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: 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) 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.). References 1. Kamitani, T., Kito, K., Nguyen, H.P. & Yeh, E.T.H. (1997) Characterization of NEDD8, 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 ReferenceUBA1 UBA2 UBA3 C-term Mono-NEDD8 Y, G – + – + [17,20, this study] Mono-Ub Y, G –––– [17] Poly-Ub chain (a-peptide linkage) Y, G + – – – [This study] Poly-Ub chain (isopeptide linkage) G –––– [This study] 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 of human 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. Ó FEBS 2004 NUB1-mediated hydrolysis of UbC1 (Eur. J. 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E.T.H & Kamitani, T (1999) Identification of NEDD8-conjugation site in human cullin-2 Biochem Biophys Res Commun 257, 100–105 6 Zheng, N., Schulman, B.A., Song, L., Miller, J.J., Jeffrey, P.D., Wang, P., Chu, C., Koepp, D.M., Elledge, S.J., Pagano, M., Conaway, R.C., Conaway, J.W., Harper, J.W & Pavletich, N.P (2002) Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex Nature 416, 703–709 . NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis Tomoaki Tanaka, Edward T. H. Yeh and Tetsu Kamitani Department of. 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