Báo cáo khóa học: NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis potx

NUB1-mediated targeting of the ubiquitin precursor UbC1 for its
C-terminal hydrolysis
Tomoaki Tanaka, Edward T. H. Yeh and Tetsu Kamitani
Department of Cardiology, University of Texas M. D. Anderson Cancer Center and Institute of Molecular Medicine,
University of Texas-Houston Health Science Center, Houston, Texas, USA
NEDD8 is a ubiquitin-like protein that controls vital bio-
logical events through its conjugation to target proteins.
Previously, we identified a negative regulator of the NEDD8
conjugation system, NEDD8 ultimate buster-1 (NUB1),
that recruits NEDD8 and its conjugates to the proteasome
for degradation. Recently, we performed yeast two-hybrid
screening with NUB1 as bait and isolated a ubiquitin pre-
cursor UbC1 that is composed of nine tandem repeats of
a ubiquitin unit through a-peptide bonds. Interestingly,
NUB1 interacted with UbC1 through its UBA domain.
Further study revealed that the UBA domain interacted with
a-peptide bond-linked polyubiquitin, but not with isopep-
tide bond-linked polyubiquitin, indicating that the UBA
domain of NUB1 is a specific acceptor for the linear
ubiquitin precursor. A functional study revealed that an
unidentified protein that was immunoprecipitated with
NUB1 served as a ubiquitin C-terminal hydrolase for
UbC1. Thus, NUB1 seems to form a protein complex with
the unidentified ubiquitin C-terminal hydrolase and recruit
UbC1 to this complex. This might allow the ubiquitin
C-terminal hydrolase to hydrolyze UbC1, in order to gen-
erate ubiquitin monomers. Northern blot analysis showed
that the mRNAs of both NUB1 and UbC1 were enriched
in the testis. Furthermore, in situ hybridization showed
that both mRNAs were strongly expressed in seminiferous
tubules of the testis. These results may imply that the UbC1

hydrolysis mediated by NUB1 is involved in cellular func-
tions in the seminiferous tubules such as spermatogenesis.
Keywords: NUB1; ubiquitin; UBA; ubiquitin C-terminal
hydrolase.
NEDD8isan81-aminoacidproteinthatshares60%
identity and 80% homology with ubiquitin. NEDD8
conjugates to a large number of target proteins [1], and
this conjugation is thought to be catalyzed by four enzymes,
NEDD8-carboxyl-terminal hydrolase [2], NEDD8-activa-
ting enzyme, NEDD8-conjugating enzyme, and NEDD8-
ligating enzyme, in a manner analogous to ubiquitination
and sentrinization (also known as SUMO conjugation) [3].
So far, all of the known targets of NEDD8 are cullin family
members, and these include Cul1, -2, -3, -4A, -4B, and -5
[4,5]. Each cullin family member appears to be a component
of the SCF (or SCF-like complex), a ubiquitin E3 ligase
composed of Skp1 (or Skp1-like protein), Cullin, F box
protein (or F box-like protein), and Roc1 [3,6]. For
example, Cul1 is a major component of an SCF complex
that catalyzes the ubiquitination of IjBa, b-catenin, and p27
(Kip1) [7–9] and controls many biological events, such
as cell-cycle transition, inflammation, and tumorigenesis.
Recently, several groups reported that NEDD8 conjugation
to Cul1 is required for the ubiquitin-ligase activity of the
Cul1-containing SCF complex [10–13]. These observations
suggested that the NEDD8 conjugation system is involved
in many important biological functions. Indeed, the
NEDD8 conjugation system has been shown to be essential
for cell-cycle progression and morphogenesis in mice [14]
and for eye development in Drosophila [15].

Recently, we identified a novel down-regulator of the
NEDD8 conjugation system, NEDD8 ultimate buster-1
(NUB1), using a yeast two-hybrid system with NEDD8 as
bait [16]. NUB1 is a NEDD8-interacting protein composed
of 601 amino acid residues with a calculated molecular mass
of 69.1 kDa. It possesses a ubiquitin-like (UBL) domain at
the N-terminal region and two ubiquitin-associated (UBA)
domains at the C-terminal region. It is an interferon-
inducible protein and predominantly localizes in the nucleus.
In a biochemical analysis, we found that NUB1 overexpres-
sion led to a severe reduction in the NEDD8 monomer and
its conjugates in cells [16]. Surprisingly, this reduction was
completely blocked by proteasome inhibitors [17]. Further-
more, we found that NUB1 was cofractionated with the 19S
proteasome activator (PA700) [17]. These results strongly
suggested that NUB1 recruits NEDD8 and its conjugates
to the proteasome for degradation, making NUB1 a
down-regulator in the NEDD8 conjugation system.
The UBA domain is a small motif of about 40 residues that
was initially identified in ubiquitination enzymes, including
E2s and E3s and other proteins linked to ubiquitination
Correspondence to T. Kamitani, Department of Cardiology,
The University of Texas M.D. Anderson Cancer Center,
1515 Holcombe Blvd., Box449, Houston, TX 77030.
Fax: + 1 713 745 1942, Tel.: + 1 713 792 6242,
E-mail:
Abbreviations: Cul, human cullin; DUB, deubiquitinating enzyme;
GST, glutathione S-transferase; HHR23, human homologue of
RAD23; HRP, horseradish peroxidase; NEM, N-ethylmaleimide;
NUB-1, NEDD8 ultimate buster-1; RH, RGS-poly His; SCF,

Skp1-Cullin-F-box protein; UBA, ubiquitin associated domain;
UCH, ubiquitin C-terminal hydrolase.
(Received 1 October 2003, revised 13 November 2003,
accepted 19 January 2004)
Eur. J. Biochem. 271, 972–982 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03999.x
[18,19]. Human NUB1 has two UBA domains, whereas
NUB1 homologues of other species such as mouse, cow,
Drosophila,andArabidopsis have three UBA domains [20].
Most recently, we identified a splicing variant of the human
NUB1 gene that encodes a longer protein, termed NUB1L
(accession number: AF459743) [20]. It possesses an insertion
of 14 amino acids that codes for an additional UBA domain
between two original UBA domains. Thus, NUB1L is
structurally more conserved among species than NUB1
because it possesses three UBA domains at the C-terminal
region. Importantly, NUB1 has a NEDD8-binding site at the
C-terminus, whereas NUB1L has an additional site at the
newly generated UBA domain [20].
In the study described here, we demonstrated that NUB1
and NUB1L interact with a ubiquitin precursor UbC1
through their first UBA domain, resulting in the hydrolysis
of UbC1 by an unidentified ubiquitin C-terminal hydrolase
(UCH). The recruitment of UbC1 to the UCH appears
to be another function of NUB1 and NUB1L.
Experimental procedures
Cell culture
COS-M6 cells were a generous gift from Steve Goldring
(Harvard Medical School). These cells were maintained in
Dulbecco’s Modified Eagle’s Medium supplemented with
10% (v/v) fetal bovine serum, 100 UÆmL

)1
penicillin B,
100 mgÆmL
)1
streptomycin sulfate and 0.25 mgÆmL
)1
of
amphotericin B.
Antibodies
Mouse anti-RH Ig (specific for the amino acid sequences
RGSHHHH and GGSHHHH) was purchased from
Qiagen (Santa Clara, CA, USA). Mouse anti-ubiquitin Ig
(1B3) was purchased from MBL (Nagoya, Japan). Mouse
anti-GST Ig (GST-12) was purchased from Santa Cruz
Biotechnology. Rabbit anti-human NUB1 serum was
generated by immunization with a GST-fusion protein of
the NUB1 fragment corresponding to amino acids 432–601
[16]. Rabbit anti-FLAG Ig was purchased from Sigma.
Construction of prokaryotic expression plasmids
To express GST fusion proteins in Escherichia coli BL21,
cDNAs of the ubiquitin monomer (Ub)1, dimer (Ub)2,
trimer (Ub)3, and nanomer (Ub)9 were subcloned into the
pGEX-2TK plasmid (Amersham Pharmacia Biotech). The
cDNAs of (Ub)9 (UbC1; accession number: AB009010)
and (Ub)3 (UbB; accession number: XM_018032) were
isolated by PCR from a human testis cDNA library
(Clontech, Palo Alto, CA, USA). To make the cDNA of
(Ub)2, the cDNA of (Ub)3 was digested with BstXI, the
cDNA of a single ubiquitin unit was removed from that of
(Ub)3, and the cDNA was ligated.

Construction of mammalian expression plasmids
and transfection
To express proteins tagged with an epitope at the N-terminus
in mammalian cells, pcDNA3/FLAG-N [17] and pcDNA3/
RH-N [21] were used. The human cDNAs used in the present
study were described previously. These include ubiquitin [22],
NEDD8 [1], NUB1 [16], HHR23B [23], AIPL1 [24], Ubc9
[25], UCH-L1 [2], and UCH-L3 [2]. These cDNAs were inser-
ted into the aforementioned plasmid vectors, and the plas-
mids were transfected into COS-M6 cells using FuGENE6
(Roche Molecular Biochemicals). The transfected cells were
then harvested for immunoprecipitation, GST pull-down
assay, or Western blot analysis 20 h after transfection.
Yeast two-hybrid assay for screening of the human
cDNA library
The yeast strain L40 was purchased from Invitrogen. The
prey vector pGAD10 was purchased from Clontech. The bait
plasmid pHybLex/NUB1 was generated by inserting the
entire coding region of NUB1 cDNA [16] into pHybLex
(Invitrogen). The pHybLex/NUB1 plasmid was then trans-
formed into L40 using the lithium acetate method [26]. The
transformants were plated on YPD medium containing
0.1% adenosine and 300 lgÆmL
)1
. Zeocin (YPAD/Zeo) and
selected for 2 days at 30 °C. The L40 clone carrying the
pHybLex/NUB1 plasmid was cultured in YPAD/Zeo
medium and sequentially transformed with 500 lgofthe
Gal4 DNA-activating domain vector, pGAD10, in which
the human testis cDNA library (Clontech) was inserted. The

transformed cells were incubated for 6 days at 30 °Con
selection plates (Ura
–
/Lys
–
/His
–
/Leu
–
/Zeocin
+
). The posit-
ive colonies were then picked and replated on selection
plates (Ura
–
/Lys
–
/His
–
/Leu
–
/Zeocin
+
)andtheb-galactosi-
dase activity on filter papers was determined as described in
the Clontech protocol.
Yeast two-hybrid assay for the interaction of UbC1
with truncated NUB1 and NUB1L
Using PCR with appropriate primers, we prepared cDNAs
of UbC1 [27] and the truncated NUB1 and NUB1L shown

below. To examine the in vivo interaction of UbC1 with
these mutants, the yeast MATCHMAKER two-hybrid
system 3 (Clontech) was used. The cDNA of UbC1 was
subcloned into pGADT7 (Gal4 DNA-activating domain
vector for Gal4-AD fusion), and the cDNA of each mutant
of NUB1 and NUB1L was subcloned into pGBKT7 (Gal4
DNA-binding domain vector for Gal4-BD fusion). The
plasmids of the two fusion constructs were then cotrans-
fected into AH109 yeast cells using the lithium acetate
method [26]. Transformed yeast cells were grown on a His
–
/
Trp
–
/Leu
–
synthetic agar plate for 3 days at 30 °C. The
specific protein–protein interaction was determined by the
growth of the cells on the selection plate.
Western blot analysis
Protein samples were treated for 1 h at 45 °Cin2%(v/v)
SDS treating solution containing 5% (v/v) 2-mercapto-
ethanol. After SDS/PAGE, Western blot analysis was
performed according to the protocol provided with the ECL
detection system (Amersham Pharmacia Biotech). Horse-
radish peroxidase (HRP)-conjugated anti-(mouse IgG) Ig
or anti-(rabbit IgG) Ig (Santa Cruz Biotechnology) was
used as a secondary antibody.
Ó FEBS 2004 NUB1-mediated hydrolysis of UbC1 (Eur. J. Biochem. 271) 973
Site-directed mutagenesis

CysfiAla and HisfiAla substitutions were made in NUB1
at Cys313 and His352, respectively. The cDNA of wild-type
NUB1 was mutated by PCR-based site-directed mutagen-
esis, as described previously [28]. The mutated cDNAs were
then subcloned into pcDNA3/FLAG-N.
GST pull-down assay for proteins expressed in bacteria
RH-tagged proteins and GST fusion proteins were
expressed in E. coli BL21 by transformation with the
pTrcHis plasmid (Invitrogen) and pGEX-2TK plasmid
(Amersham Pharmacia Biotech), respectively. Cells were
resuspended in the lysis buffer [50 m
M
Tris/HCl, pH 7.5,
100 m
M
NaCl, and 0.1% (v/v) NP-40] containing the
protease inhibitor cocktail (Roche) and then lysed by brief
sonication. The GST fusion proteins were purified using
glutathione-Sepharose beads (Amersham Pharmacia Bio-
tech) as described previously [28]. The bacterial crude lysate
containing RH-tagged proteins was centrifuged at 14 000 g
for 5 min, and the supernatant was incubated for 3 h at
room temperature with GST fusion proteins immobilized
on glutathione-Sepharose beads. The beads were then
washed four times with the lysis buffer. The precipitated
proteins on the beads were solubilized in 2% SDS treating
solution containing 5% (v/v) 2-mercaptoethanol, followed
by Western blot analysis using anti-RH Ig.
GST pull-down assay for proteins expressed in COS cells
COS cells were cultured to 60% confluency in a 6-cm

plate and transfected to express RH-tagged proteins.
Twenty hours after transfection, the COS cells were
harvested and lysed in 1 mL of lysis buffer [50 m
M
Tris/
HCl, pH 7.5, 100 m
M
NaCl, and 0.1% (v/v) NP-40]
containing the protease inhibitor cocktail. The cell lysate
was passed through a 22G needle five times, to shear off
the DNA, and then centrifuged at 100 000 g for 30 min at
4 °C. After centrifugation, the supernatant was incubated
with GST fusion proteins immobilized on glutathione-
Sepharose beads for 3 h at 4 °C. The beads were then
washed four times with the lysis buffer, and the precipi-
tated proteins on the beads were solubilized in 2% (v/v)
SDS treating solution containing 5% (v/v) 2-mercapto-
ethanol. This was followed by Western blot analysis using
anti-RH Ig.
GST pull-down assay for tetra-ubiquitin
Tetra-ubiquitin linked by isopeptide bonds was purchased
from Affinity Research Products (Mamhead, UK). The
tetra-ubiquitin was diluted in lysis buffer [50 m
M
Tris/
HCl, pH 7.5, 100 m
M
NaCl, and 0.1% (v/v) NP-40]
containing the protease inhibitor cocktail and incubated
with GST fusion proteins immobilized on glutathione-

Sepharose beads for 3 h at 4 °C. The beads were then
washed four times with the lysis buffer. The precipitated
proteins on the beads were solubilized in 2% (v/v) SDS
solution containing 5% (v/v) 2-mercaptoethanol, followed
by Western blot analysis using anti-ubiquitin Ig 1B3
(MBL).
Immunoprecipitation studies
COS cells were cultured to 60% confluency in a 6-cm
plate and transfected to express FLAG-tagged proteins.
Twenty hours after transfection, the COS cells were
harvested and lysed in 1 mL of lysis buffer (50 m
M
Tris/
HCl, pH 7.5, 100 m
M
NaCl, and 0.1% NP-40) contain-
ing the protease inhibitor cocktail. The cell lysate was
passed through a 22G needle five times to shear off the
DNA and then centrifuged at 100 000 g for 30 min at
4 °C. After centrifugation, the supernatant was incubated
for 2 h at 4 °Cwith40lL of anti-FLAG M2 beads
(Sigma). The beads coated with immunoprecipitates were
washed three times with the lysis buffer and used for the
in vitro hydrolysis assay.
In vitro
hydrolysis assay for a-peptidase activity
A substrate, GST-UbC1, was purified using the glutathi-
one-Sepharose beads, as described previously [28], and
eluted in the GST elution buffer (50 m
M

Tris/HCl,
pH 7.5, 100 m
M
NaCl, and 10 m
M
reduced glutathione).
Enzymes used were immunoprecipitates that were immo-
bilized on anti-FLAG M2 beads (Immunoprecipitation
studies). For the in vitro hydrolysis assay, the substrate
GST-UbC1 was mixed with the beads coated with the
immunoprecipitates and incubated at 37 °Cinthereac-
tion buffer [50 m
M
Tris/HCl, pH 7.5, 100 m
M
NaCl,
0.2% (v/v) NP-40, and 1 m
M
dithiothreitol] for varying
amounts of time. After the hydrolysis reaction, the
solution was centrifuged. The supernatant containing
GST-UbC1 was treated in 2% (v/v) SDS treating solution
containing 5% (v/v) 2-mercaptoethanol and analyzed by
Western blotting using anti-GST Ig to detect the sub-
strate. As markers for the hydrolyzed substrate, we used
undigested GST-(Ub)1 (ubiquitin monomer), GST-(Ub)3
(ubiquitin trimer), and GST-UbC1 (ubiquitin nanomer).
The ubiquitin trimer and nanomer were polyubiquitin
linked by a-peptide bonds.
Treatment with a thiol-blocking reagent

To inhibit the activities of C-terminal hydrolases,
N-ethylmaleimide (NEM) [29,30] was used. As a control,
a serine protease inhibitor, phenylmethylsulfonyl fluoride,
was used. These reagents were purchased from Sigma. A
substrate, GST-UbC1, was purified as described above and
incubated with immunoprecipitates for 120 min at 37 °Cin
the reaction buffer in the absence or presence of 2 m
M
NEM
or 2 m
M
phenylmethylsulfonyl fluoride. After the hydrolysis
reaction, the solution was centrifuged. The supernatant
containing GST-UbC1 was treated in 2% (v/v) SDS
treating solution containing 5% (v/v) 2-mercaptoethanol
and analyzed by Western blotting using anti-GST Ig to
detect the substrate.
Northern blot analysis
Northern blotting was performed to show the mRNA
expression of NUB1 and UbC1 in various human tissues.
For a probe of NUB1, we chose a sequence located in the
coding region of NUB1 between nucleotides 450 and 900.
974 T. Tanaka et al. (Eur. J. Biochem. 271) Ó FEBS 2004
This sequence is also shared by NUB1L. This cDNA
fragment was amplified by PCR using pcDNA3/RH-
NUB1 [16] as the template and primers (forward primer,
5¢-GTGAAAGCGATGGTGCTTGA-3¢; reverse primer,
5¢-AAGGCATTCCAGCTGTTCCA-3¢) and subcloned
into the pGEM-T plasmid (Promega). The insert was then
cut out and labeled with [

32
P]dCTP[aP] by the Ready-To-
Go DNA labeling kit (Amersham Pharmacia Biotech). The
radioactive probe was then hybridized with a human
multiple-tissue Northern blot (Clontech) in the ExpressHyb
solution (Clontech) at 68 °C for 1 h, followed by washing at
50 °C in the washing solution [300 m
M
NaCl, 30 m
M
sodium citrate, and 0.1% (v/v) SDS]. After this, the blot
membrane was exposed to a film. For the Northern blot
analysis of UbC1, we synthesized the oligonucleotide probe
of 42 bases (5¢-GATTTGGGTCGCGGTTCTTGTTT
GTGGATCGCTGTGATCGTC-3¢), which is derived
from the 5¢-terminal noncoding region of UbC1 (accession
number: AB009010) [27]. The probe was labeled with
[
32
P]ATP[cP] by T4 polynucleotide kinase (New England
BioLabs). The radioactive probe was then hybridized with
the same blot, as above, in ExpressHyb solution at 37 °Cfor
1 h, followed by washing at room temperature in the
washing solution described above. The blot membrane was
then exposed to a film.
In situ
hybridization
Paraffin-embedded tissue sections (thickness, 5 lm) of
human adult testis were purchased from BIOCHAIN
(Hayward, CA, USA). First, these tissue sections were

dewaxed and rehydrated. The sections were then fixed
with 4% (v/v) formaldehyde, digested with 10 lgÆmL
)1
proteinase K, acetylated by exposure to acetic acid
anhydrate, dehydrated, and dried. For the in situ hybrid-
ization of NUB1, the pGEM-T vector inserted with the
cDNA fragment of NUB1 was used again (Northern blot
analysis section). Using the linearized plasmid as the
template, sense and antisense probes of single-stranded
RNA were generated. At this step, probes were labeled
with [
35
S]UTP[aS] by T7 RNA polymerase (Promega).
The antisense and sense RNA probes labeled with
[
35
S]UTP[aS] were then denatured and mounted on tissue
sections (5 · 10
5
c.p.m. per slide). After incubation at
55 °C for 16 h, tissue sections were washed at 65 °Cand
treated with RNase A to remove unhybridized RNA
probe. Hybridization signals were visualized by autoradio-
graphy using Hypercoat emulsions (Amersham Pharmacia
Biotech). To examine a specific distribution of UbC1 in
human testis, we generated DNA probes from the same
synthetic oligonucleotide as described in the Northern
Blot Analysis section for UbC1. The probes were labeled
with [
35

S]dATP[aS] at the 3¢-end using terminal deoxy-
nucleotidyl transferase (TdT) (Promega). Tissue sections
were treated with the probes in basically the same way as
with the cRNA probes, excluding the proteinase K
treatment. [
35
S]dATP[aS]-labeled oligonucleotide probes
were added to the tissue sections (5 · 10
5
c.p.m. per slide).
After incubation at 37 °C for 16 h, tissue sections were
washed at a high-stringency temperature of 55 °C.
Hybridization signals were visualized using the same
method as used for the RNA probe.
Results
NUB1 interacts with a-peptide bond-linked polyubiquitin
such as UbC1 in yeast
We have previously found a novel NEDD8-interacting
protein, NUB1, using yeast two-hybrid screening. To
determine the molecular function of NUB1, we further
performed yeast two-hybrid screening with NUB1 as bait.
The yeast strain L40, which contains LexA DNA-binding
sites as upstream activating sequences and two reporter
genes, HIS3 and lacZ, was initially transformed with the
bait plasmid pHybLex/NUB1. The resulting transformant
was used as a host for the following transformation with the
prey plasmid pGAD10 containing the cDNA library. As
the mRNA of NUB1 is highly enriched in the testis [16],
a human testis cDNA library was used for the screening.
Approximately 2 · 10

6
primary library transformants were
inoculated onto plates lacking histidine and leucine and
containing Zeocin. A total of 84 colonies grew on the
selection plates, 40 of which stained positive when tested for
b-galactosidase expression. Subsequent DNA sequencing of
the positive clones showed that 25 clones encoded UbC1
(accession number: AB009010). Interestingly, UbC1 is a
ubiquitin precursor composed of nine tandem repeats of a
ubiquitin unit linked by a-peptide bonds [27,31]. UbC1
generates nine ubiquitin monomers by the C-terminal
hydrolysis.
In yeast cells, ubiquitin fusions appear to be efficiently
processed. Why could we detect the interaction between
NUB1 and UbC1 in yeast cells? We believe that the UbC1
or its fragments might quickly interact with NUB1 before it
was completely processed to ubiquitin monomers. Other-
wise, the interaction of UbC1 with NUB1 might impede the
processing of UbC1.
NUB1 directly interacts with polyubiquitin linked by
a-peptide bonds, but not with the ubiquitin monomer
Although the yeast two-hybrid system showed that NUB1
interacted with UbC1, there were three possible interpreta-
tions of this finding: (a) NUB1 might directly interact with
UbC1 before its hydrolysis in yeast cells; (b) NUB1 might
directly interact with ubiquitin monomer units before or
after the hydrolysis of UbC1 or (c) NUB1 might indirectly
interact with UbC1 via other proteins.
To rule out the second possibility, we examined the
interaction between the ubiquitin monomer and NUB1

using the yeast two-hybrid system. For this assay, we used a
mutant of ubiquitin monomer to prevent forming poly-
ubiquitin as much as possible. The mutant of ubiquitin
monomer, in which Gly76 was removed and Lys48 was
substituted to Arg, was fused with the Gal4 DNA-activating
domain. NUB1 was fused with the Gal4 DNA-binding
domain. These proteins were expressed in yeast cells. This
showed no interaction (data not shown), indicating that
NUB1 does not interact with the ubiquitin monomer in
yeast cells. We had also previously examined the in vitro
interaction of NUB1 with the wild-type ubiquitin monomer,
NEDD8 monomer and sentrin/SUMO1 monomer, and
found that NUB1 interacted only with the NEDD8
monomer but not with the ubiquitin monomer or sentrin
Ó FEBS 2004 NUB1-mediated hydrolysis of UbC1 (Eur. J. Biochem. 271) 975
monomer [17] (Fig. 1A, upper panel, lane 3). Thus, based
on our assays, NUB1 does not interact with the ubiquitin
monomer. This conclusion is also supported by the fact that
our yeast two-hybrid screening by NUB1 bait failed to
detect the natural ubiquitin-ribosomal peptide fusions.
To investigate the first and third possibilities, an in vitro
interaction assay was performed. RH-tagged wild-type
NUB1 was expressed in bacteria. The bacterial lysate
containing RH-NUB1 was then incubated with GST alone,
or with GST-fused ubiquitin monomer (negative control),
dimer, trimer, or nanomer (UbC1) and precipitated by the
GST pull-down method. The precipitate was then analyzed
by Western blotting using anti-RH Ig. As shown in the
upper panel of Fig. 1A, RH-NUB1 was precipitated with
the GST-fused ubiquitin dimer, trimer, and UbC1 (lanes 4–

6), but not with GST alone or the GST-fused ubiquitin
monomer (lanes 2 and 3). These results indicated that
NUB1 directly interacted not only with UbC1 but also with
other a-peptide bond-linked polyubiquitins, whereas NUB1
does not interact with the ubiquitin monomer. Thus, we
concluded that only the first possibility held true.
NUB1 does not interact with isopeptide bond-linked
polyubiquitin chain
As NUB1 interacted with polyubiquitin linked with
a-peptide bonds, including the ubiquitin dimer, trimer and
UbC1, we examined whether NUB1 also interacted with the
polyubiquitin chain linked by isopeptide bonds. To investi-
gate the interaction, a GST pull-down assay was performed.
As a positive control, we used the human homologue
RAD23 (HHR23), because RAD23/HHR23 has been
reported to interact with the polyubiquitin chain linked by
isopeptide bonds [32]. In this experiment, GST alone, GST-
fused NUB1, and HHR23 were expressed in bacteria and
purified by glutathione-Sepharose beads. The beads were
used for the precipitation of three different targets. First,
Fig. 1. In vitro interaction of NUB1 with two different types of poly-
ubiquitin. (A) Interaction of NUB1 with polyubiquitin linked by
a-peptide bonds. GST and GST-fused ubiquitin monomer and poly-
ubiquitins (a-peptide linkage) were expressed in bacteria and purified
using glutathione-Sepharose beads. RH-tagged NUB1 expressed in
bacteria was then precipitated with these beads, which were coated
with GST alone (lane 2), GST-ubiquitin monomer (lane 3), GST-
ubiquitin dimer (lane 4), GST-ubiquitin trimer (lane 5), or
GST-ubiquitin nanomer (human UbC1; lane 6). The precipitates were
analyzed by Western blotting using anti-RH Ig to detect RH-NUB1

(upper panel) and anti-GST Ig to detect immobilized GST, GST-
ubiquitin monomer, or GST-polyubiquitin (lower panel). (B) Inter-
action of NUB1 with polyubiquitin linked by isopeptide bonds. GST,
GST-NUB1, and GST-HHR23 were expressed in bacteria and
purified using glutathione-Sepharose beads. These beads were used for
GST pull-down assays. In the upper panel, RH-AIPL1 (positive
control) was expressed in bacteria, precipitated by beads coated with
GST, GST-NUB1, or GST-HHR23, and detected by Western blotting
using anti-RH Ig. In the middle panel, tetra-ubiquitin (isopeptide
linkage) was precipitated by beads coated with GST, GST-NUB1, or
GST-HHR23 and detected by Western blotting using anti-ubiquitin
Ig. In the lower panel, RH-ubiquitin was overexpressed in COS cells.
The RH-ubiquitin monomer and polyubiquitinated proteins in the
total cell lysate were precipitated by beads coated with GST, GST-
NUB1, or GST-HHR23 and detected by Western blotting using anti-
RH Ig. The identity of each band is indicated in the right-hand side.
Molecular size markers are shown in kilodaltons.
976 T. Tanaka et al. (Eur. J. Biochem. 271) Ó FEBS 2004
RH-tagged AIPL1 [24] was used as a positive control for the
interaction with NUB1 (Fig. 1B, upper panel). As expected,
RH-AIPL1 was precipitated by GST-NUB1 (lane 3), but
not by GST alone (lane 2) or GST-HHR23 (lane 4). Next,
we used a tetra-ubiquitin linked by isopeptide bonds. As
shown in Fig. 1B (middle panel), the tetra-ubiquitin was
precipitated by GST-HHR23 (lane 4), but not by GST
alone (lane 2) or GST-NUB1 (lane 3). Finally, we used
polyubiquitinated cellular proteins. In this experiment, RH-
tagged wild-type ubiquitin was overexpressed in COS cells
to generate polyubiquitinated proteins in the cells. The total
cell lysate was then incubated with GST-fusion proteins

immobilized on beads for the GST pull-down assay. As
shown in Fig. 1B (lower panel), polyubiquitinated proteins
could be precipitated by GST-HHR23 (lane 4), but not by
GST alone (lane 2) or GST-NUB1 (lane 3). Thus, we found
that NUB1 did not interact with the polyubiquitin chain
linked by isopeptide bonds.
NUB1 interacts with a-peptide bond-linked polyubiquitin
through its UBA1 domain
In this section, we identified precisely the binding site of
a-peptide bond-linked polyubiquitin on NUB1. We nar-
rowed down the binding area by first performing a yeast two-
hybrid assay using deletion mutants of NUB1. As shown in
Fig. 2A, we generated six mutants of NUB1 (M1–M6) to
examine the interaction with UbC1. Each mutant had a
C-terminal deletion and/or an N-terminal deletion. For
example, M1 had a C-terminal deletion from Lys371 to
Asn601, resulting in the loss of two UBA domains (UBA1
and UBA3) and a PEST domain. M3 had an N-terminal
deletion from Met1 to Phe370, resulting in the loss of a UBL
domain. Using these mutants and a wild-type NUB1, we
then examined the interaction with UbC1 in yeast cells. In the
assay, UbC1 fused to the Gal4 DNA-activation domain was
used for the interaction with a panel of NUB1 mutants fused
to the Gal4 DNA-binding domain. As shown in Fig. 2A,
UbC1 interacted with wild-type NUB1 (WT), NUB1(1–418)
(M2), NUB1(371–601) (M3), and NUB1(371–418) (M6),
but not with NUB1(1–370) (M1), NUB1(427–601) (M4), or
NUB1 (515–601) (M5). These results indicated that a UbC1
binding site was located between amino acid residues 371 and
418 of NUB1. Interestingly, this region contains the entire

UBA1 domain of NUB1, which is located between amino
acid residues 376 and 413.
We also investigated the UbC1 binding sites on a splicing
variant NUB1L [20] using the yeast two-hybrid assay. As the
difference between NUB1L and NUB1 is the 14-amino-acid
insertion that generates an additional UBA domain (UBA2)
[20], we examined whether the UBA2-containing fragment
interacted with UbC1. As shown in Fig. 2A, we made one
NUB1L mutant, LM1, which possessed two UBA domains
(UBA2 and UBA3) and a PEST domain, but not UBA1
domain. UbC1 interacted with wild-type NUB1L (LWT),
but not with NUB1L(427–615) (LM1), indicating that the
UBA2 domain of NUB1L did not contribute to the
interaction with UbC1. We concluded therefore that NUB1
and NUB1L interacted with UbC1 at their UBA1 domain.
We then performed an in vitro interaction assay to
confirm the results of the yeast two-hybrid assay. In this
experiment, RH-tagged wild-type NUB1 (WT) and its
truncated mutants (M1-6) (Fig. 2A) were first expressed in
bacteria. The bacterial lysates containing the RH-tagged
proteins were incubated with GST-fused UbC1 and
Fig. 2. Mapping of UbC1-binding site on NUB1 and NUB1L. (A)
Interaction of human UbC1 with mutant NUB1 and NUB1L in yeast
two-hybrid system. The yeast strain AH109 was transformed with
pGADT7/UbC1 and the pGBKT7 construct expressing wild-type
NUB1 (WT), mutant NUB1 (M1–6), wild-type NUB1L (LWT), or
mutant NUB1L (LM1). Transformed yeast cells were grown on a His
–
/
Trp

–
/Leu
–
synthetic agar plate for 3 days at 30 °C. The specific protein–
protein interaction was determined by the growth of the cells on the
selection plate. (B) GST pull-down assay to detect the interaction
between human UbC1 and NUB1 possessing various deletions. Wild-
type NUB1 (lanes 1 and 8) and mutant NUB1 with deletions (lanes 2–7
and 9–14) were tagged with RH-epitope and expressed in bacteria. The
bacteriallysates wereused forWesternblottingwithanti-RHIg(lanes 1–
7) or for GST pull-down assay (lanes 8–14). For the pull-down assay, the
lysates were precipitated by GST-fused UbC1 and analyzed by Western
blotting using anti-RH Ig to detect the wild-type and deletion mutants of
RH-NUB1. Molecular size markers are shown in kilodaltons.
Ó FEBS 2004 NUB1-mediated hydrolysis of UbC1 (Eur. J. Biochem. 271) 977
precipitated by the GST pull-down method. The precipi-
tates were then analyzed by Western blotting using anti-RH
Ig. As shown in Fig. 2B, GST-UbC1 precipitated RH-
tagged wild-type NUB1 (WT), NUB1(1–418) (M2),
NUB1(371–601) (M3), and NUB1(371–418) (M6), but not
NUB1(1–370) (M1), NUB1(427–601) (M4) or NUB1(515–
601) (M5). These results were consistent with those of the
yeast two-hybrid assay shown in Fig. 2A, indicating that
NUB1 directly interacted with UbC1 through its UBA1
domain.
UbC1 is hydrolyzed by immunoprecipitates of NUB1
At the C-terminal region, NUB1 possesses two UBA
domains, that are found in proteins involved in the
ubiquitination pathway, including E2 ubiquitin conjugating
enzymes, E3 ubiquitin ligases, and deubiquitinating

enzymes (DUBs, also called UBPs) [18]. Recently, we found
two additional sequences, a Cys box-like sequence (Lys306
to Glu320) and a His box-like sequence (Tyr343 to Tyr362),
in the central region of NUB1 (see below). The Cys box and
His box are commonly found in sequences of DUBs [33].
DUBs are ubiquitin-specific thiol-proteases that include
ubiquitin C-terminal hydrolases (UCHs) and ubiquitin
isopeptidases. UCHs cleave the linear ubiquitin precursor
linked by a-peptide bonds, such as UbC1, while ubiquitin
isopeptidases cleave the polyubiquitin chain linked by
isopeptide bonds, of ubiquitin conjugates. The catalytic
cysteine of DUBs is located in their Cys box [33].
As NUB1 has two UBA domains, a Cys box-like
sequence, and a His box-like sequence and interacts with
a linear ubiquitin precursor, UbC1, we hypothesized that
NUB1 is a member of DUB family and has the enzymatic
Fig. 3. C-terminal hydrolysis of ubiquitin nanomer, UbC1. (A) In vitro
hydrolysis of UbC1 by UCH-L3. Empty vector (lane 4), FLAG-Ubc9
(lane 5), FLAG-UCH-L3 (wild-type; lane 6), or FLAG-UCH-L3 mu-
tant with a Cys95fiSer substitution at the enzymatic active site (lane 7)
was expressed in COS cells and precipitated with beads coated with anti-
FLAG Ig. The beads were incubated with purified GST-UbC1 for
120 min (lanes 3–7). After centrifugation, the supernatant containing
GST-UbC1 was treated with SDS and analyzed by Western blotting
using anti-GST Ig to detect the derivatives of GST-UbC1. (B) In vitro
hydrolysis of UbC1 by proteins coimmunoprecipitated with NUB1.
Empty vector (lane 4) or FLAG-NUB1 (lanes 5–7) was expressed in
COS cells and precipitated with beads coated with anti-FLAG Ig The
beads were incubated with purified GST-UbC1 for 30 min (lane 5),
60 min (lane 6), or 120 min (lanes 4 and 7). After centrifugation, the

supernatant containing GST-UbC1 was treated with SDS and analyzed
by Western blotting using anti-GST Ig to detect the derivatives of GST-
UbC1 (upper panel). The beads were treated separately with SDS, and
immunoprecipitated FLAG-NUB1 was analyzed by Western blotting
using anti-FLAG antibody (lower panel). (C) Inhibition of UbC1
hydrolysis by a thiol-blocking reagent, NEM. Empty vector (lane 4) or
FLAG-NUB1(lanes5–7)wasexpressedinCOScellsandprecipitated
with beads coated with anti-FLAG. The beads were incubated with
purified GST-UbC1 in the absence (lanes 4 and 5) or presence of NEM
(lane 6) or phenylmethylsulfonyl fluoride (lane 7). After centrifugation,
the supernatant containing GST-UbC1 was treated with SDS and
analyzed by Western blotting using anti-GST Ig to detect the derivatives
of GST-UbC1 (upper panel). The beads were separately treated with
SDS, and immunoprecipitated FLAG-NUB1 was analyzed by Western
blotting using anti-FLAG Ig (lower panel). The identity of each band is
indicated on the right. Molecular size markers are shown in kilodaltons
on the left.
978 T. Tanaka et al. (Eur. J. Biochem. 271) Ó FEBS 2004
activity of UCH, which cleaves ubiquitin monomers from
UbC1. To examine this possibility, we first established an
in vitro assay system using UCH-L3, which is well-charac-
terized as a UCH [34]. We overexpressed FLAG-tagged
Ubc9 (negative control), UCH-L3 wild-type, and UCH-L3
mutant lacking enzymatic activity (negative control) [2] in
COS cells. The FLAG-tagged proteins were then immuno-
precipitated. Using purified GST-UbC1 as a substrate, the
immunoprecipitated FLAG-tagged proteins were tested for
the enzymatic activity of UCH. As shown in Fig. 3A, we
successfully detected the hydrolysis of UbC1 by UCH-L3
wild-type (lane 6), but could not detect it by Ubc9 (lane 5) or

UCH-L3 mutant (lane 7). Thus, we established the assay
system. Using this assay system, we next examined whether
the immunoprecipitates of FLAG-NUB1 have the enzy-
matic activity of UCH. As shown in Fig. 3B (upper panel),
we incubated GST-UbC1 with the immunoprecipitates of
FLAG-NUB1 for 0 (lane 3), 30 (lane 5), 60 (lane 6) or
120 min (lane 7). As expected, the hydrolysis of GST-UbC1
was detected clearly and increased with time, indicating that
the immunoprecipitates of FLAG-NUB1 have the UCH
activity. Finally, we further confirmed the UCH activity of
the immunoprecipitates of FLAG-NUB1 by inhibiting the
hydrolysis with a chemical reagent. As the enzymatic activity
of UCHs can be specifically inhibited by thiol-blocking
agents such as NEM [29,30], we used NEM to inhibit the
UCH activity of the immunoprecipitates and also used a
serine protease inhibitor, phenylmethylsulfonyl fluoride, as a
negative control. As shown in Fig. 3C (upper panel), the
UCH activity of the immunoprecipitates of FLAG-NUB1
was dramatically inhibited by NEM (lane 6), but not
by phenylmethylsulfonyl fluoride (lane 7). This confirmed
that the immunoprecipitates of FLAG-NUB1 have UCH
activity.
As described above, the difference between NUB1L
and NUB1 is the insertion of a UBA2 domain between
the UBA1 domain and UBA3 domain (Fig. 2A). As
NUB1L interacted with UbC1 and its structure was
identical to that of NUB1 with the exception of the
UBA2 insertion, we hypothesized that the immunopre-
cipitates of NUB1L had the same enzymatic activity as
those of NUB1. To prove this, we performed the in vitro

hydrolysis assay and found that the immunoprecipitates
of FLAG-NUB1L also had the UCH activity for UbC1
(data not shown).
NUB1 itself does not have UCH activity
In the experiments described above, we used the immuno-
precipitates of FLAG-NUB1. Although their UCH activity
could be demonstrated in Fig. 3B,C, we did not know
whether the activity was derived from FLAG-NUB1 or the
coprecipitated proteins. To settle this point, mutational
studies were performed. We mutated the possible catalytic
residue Cys313 to Ser in the Cys box-like sequence of NUB1
(Fig. 4A). Moreover, we also mutated the His352 residue to
Ala in the His box-like sequence of NUB1 (Fig. 4A). We
did this because these Cys and His residues are conserved in
the Cys and His boxes of all UCHs and function in catalysis
[34] (Fig. 3A, lane 7). Using the in vitro assay system
described in the previous section, we determined whether
the mutation at the Cys or the His residue abolished the
hydrolase activity. As shown in Fig. 4B (upper panel),
neither mutation affected the hydrolase activity (lanes 6 and
7), suggesting that the UCH activity was not derived from
Fig. 4. Cys and His box-like sequences in NUB1. (A) Locations and
sequences of Cys and His box-like sequences in NUB1. Arrowheads
indicate the active Cys residue in the Cys box and the conserved His
residue in the His box. (B) Mutational analysis of the Cys and His box-
likesequencesinNUB1.COScellsweretransfectedtoexpressthe
empty vector (lane 4), FLAG-tagged wild-type NUB1 (lane 5), NUB1
with a CysfiAla substitution at Cys313 (lane 6), or NUB1 with a His-
to-Ala substitution at His352 (lane 7). The expressed proteins were
precipitated with beads coated with anti-FLAG Ig. The beads were

incubated with purified GST-UbC1 for 120 min (lanes 4–7). After
centrifugation, the supernatant containing GST-UbC1 was treated
with SDS and analyzed by Western blotting using anti-GST Ig to
detect the derivatives of GST-UbC1 (upper panel). The beads were
separately treated with SDS, and immunoprecipitated FLAG-NUB1
was analyzed by Western blotting using anti-FLAG Ig (lower panel).
Ó FEBS 2004 NUB1-mediated hydrolysis of UbC1 (Eur. J. Biochem. 271) 979
FLAG-NUB1 but from the coprecipitated proteins. This
was also supported by another experiment showing that the
NUB1 expressed in bacteria did not hydrolyze UbC1 in vitro
(data not shown). These results prompted us to determine
which UCH is coimmunoprecipitated with NUB1 and
hydrolyzes UbC1. To do so, we examined whether NUB1
coimmunoprecipitates with known UCHs, including UCH-
L1 and UCH-L3. We overexpressed FLAG-tagged AIPL1
[24] (positive control), Ubc9 (negative control), UCH-L1,
and UCH-L3 in COS cells and immunoprecipitated them
with anti-FLAG Ig. The immunoprecipitates were then
analyzed by Western blotting using anti-NUB1 antibody
to detect endogenous NUB1 coimmunoprecipitated with
FLAG-tagged proteins. As shown in Fig. 5 (upper panel),
the endogenous NUB1 was coimmunoprecipitated by
AIPL1 (lane 2), but not by Ubc9 (lane 3), UCH-L1 (lane
4), or UCH-L3 (lane 5), indicating that UCH-L1 and UCH-
L3 do not cooperate with NUB1 in the hydrolysis of UbC1.
Both mRNAs of NUB1 and UbC1 are enriched
in seminiferous tubules of testis
To determine the expression of UbC1 in human tissues,
Northern blot analysis was performed using [
32

P]-labeled
UbC1 cDNA as a probe. This probe was designed not to
hybridize with ubiquitin-coding mRNAs other than UbC1
mRNA. As shown in Fig. 6, UbC1 mRNA was highly
enriched in the testis, but to a much lower degree in all other
tissues (middle panel). Interestingly, NUB1 mRNA was also
strongly detected in the testis on the same blot (upper panel).
We detected two isoforms of 3.1–3.5 kb and 2.3–2.7 kb.
The shorter message, which was a major transcript in the
testis, seemed to code NUB1, not NUB1L, as described
previously [20]. Thus, both mRNAs of UbC1 and NUB1
were enriched in the testis. Next, we determined the location
of cells expressing the mRNA of UbC1 or NUB1 in human
testis using in situ hybridization with a
35
S-labeled antisense
probe. As shown in Fig. 7, both UbC1 mRNA (E and F)
and NUB1 mRNA (B and C) were strongly expressed in
seminiferous tubules.
Discussion
Recently, we isolated a splicing variant of NUB1, termed
NUB1L. It possesses an additional UBA domain (UBA2)
between original UBA domains UBA1 and UBA3 [20]. In
the study described here, yeast two-hybrid assay showed that
NUB1 and NUB1L interacted with a linear ubiquitin
precursor such as UbC1. Further study revealed that
NUB1 and NUB1L interacted directly with the a-peptide
bond-linked polyubiquitin through their UBA1 domain.
Interestingly, NUB1 did not interact with either the ubiquitin
monomer or the polyubiquitin linked by isopeptide bonds

(Table 1). In addition to studying the interaction with the
Fig. 6. Northern blot analysis of NUB1 and UbC1. mRNA expression
of NUB1, UbC1 and b-actin was examined using a variety of human
tissues. Samples of poly(A)
+
RNA (2 lg) from the indicated sources
were run on a denaturing gel, transferred to a nylon membrane, and
hybridized with a
32
P-labeled cDNA probe of NUB1 (upper panel),
UbC1 (middle panel), or b-actin (lower panel). RNA size markers are
showninkilobases(kb).
Fig. 5. Co-immunoprecipitation of NUB1 by FLAG-tagged UCH
family members in COS cells. FLAG-tagged AIPL1 (positive control;
lane 2), Ubc9 (negative control; lane 3), UCH-L1 (lane 4), and UCH-
L3 (lane 5) were overexpressed in COS cells. Total cell lysate was
incubated with mouse anti-FLAG Ig for immunoprecipitation. Co-
precipitated proteins were analyzed by Western blotting using rabbit
anti-NUB1 Ig (upper panel). Precipitated FLAG-tagged proteins were
confirmed by Western blotting using rabbit anti-FLAG Ig (lower
panel). Molecular size markers are shown in kilodaltons.
980 T. Tanaka et al. (Eur. J. Biochem. 271) Ó FEBS 2004
ubiquitin monomer and two types of polyubiquitin, we
recently investigated the interaction of NUB1 and NUB1L
with the NEDD8 monomer [20]. As summarized in Table 1,
NUB1 interacted with the NEDD8 monomer through the
C-terminus, while NUB1L interacted with the NEDD8
monomer through the C-terminus and the UBA2 domain
[20]. Therefore, we conclude that the UBA1 domain in
NUB1 and NUB1L is utilized for binding with a-peptide

bond-linked polyubiquitin but not with isopeptide bond-
linked polyubiquitin. As the UBA1 domain seems to
recognize the structural differences between a-peptide-bond
linkage and isopeptide-bond linkage, the UBA1 domain
would appear to serve as a specific acceptor for a-peptide
bond-linked polyubiquitin. In contrast, the UBA2 domain is
utilized for binding with the NEDD8 monomer, while the
UBA3 domainis notutilized for binding with either ubiquitin
or NEDD8. These findings therefore indicate that each UBA
domain in NUB1 and NUB1L has a distinct binding ability.
In this study, we further defined the biological relevance
of the interaction between the UBA1 domain and a-peptide
bond-linked polyubiquitin. In particular, we demonstrated
that a ubiquitin precursor, UbC1, which is composed of
nine tandem repeats of the ubiquitin unit through a-peptide
bond, is hydrolyzed by immunoprecipitates of NUB1. On
the basis of these observations, we made a model of the
UbC1 hydrolysis mediated by NUB1. In this model, NUB1
forms a protein complex with a UCH that is an unidentified
enzyme other than UCH-L1 and UCH-L3. In this complex,
NUB1 is a subunit that binds to UbC1, while the
unidentified UCH is a catalytic subunit that hydrolyzes
UbC1. NUB1 recruits UbC1 to this complex, and subse-
quently the UCH hydrolyzes UbC1 to generate nine
ubiquitin monomers. As NUB1 does not interact with the
ubiquitin monomer, the ubiquitin monomers generated are
released from the protein complex and are then utilized for
the polyubiquitination of target proteins through the
linkage of isopeptide bonds. As NUB1 does not interact
with polyubiquitin linked with isopeptide bonds, NUB1

does not interfere with the polyubiquitination. Although
this model appears to be true, the UCH binding to NUB1
needs to be isolated and characterized. The UbC1 hydro-
lysis mediated by NUB1 may be specific to the testis,
because both mRNAs of NUB1 and UbC1 are highly
enriched in the testis. Interestingly, both mRNAs were
strongly expressed in the seminiferous tubules of the testis.
These results might imply that the UbC1 hydrolysis
mediated by NUB1 is involved in the cellular functions of
the seminiferous tubules such as spermatogenesis.
Acknowledgements
We thank Mr Hung Phi Nguyen for technical and editorial assistance.
This work was supported by National Institutes of Health Grant R01
DK56298 (to T. K.).
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