi:10.1371/journal.pone.0038570.g004
Table 1. Amino acid identity across several functional
domains between human USP1 protein and USP1 proteins
from the mouse, the clawed frog Xenopus laevi (xenla), and
the zebrafish Danio rerio (danre).
% amino acid identity (ClustalW score)
human USP1 domain* mouse USP1 xenla USP1 danre USP1
Overall (1–785) 88 54 49
UAF1-binding (420–520) 100 81 79
Cys (82–99) 100 100 100
Asp (197–213) 100 100 94
His (576–784) 89 48 37
*Cys, Asp and His domains as described in [10].
doi:10.1371/journal.pone.0038570.t001
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translocated to the nucleus through active nuclear import
mediated by the two USP1 NLSs (Figure 3D). Such a model,
obviously, implies that USP1 can bind simultaneously to the
nuclear import receptors and to UAF1. The region of USP1 that
mediates its interaction with UAF1, however, remains to be
identified.
Mapping the UAF1-interacting Domain of Human USP1
In order to map the UAF1-binding domain in USP1 we used a
cellular relocation assay based on the change that USP1 co-
expression causes in the nucleocytoplasmic localization of Xpress-
UAF1.
We generated a series of seven YFP- or GFP-tagged USP1
deletion mutants lacking different amino-terminal, carboxy-
terminal, or interstitial amino acid segments (Figure 4A). The
NLS1 sequence was added to the amino-terminal end of those
USP1 fragments lacking the NLSs (420–785, 450–785 and 420–
520) in order to force their nuclear localization. The expression
and size of each USP1 mutant was confirmed by immunoblot
(Figure 4B). Xpress-UAF1 was co-expressed with each of the
USP1 deletion mutants, and the localization of both proteins was
examined in transfected 293T cells. The results are summarized in
Figure 4A, and illustrated in Figure 4C. As expected, all the USP1
mutants were localized in the nucleus. The fragment 1–672,
mimicking USP1 autocatalytic cleavage product [10], and the
fragment 1–520 induced nuclear relocation of UAF1. In contrast,
UAF1 was cytoplasmic when co-expressed with the USP1
fragment 1–500, suggesting that this mutant was unable to bind
UAF1. On the other hand, the USP1 fragment 420–785, but not
the fragment 450–785, was able to relocate UAF1 to the nucleus.
The 100 amino acid fragment 420–520 readily induced nuclear
accumulation of UAF1, whereas deletion of this fragment in the
mutant Del(420–520) abolished nuclear relocation of co-expressed
UAF1. Combined, these results indicate that USP1 region 420–
520 mediates its interaction with UAF1. In fact, a semiquantitative
analysis of Xpress-UAF1 nucleocytoplasmic distribution
(Figure 4D) showed that the 420–520 fragment was even more
efficient than full-length USP1 in promoting nuclear accumulation
of UAF1. This observation raises the possibility that UAF1
interaction may be negatively regulated by USP1 sequences
outside the UAF1-binding domain.
The role of the USP1 fragment (420–520) in mediating UAF1
binding was further confirmed by co-immunoprecipitation anal-
ysis. As shown in Figure 4E, full-length USP1 and the (420–520)
fragment, but not the USP1 Del(420–520) mutant, interact with
Xpress-UAF1 in co-transfected 293T cells.Alignment analysis of
USP1 proteins from human, mouse, frog and fish (Figure 4F and
Table 1) revealed that the UAF1-binding domain is highly
conserved among different vertebrate species. For example,
whereas the overall amino acid identity between human and
zebrafish USP1 is 49%, the identity within the UAF1-binding
domain reaches 79%. Although this high degree of conservation
may be due to a variety of reasons, it is entirely consistent with the
possibility that the UAF1 interacting domain identified here is
functionally relevant.
It has been previously shown that a short amino acid motif of
HPV E1 protein (VEAIV) is critical for UAF1 binding [17].
Double amino acid substitutions in this motif (VE or IV to AA)
effectively abrogate E1/UAF1 interaction. We noted the presence
of a similar motif (VERIV), located at amino-acid position 495–
499 within the UAF1-binding domain of USP1 (Figure 4F).
However, mutation of this sequence to AARAA did not abrogate
UAF1 interaction (data not shown), indicating that the VERIV
motif is not essential for USP1/UAF1 complex formation.
The identification of the UAF1-binding domain in USP1 may
be of clinical relevance in the light of the recent observation that
interfering with the activity of the USP1/UAF1 complex sensitizes
non-small cell lung cancer cells to DNA damaging agents [20].
Figure 5. A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation. In the upper part, the
formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation
of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization
of Xpress-UAF1.
doi:10.1371/journal.pone.0038570.g005
Nuclear Import of USP1/UAF1 Complex by UPS1 NLSs
PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38570
This observation suggests that the USP1/UAF1 complex may be
an important novel target in cancer treatment and thus, interfering
with the formation and/or inducing the dissociation of the
complex may represent a potential therapeutic strategy. In this
regard, previous studies have shown that the USP1-interacting
region of UAF1 encompasses a large and ill-defined region [15],
which would not be easy to target. In contrast, our findings reveal
a discrete UAF1-binding interface in USP1, which could be more
easily targeted and thus, more relevant from a therapeutic point of
view.
Importantly, the striking change in the nucleocytoplasmic
localization of Xpress-UAF1 that results from GFP-USP1 co-
expression provides the basis for a relocation assay to test USP1/
UAF1 complex formation in cells. Here, we have used this assay to
map the UAF1-binding motif in USP1, but such an assay can also
be applied to evaluate the ability of different drug treatments to
interfere with the formation of the USP1/UAF1 complex
(Figure 5). Furthermore, by genetically tagging UAF1 with a
fluorescent protein, such as RFP, and using automated microscopy
procedures, the assay can be readily adapted to a high throughput
format in order to allow a high content screening for molecules
that interfere with USP1/UAF1 complex formation.
Acknowledgments
We thank Dr. Ana Zubiaga for her continuous support and encourage-
ment. We appreciate the generous gift of plasmids from the following
investigators: Dr Beric Henderson (University of Sydney, Sydney,
Australia), Dr Rene Bernards (Netherlands Cancer Institute, Amsterdam,
The Netherlands), and Dr. Jae Jung (University of Southern California, Los
Angeles, USA). We appreciate the technical support by the staff from the
High Resolution Microscopy Facility (Sgiker-UPV/EHU).
Author Contributions
Conceived and designed the experiments: IG-S JAR. Performed the
experiments: IG-S JAR. Analyzed the data: IG-S JAR. Contributed
reagents/materials/analysis tools: KZ. Wrote the paper: JAR.
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