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

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 ofUbC1 by an unidenti

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 ofthe 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 ofnine 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 ofNUB1 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 ofboth NUB1 and UbC1 were enriched

in the testis Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules ofthe 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

NEDD8 is an 81-amino acid protein that shares 60%

identity and 80% homology with ubiquitin NEDD8

conjugates to a large number oftarget proteins [1], and

this conjugation is thought to be catalyzed by four enzymes,

NEDD8-carboxyl-terminal hydrolase [2],

activa-ting enzyme, conjugaactiva-ting enzyme, and

NEDD8-ligating enzyme, in a manner analogous to ubiquitination

and sentrinization (also known as SUMO conjugation) [3]

So far, all ofthe known targets ofNEDD8 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

ofthe SCF (or SCF-like complex), a ubiquitin E3 ligase

composed ofSkp1 (or Skp1-like protein), Cullin, F box

protein (or F box-like protein), and Roc1 [3,6] For

example, Cul1 is a major component ofan SCF complex

that catalyzes the ubiquitination ofIjBa, 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 ofthe 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 of601 amino acid residues with a calculated molecular mass of69.1 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 motifofabout 40 residues that was initially identified in ubiquitination enzymes, including E2s and E3s and other proteins linked to ubiquitination

Correspondence to T Kamitani, Department ofCardiology,

The University ofTexas M.D Anderson Cancer Center,

1515 Holcombe Blvd., Box449, Houston, TX 77030.

Fax: + 1 713 745 1942, Tel.: + 1 713 792 6242,

E-mail: tkamitani@mdanderson.org

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)

[18,19] Human NUB1 has two UBA domains, whereas

NUB1 homologues ofother species such as mouse, cow,

Drosophila, and Arabidopsis have three UBA domains [20]

Most recently, we identified a splicing variant ofthe human

NUB1gene that encodes a longer protein, termed NUB1L

(accession number: AF459743) [20] It possesses an insertion

of14 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

ofUbC1 by an unidentified ubiquitin C-terminal hydrolase

(UCH) The recruitment ofUbC1 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 ofthe 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 ofa 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 ofNUB1 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 30C The L40 clone carrying the pHybLex/NUB1 plasmid was cultured in YPAD/Zeo medium and sequentially transformed with 500 lg of the 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 30C on selection plates (Ura–/Lys–/His–/Leu–/Zeocin+) The posit-ive colonies were then picked and replated on selection plates (Ura–/Lys–/His–/Leu–/Zeocin+) and the b-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 ofUbC1 [27] and the truncated NUB1 and NUB1L shown below To examine the in vivo interaction ofUbC1 with these mutants, the yeast MATCHMAKER two-hybrid system 3 (Clontech) was used The cDNA ofUbC1 was subcloned into pGADT7 (Gal4 DNA-activating domain vector for Gal4-AD fusion), and the cDNA of each mutant ofNUB1 and NUB1L was subcloned into pGBKT7 (Gal4 DNA-binding domain vector for Gal4-BD fusion) The plasmids ofthe 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 30C The specific protein–protein interaction was determined by the growth ofthe cells on the selection plate

Western blot analysis Protein samples were treated for 1 h at 45C in 2% (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

Site-directed mutagenesis

CysfiAla and HisfiAla substitutions were made in NUB1

at Cys313 and His352, respectively The cDNA ofwild-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 mM Tris/HCl, pH 7.5,

100 mM 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 oflysis buffer [50 mM Tris/

HCl, pH 7.5, 100 mM 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

4C After centrifugation, the supernatant was incubated

with GST fusion proteins immobilized on

glutathione-Sepharose beads for 3 h at 4C 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 mM Tris/

HCl, pH 7.5, 100 mM 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 4C 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 oflysis buffer (50 mM Tris/ HCl, pH 7.5, 100 mM 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

4C After centrifugation, the supernatant was incubated for 2 h at 4C with 40 lL ofanti-FLAG M2 beads (Sigma) The beads coated with immunoprecipitates were washed three times with the lysis buffer and used for the

in vitrohydrolysis 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 mM Tris/HCl,

pH 7.5, 100 mM NaCl, and 10 mM 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 37C in the reac-tion buffer [50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.2% (v/v) NP-40, and 1 mM dithiothreitol] for varying amounts oftime 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 ofC-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 37C in the reaction buffer in the absence or presence of 2 mMNEM

or 2 mMphenylmethylsulfonyl 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 ofNUB1 and UbC1 in various human tissues For a probe ofNUB1, we chose a sequence located in the coding region ofNUB1 between nucleotides 450 and 900

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 [32P]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 68C for 1 h, followed by washing at

50C in the washing solution [300 mM NaCl, 30 mM

sodium citrate, and 0.1% (v/v) SDS] After this, the blot

membrane was exposed to a film For the Northern blot

analysis ofUbC1, we synthesized the oligonucleotide probe

of42 bases (5¢-GATTTGGGTCGCGGTTCTTGTTT

GTGGATCGCTGTGATCGTC-3¢), which is derived

from the 5¢-terminal noncoding region ofUbC1 (accession

number: AB009010) [27] The probe was labeled with

[32P]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 37C f or

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 ofNUB1, 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 ofsingle-stranded

RNA were generated At this step, probes were labeled

with [35S]UTP[aS] by T7 RNA polymerase (Promega)

The antisense and sense RNA probes labeled with

[35S]UTP[aS] were then denatured and mounted on tissue

sections (5· 105 c.p.m per slide) After incubation at

55C for 16 h, tissue sections were washed at 65 C and

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 ofUbC1 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 [35S]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 [35S]dATP[aS]-labeled oligonucleotide probes

were added to the tissue sections (5· 105c.p.m per slide)

After incubation at 37C for 16 h, tissue sections were

washed at a high-stringency temperature of55C

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 ofNUB1 is highly enriched in the testis [16],

a human testis cDNA library was used for the screening Approximately 2· 106primary library transformants were inoculated onto plates lacking histidine and leucine and containing Zeocin A total of84 colonies grew on the selection plates, 40 ofwhich 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 ofnine tandem repeats ofa 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 ofUbC1 with NUB1 might impede the processing ofUbC1

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 ofthis 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 ofubiquitin monomer to prevent forming poly-ubiquitin as much as possible The mutant ofpoly-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 ofNUB1 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

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 ofFig 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 ofNUB1 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), ubiquitin monomer (lane 3), GST-ubiquitin dimer (lane 4), GST-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-polyGST-ubiquitin (lower panel) (B) Inter-action ofNUB1 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 ofeach band is indicated in the right-hand side Molecular size markers are shown in kilodaltons.

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 ofNUB1 As shown in

Fig 2A, we generated six mutants ofNUB1 (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 oftwo 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 ofNUB1 Interestingly, this region contains the entire

UBA1 domain ofNUB1, 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 ofNUB1L 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 ofthe 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 ofhuman 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 ofthe 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 bacterial lysates were used for Western blotting with anti-RH Ig (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.

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 ofthe 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 ofNUB1 (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, ofubiquitin conjugates The catalytic cysteine ofDUBs 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 ofDUB family and has the enzymatic

Fig 3 C-terminal hydrolysis of ubiquitin nanomer, UbC1 (A) In vitro hydrolysis ofUbC1 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 ofGST-UbC1 (B) In vitro hydrolysis ofUbC1 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 ofGST-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 ofUbC1 hydrolysis by a thiol-blocking reagent, NEM Empty vector (lane 4) or FLAG-NUB1 (lanes 5–7) was expressed in COS cells and precipitated with beads coated with anti-FLAG The beads were incubated with purified GST-UbC1 in the absence (lanes 4 and 5) or presence ofNEM (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 ofGST-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 ofeach band is indicated on the right Molecular size markers are shown in kilodaltons

on the left.

activity ofUCH, which cleaves ubiquitin monomers from

UbC1 To examine this possibility, we first established an

in vitroassay 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 ofUCH 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 ofFLAG-NUB1 have the

enzy-matic activity ofUCH 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 ofGST-UbC1

was detected clearly and increased with time, indicating that

the immunoprecipitates ofFLAG-NUB1 have the UCH

activity Finally, we further confirmed the UCH activity of

the immunoprecipitates ofFLAG-NUB1 by inhibiting the

hydrolysis with a chemical reagent As the enzymatic activity

ofUCHs can be specifically inhibited by thiol-blocking

agents such as NEM [29,30], we used NEM to inhibit the

UCH activity ofthe immunoprecipitates and also used a

serine protease inhibitor, phenylmethylsulfonyl fluoride, as a

negative control As shown in Fig 3C (upper panel), the

UCH activity ofthe immunoprecipitates ofFLAG-NUB1

was dramatically inhibited by NEM (lane 6), but not

by phenylmethylsulfonyl fluoride (lane 7) This confirmed

that the immunoprecipitates ofFLAG-NUB1 have UCH

activity

As described above, the difference between NUB1L

and NUB1 is the insertion ofa UBA2 domain between

the UBA1 domain and UBA3 domain (Fig 2A) As

NUB1L interacted with UbC1 and its structure was

identical to that ofNUB1 with the exception ofthe

UBA2 insertion, we hypothesized that the

immunopre-cipitates ofNUB1L had the same enzymatic activity as

those ofNUB1 To prove this, we performed the in vitro

hydrolysis assay and found that the immunoprecipitates

ofFLAG-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 ofFLAG-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 ofNUB1

(Fig 4A) Moreover, we also mutated the His352 residue to

Ala in the His box-like sequence ofNUB1 (Fig 4A) We

did this because these Cys and His residues are conserved in

the Cys and His boxes ofall 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 ofCys 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 ofthe Cys and His box-like sequences in NUB1 COS cells were transf ected to express the 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 ofGST-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).

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 AIPUCH-L1

[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 L3 (lane 5), indicating that L1 and

UCH-L3 do not cooperate with NUB1 in the hydrolysis ofUbC1

Both mRNAs of NUB1 and UbC1 are enriched

in seminiferous tubules of testis

To determine the expression ofUbC1 in human tissues,

Northern blot analysis was performed using [32P]-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 ofUbC1 and NUB1 were enriched in the testis Next, we determined the location ofcells expressing the mRNA ofUbC1 or NUB1 in human testis using in situ hybridization with a35S-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 ofNUB1, 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 ofhuman tissues Samples ofpoly(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 ofNUB1 (upper panel), UbC1 (middle panel), or b-actin (lower panel) RNA size markers are shown in kilobases (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), 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.

ubiquitin monomer and two types ofpolyubiquitin, we

recently investigated the interaction ofNUB1 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

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 domain is not utilized 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

ofthe 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 ofthe ubiquitin unit through a-peptide

bond, is hydrolyzed by immunoprecipitates ofNUB1 On

the basis ofthese observations, we made a model ofthe

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 oftarget proteins through the linkage ofisopeptide 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 ofNUB1 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 ofHealth Grant R01 DK56298 (to T K.).

References

1 Kamitani, T., Kito, K., Nguyen, H.P & Yeh, E.T.H (1997) Characterization ofNEDD8, a developmentally down-regulated ubiquitin-like molecule J Biol Chem 272, 28557–28562.

Table 1 Interaction of NUB1/NUB1L with mono and polyubiquitins and mono-NEDD8 Y, Yeast two-hybrid; G, GST pull-down.

Assay

NUB1/NUB1L

Reference

Fig 7 In situ hybridization of NUB1 and

UbC1 mRNA with 35

S-labeled antisense probes

in human testis mRNA expression of NUB1

(A, B and C) and UbC1 (D, E and F) was

examined in tissue sections ofhuman testis.

Sections incubated with sense (A and D) or

antisense probes (B, C, E and F) were

photo-graphed against a dark field using low

magnification (A, B, D and E) and high

magnification (C and F) Tissue sections of

human testis were also stained by

hematoxy-lin/eosin (G and H) The bar represents 50 lm

for all panels.