- Email: [email protected]
UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation
Biochemical and Biophysical Research Communications 382 (2009) 303–308
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation Masami Nagahama a,*, Machi Ohnishi a, Yumiko Kawate b, Takayuki Matsui a, Hitomi Miyake a, Keizo Yuasa a, Katsuko Tani b, Mitsuo Tagaya b, Akihiko Tsuji a a b
Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
a r t i c l e
i n f o
Article history: Received 27 February 2009 Available online 9 March 2009
Keywords: AAA ATPase ERAD UBX domain VCP
a b s t r a c t AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1–Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domaincontaining proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Eukaryotes have an elaborate endoplasmic reticulum (ER) quality control system to ensure that misfolded or unassembled proteins are retained in the ER and then directed for degradation by the pathway called ER-associated degradation (ERAD) [1,2]. ERAD consists of multi-step processes involving misfolded protein recognition, polyubiquitination, dislocation into the cytosol, and proteasomal degradation. These steps appear to be tightly coupled. The cytoplasmic ATPase valosin containing protein (VCP; p97; and Cdc48 in yeast) is likely involved in the dislocation of the substrates using energy provided by ATP hydrolysis in association with other ERAD components such as ubiquitin ligases and a putative dislocation channel component Derlin-1. VCP is a member of the AAA (ATPase associated with various cellular activities) protein family, and is proposed to serve as a molecular chaperone in the ubiquitin signaling pathway, through which it participates in a wide range of cellular activities, including organelle membrane fusion, protein degradation, transcriptional activation, cell-cycle control, apoptosis, and DNA repair [3,4]. The functional diversity of VCP is partly explained by its interaction with a variety of cofactors or adaptors, which direct VCP into different cellular pathways. In the ERAD pathway, VCP forms a com* Corresponding author. Fax: +81 88 655 3161. E-mail address: [email protected] (M. Nagahama). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.012
plex with the Ufd1–Npl4 heterodimer. In addition, activities of VCP in ERAD are known to be regulated by several other cofactors. We previously identified the small VCP-interacting protein (SVIP) [5]. Overexpression of SVIP in cells forms large ER membrane-derived vacuoles. The VCP-binding sites of SVIP and ERAD E3 gp78 share significant sequence similarity, which are termed VCP-interacting motifs (VIM) [6]. By negatively regulating the assembly of the gp78–VCP–Derlin-1 complex, SVIP acts as an endogenous inhibitor of ERAD [7]. Roles of proteins containing an ubiquitin regulatory X (UBX) domain in ERAD have also been reported recently. The UBX domain has a tertiary structure similar to that of ubiquitin and is considered to be a general VCP-binding module [8]. In the yeast Saccharomyces cerevisiae, seven UBX domain-containing proteins have been identified and all the members were confirmed to bind Cdc48. In addition, most of them were shown to be involved in intracellular proteolysis [9,10]. In particular, the participation of a membrane-bound UBX protein, Ubx2, in ERAD has been studied in detail [11,12]. On the ER membrane, Ubx2 interacts with Cdc48 and membrane-integrated ubiquitin ligases via its UBX and UBA domains, respectively. Thereby, it spatially links those activities at the site of ERAD. In mammals, the involvement of erasin (UBXD2) in ERAD has been reported [13]. Erasin is an ER membrane-integrated protein, which binds VCP via its UBX domain. In addition, it is in a complex with gp78 and Derlin-1, and is required for the degradation of the model substrate CD3-d by ERAD.
304
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
Materials and methods Plasmid construction. To construct pFT-GFP-CFTR4F508 for stably expressing GFP-CFTR4F508 in the Flp-In T-REx system (Invitrogen, Carlsbad, CA), a cDNA fragment encoding GFP-CFTR4F508 [14] was inserted into pcDNA5/FRT/TO (Invitrogen). pFT103 was constructed by inserting a fragment encoding One-STrEP-tag (a modified Strep-tagII), which was excised from pEXPR-IBA103 (IBA, St. Louis, MO), into the multiple cloning site of pcDNA5/ FRT/TO. Full-length cDNA for rat and human UBXD1 was amplified respectively from a rat liver and a human kidney cDNA library (Clontech, Palo Alto, CA) by PCR. The rat UBXD1 cDNA was subcloned into pFLAG-CMV-6 (Sigma–Aldrich, St. Louis, MO) to express FLAG-tagged UBXD1, and into pcDNA5/FRT/TO (Invitrogen) to express UBXD1 in the doxycycline (a tetracycline derivative) inducible Flp-In T-REx system. The human UBXD1 cDNA was subcloned into pGEX-6P-1 (GE Healthcare, Piscataway, NJ) to express UBXD1 as a glutathione-S-transferase (GST)-fused protein, and into pFT103 to express strep-tagged UBXD1. Expression plasmids for the GST- or FLAG-tagged SVIP, Ufd1, and p47 were described previously [5]. Antibodies. Rabbit anti-VCP and anti-SVIP antibodies have been described previously [5]. The monoclonal anti-FLAG M2, anti-strep, and anti-GFP antibodies were purchased from Sigma–Aldrich, Qiagen (Chatworth, CA), and Covance (Berkeley, CA), respectively. The monoclonal anti-Ufd1 and anti-calnexin antibodies were purchased from BD Biosciences (San Jose, CA). The polyclonal anti-Derlin-1 and anti-VIMP antibodies were purchased from Sigma–Aldrich. To generate the polyclonal anti-UBXD1 antibody, rabbits were immunized with GST-fused full-length UBXD1 expressed in Escherichia coli. Antibodies reactive to GST in the antiserum were removed by passage through a column of GST-immobilized Sepharose beads and the anti-UBXD1 antibody were affinity purified using GST-UBXD1-immobilized beads. Cell culture, transfection, and immunofluorescence. HeLa cells were cultured in a-minimum essential medium supplemented with 10% fetal bovine serum. Human embryonic kidney 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Flp-In T-Rex-293 cells (Invitrogen) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 lg/ml Zeocin (InvivoGen, San Diego, CA), and 15 lg/ml blastcidin S (Kaken Pharma, Tokyo, Japan). Transfection was performed with LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. To generate stable cells exhibiting doxycycline-inducible expression of cDNAs, Flp-In T-Rex-293 cells were cotransfected with pFT-GFP-CFTR4F508 or pFT103-UBXD1 and the Flp recombinase plasmid pOG44. Stable integrants were selected by culture in hygromycin B (100 lg/ml) (Wako, Osaka, Japan) and blastcidin S (15 lg/ml) and maintained under the same conditions. Cells were induced for the cDNA expression with varying concentrations of doxycycline (Sigma–Aldrich) for 24 h before use in the experiments. Immunofluorescence staining of cells was performed as described previously [15]. RNA interference. Synthetic small interference RNA (siRNA) was purchased from B-Bridge International (Mountain View, CA). The siRNA sequences targeting UBXD1 and SVIP were siUBXD1–302, 5’-GGACCAACGUGGUAUCUGA-dTdT-3’; siUBXD1–1312, 5’-GAGAA GCUCUUGUGAAAUA-dTdT-3’; and siSVIP, 5’-GGTGGACTTAGG TGGACAG-dTdT-3’. The siRNA targeting luciferase (GL3) was used as a negative control. The siRNAs were transfected with LipofectAMINE 2000 as described by the manufacturer. Pull-down assays. For GST pull-down assays, cells expressing GST-fusion proteins were lysed for 20 min at 4 °C in lysis buffer [20 mM Tris–HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 5 mM MgCl2, 10 lg/ml leupeptin, 1 lg/ml pepstatin A,
and 1 mM phenylmethyl sulfonylfluoride (PMSF)]. The lysate was then centrifuged at 14,000 rpm for 20 min at 4 °C, and supernatant was incubated with glutathione–Sepharose 4B beads (GE Healthcare) for 2 h at 4 °C with gentle rotation. After incubation, the beads were collected by centrifugation, washed four times with wash buffer (lysis buffer containing 0.05% Triton X-100 instead of 0.5%), and then treated with SDS–PAGE sample buffer. Proteins in the precipitates were analyzed by SDS–PAGE and Western blotting with an enhanced chemiluminescence reagent (Pierce Chemical, Rockford, IL). For strep pull-down assay, lysate from cells transiently expressing strep-tagged UBXD1 was incubated with Strep-Tactin beads (IBA) for 2 h at 4 °C and processed as for GST pull-down analysis. For pull-down experiments with purified proteins, 2 lg of GST-fusion proteins was incubated with 6 lg of Histagged proteins in a buffer consisting of 20 mM HEPES–KOH (pH 7.4), 100 mM KCl, 0.5% Triton X-100, 1 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, and 5% glycerol and then pulled down with glutathione–Sepharose 4B. The precipitated materials were separated by SDS–PAGE and detected by Coomassie brilliant blue (CBB) staining. Results Identification of UBXD1 as a VCP-interacting protein To identify proteins that interact with SVIP, we conducted a yeast two-hybrid screen using a rat liver cDNA library and the full-length SVIP as bait. A positive clone encoded a UBX domaincontaining protein UBXD1. The UBXD1 gene was previously identified but its biochemical and cell biological study remains sparse [16]. To examine whether the interaction found by the yeast two-hybrid system exists in mammalian cells, GST-fusion proteins of SVIP, p47, Ufd1, and VCP were expressed in 293 cells, and their interaction with endogenous UBXD1 was tested by GST pull-down with glutathione beads. As shown in Fig. 1A, the endogenous UBXD1 did not coprecipitate with SVIP or other VCP adaptors, although it did coprecipitate with VCP. These results suggest that UBXD1 directly binds VCP rather than SVIP. To further confirm the above result, strep-tagged UBXD1 (UBXD1-str) was expressed in cells, and pull-down analysis using Strep-Tactin beads was conducted (Fig. 1B). The results revealed that UBXD1-str interacted with endogenous VCP but not with SVIP or Ufd1. Remarkably, UBXD1 also associated with Derlin-1, suggesting the potential involvement of UBXD1 in ERAD. We interpret the above results to indicate that UBXD1 directly interacts with VCP, and that the SVIP-UBXD1 interaction observed in the yeast two-hybrid system may be an indirect one, probably mediated by the yeast VCP ortholog Cdc48. To confirm the direct interaction of UBXD1 with VCP, we conducted in vitro binding assays using recombinant proteins. In the first experiment, recombinant His-tagged VCP was incubated with GST-fused UBXD1 or GST as a control, and a GST pull-down assay was performed using glutathione beads. The results showed that His-VCP coprecipitated with GST-UBXD1 but not with control GST (Fig. 1C). Next, we performed the reverse experiment using His-tagged UBXD1 and GST fusion proteins of VCP or its cofactors (Fig. 1D). The results showed that His-UBXD1 coprecipitated with GST-VCP, but not with GST-fusion proteins of the cofactors including SVIP. Thus, these in vitro binding assays indicate that UBXD1 directly interacts with VCP but not with its cofactors. Overexpression of UBXD1 inhibits interaction between VCP and Ufd1 We next asked whether UBXD1 could influence the interaction of VCP with other cofactors. To investigate this possibility,
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
305
Fig. 1. UBXD1 interacts with VCP and Derlin-1. (A) Human embryonic kidney 293 cells were transfected with indicated GST-fusion proteins. After 24 h, cell lysates were subjected to pull-down with glutathione beads. The precipitates (Pull-down) and 4% of the starting material (Input) were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Human embryonic kidney 293 cells were transfected with strep-tagged UBXD1 (UBXD1-str) or an empty vector as a control. After 24 h, cell lysates were subjected to pull-down with Strep-Tactin beads. (C) Recombinant GST-UBXD1 or GST as a control was incubated with His-VCP and subjected to pull-down with glutathione beads. The precipitated proteins were detected by SDS–PAGE followed by CBB staining. The amounts of 10% of the His-VCP added in the reaction mixtures are shown in Input. (D) Indicated recombinant GST-fusion proteins were incubated with His-UBXD1 and subjected to pull-down with glutathione beads. The reaction with GST-Ufd1 was added with His-tagged Npl4. The precipitated proteins were analyzed by SDS–PAGE followed by CBB staining. The amounts of 10% of the His-UBXD1 added in the reaction mixtures are shown in Input.
GST-fusion proteins of the various cofactors were expressed in cells in the absence or presence of FLAG-UBXD1. The cell lysates were then prepared and subjected to a GST pull-down assay to monitor the interaction of the GST-fused cofactors with endogenous VCP (Fig. 2A). In the absence of FLAG-UBXD1 expression, interactions of VCP with GST-SVIP, GST-Ufd1, and GST-p47 were observed with similar efficiency (Fig. 2A, lanes 8–10). Interestingly, in the presence of FLAG-UBXD1 expression as shown in the right panel, the interaction of VCP with GST-Ufd1 was dramatically inhibited (Fig. 2A, lane 19), whereas that with GST-SVIP or GST-p47 was not affected. These results suggest that UBXD1 specifically attenuates the interaction between VCP and Ufd1. FLAG-UBXD1 was also coprecipitated with GST-SVIP and GSTp47, which is probably mediated by VCP interactions (Fig. 2A, lanes 18 and 20).
The above results were further confirmed in cells where the expression of UBXD1 is under the control of a doxycycline-inducible promoter. The cells were transfected with the expression plasmid for GST-Ufd1 and then grown with different concentrations of doxycycline to modulate the level of UBXD1 expression. The cellular extracts were then subjected to a GST pull-down assay to monitor the interaction between VCP and GST-Ufd1. The results showed that the increase of UBXD1 expression decreased the VCP binding to GST-Ufd1 in a dose-dependent manner (Fig. 2B). UBXD1 is a cytosolic protein whose overexpression causes cell vacuolation To investigate the intracellular localization of UBXD1, 293 cells were processed for fractionation into cytosol and microsomes, and
306
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
Fig. 2. Overexpression of UBXD1 causes the dissociation of VCP and Ufd1. (A) Human embryonic kidney 293 cells transiently transfected as indicated were processed for GST pull-down analysis. The precipitates (Pull-down) and 4% of the starting material (Input) were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Doxycycline (Dox)-inducible 293 cells that stably express UBXD1 were transiently transfected with GST-UBXD1 and then treated with various concentrations of Dox to induce UBXD1 expression for 24 h. Extracts were prepared and subjected to GST pull-down analysis.
proteins were detected by Western blotting. The membrane-anchored protein SVIP and the membrane-spanning proteins calnexin and Delin-1 were detected only in the microsomal membranes. Endogenous UBXD1 was detected in both the cytosol and membrane fraction (Fig. 3A). Next, to examine how UBXD1 associates with the membrane, the membrane fraction was subjected to alkaline extraction with Na2CO3. As in the case of VCP, which is known to be peripherally associated with the microsomes, a significant amount of UBXD1 was released into the supernatant by the treatment, whereas the treatment had no effect on the membrane association of SVIP, calnexin, and Delin-1. These data suggest that, similar to VCP, UBXD1 is a cytosolic protein and a part of it peripherally associates with microsomal membranes. To confirm the intracellular localization of UBXD1, FLAG-tagged UBXD1 was expressed in cells and processed for immunofluorescence analysis (Fig. 3B). The results revealed that UBXD1 was distributed throughout the cytoplasm. Interestingly, we noticed that large, aberrant vacuoles formed in cells expressing FLAG-UBXD1. Similar vacuoles are observed in cells overexpressing SVIP or a dominant negative mutant of VCP, and they have been shown to be derived from the ER and might be caused by accumulation of misfolded proteins [5,17]. These observations suggest that UBXD1 may be involved in a quality control system of the ER by regulating the function of VCP.
UBXD1 is required for efficient degradation of CFTRDF508 by ERAD To assess the involvement of UBXD1 in ERAD, we established 293 cells that express a mutant form of CFTR (CFTR4F508), a widely used ERAD substrate, as a GFP-fusion protein. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of cycloheximide, a protein synthesis inhibitor. As shown in Fig. 4A, GFP-CFTR4F508 was rapidly degraded in cells transfected with the empty vector as a control (lanes 1–4). In contrast, overexpression of UBXD1 gave a significant increase of the steady-state level of the substrate and its stability during the cycloheximide chase (lanes 5–8), indicating that the degradation of GFPCFTR4F508 was inhibited in these cells. In a parallel experiment, overexpression of SVIP, which has been shown to function as an endogenous inhibitor of ERAD by using CD3-d as a substrate [17], increased the steady-state level and stability of GFP-CFTR4F508 (lanes 9–12). Furthermore, we investigated the requirement of UBXD1 in ERAD in GFP-CFTR4F508 expressing cells via RNA interference mediated silencing of UBXD1 expression. A significant reduction in the level of UBXD1 protein was achieved by transfection with two specific siRNAs (UBXD1–302 and UBXD1–1312) (Fig. 4B). The UBXD1 knockdown led to a marked increase of the steadystate level of GFP-CFTR4F508 (lanes 4 and 7). A cycloheximide
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
307
of GFP-CFTR4F508 (lanes 10–12). This is consistent with a previous report that SVIP knockdown enhances turnover of CD3-d [7]. These data suggest that, in contrast to the inhibitory role of SVIP in ERAD, UBXD1 is a regulatory component required for efficient progression of the ERAD pathway. Discussion
Fig. 3. (A) Subcellular fractionation shows cytosolic localization of UBXD1. Human embryonic kidney 293 cells were homogenized and the cytosol (Cyt) and microsomes (Mem) were isolated from the post-nuclear supernatant. For alkaline extraction, the microsomes were extracted with Na2CO3 (pH 12) or Tris–HCl (pH 7.4) as a control. The membranes (ppt) and supernatants (sup) fractions were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Overexpression of UBXD1 causes cell vacuolation. The FLAG-tagged UBXD1 was expressed in HeLa cells. The cells were double immunostained with antibodies against FLAG and calnexin.
UBX domain-containing proteins constitute a large and widespread family of diverse VCP cofactors. They are thought to be involved in substrate recruitment to VCP or in the temporal and spatial regulation of its activity. The Saccharomyces cerevisiae genome encodes seven UBX domain-containing proteins, which have been shown to be required for sporulation or protein degradation [9,10]. In humans, 13 UBX domain-containing proteins have been identified, including erasin/UBXD2, which is involved in ERAD [13]. However, the functional correlations between yeast and human protein members are obscure. The primary structure of UBXD1 is characterized by two recognizable protein domains, UBX and PUB domains, both of which are considered to be involved in the ubiquitin-related pathways. The PUB domain was first identified in peptide N-glycanase, which cleaves N-glycan chains from misfolded glycoproteins during ERAD, and is thought to function as a protein-protein interaction domain in this process [18]. These structural features of UBXD1 suggest its involvement in ERAD via interaction with VCP and other ERAD-related proteins. UBXD1 is found in eukaryotes except yeast and shows a high degree of sequence conservation among species [16], suggesting that this protein participates in an important function specific for multicellular organisms. We showed that both overexpression and knockdown of UBXD1 in cells cause defects in the degradation of CFTR4F508 by ERAD (Fig. 4). These results suggest that UBXD1 is a regulatory component in the ERAD pathway, and that its appropriate expression level is critical for efficient substrate degradation. Consistent with these observations, we found that overexpression of UBXD1 induces specific dissociation of Ufd1, the adaptor for the ERAD pathway, from the VCP complex (Fig. 2). This finding suggests a regulatory role of UBXD1 in the VCP-Ufd1 interaction, although further evidences are required to support a physiological significance of this effect in the ERAD regulation. Recently, interactions of UBXD1 with VCP and other ERAD components such as Hrd1 and HERP were reported [19,20]; however, siRNA-mediated depletion of UBXD1 did not affect the ERAD of CD3-d [20]. This discrepancy may be due to the different experimental systems employed. UBXD1 may be involved in degradation of particular substrates or it may function in particular cellular environments. Acknowledgments
Fig. 4. (A) Effects of UBXD1 overexpression on ERAD. Doxycycline (Dox)-inducible 293 cells that stably express GFP-CFTR4F508 were transiently transfected with FLAG-tagged UBXD1, FLAG-tagged SVIP, or vector as a control. The cells were then treated with 0.1 lg/ml Dox for 24 h to induce GFP-CFTR4F508 expression. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of 50 lg/ml cycloheximide (CHX). The cell extracts were subjected to SDS– PAGE and then analyzed by Western blotting. (B) Effect of UBXD1 knockdown on ERAD. Dox-inducible 293 cells that stably express GFP-CFTR4F508 were transfected with 100 nM control siRNA or siRNAs directed against UBXD1 (siUBXD1–302 or siUBXD1–1312) or SVIP for 72 h, followed by induction with 0.1 lg/ml Dox to induce GFP-CFTR4F508 expression for 24 h. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of 50 lg/ml CHX.
chase analysis showed that degradation of GFP-CFTR4F508 is remarkably delayed in UBXD1 knockdown cells (lanes 4–9). In a parallel experiment, SVIP knockdown led to enhanced degradation
We are grateful to Dr. G. Warren and Dr. R. Kopito for the kind gifts of the plasmids. This work was supported in part by Grantsin-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and by a grant from the Ichiro Kanehara Foundation. References [1] B. Meussr, C. Hirsch, E. Jarosch, T. Sommer, ERAD: the long road to destruction, Nat. Cell Biol. 7 (2005) 766–772. [2] K. Römisch, Endoplasmic reticulum associated degradation, Annu. Rev. Cell Dev. Biol. 21 (2005) 435–456. [3] P.G. Woodman, p97, a protein coping with multiple identities, J. Cell Sci. 116 (2003) 4283–4290. [4] Q. Wang, C. Song, C.C. Li, Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions, Struct. Biol. 146 (2004) 44–57.
308
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
[5] M. Nagahama, M. Suzuki, Y. Hamada, K. Hatsuzawa, K. Tani, A. Yamamoto, M. Tagaya, SVIP is a novel VCP-interacting protein whose expression causes cell vacuolation, Mol. Biol. Cell 14 (2003) 262–273. [6] P. Ballar, Y. Shen, H. Yang, S. Fang, The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation, J. Biol. Chem. 281 (2006) 35359–35368. [7] P. Ballar, Y. Zhong, M. Nagahama, M. Tagaya, Y. Shen, S. Fang, Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation, J. Biol. Chem. 282 (2007) 33908–33914. [8] C. Schuberth, A. Buchberger, UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97, Cell. Mol. Life Sci. 65 (2008) 2360–2371. [9] A. Decottignies, A. Evain, M. Ghislain, Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation, Yeast 21 (2004) 127–139. [10] C. Schuberth, H. Richly, S. Rumpf, A. Buchberger, Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation, EMBO Rep. 5 (2004) 818–824. [11] O. Neuber, E. Jarosch, C. Volkwein, J. Walter, T. Sommer, Ubx2 links the Cdc48 complex to ER-associated protein degradation, Nat. Cell Biol. 7 (2005) 993–998. [12] C. Schuberth, A. Buchberger, Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation, Nat. Cell Biol. 7 (2005) 999–1006. [13] J. Liang, C. Yin, H. Doong, S. Fang, C. Peterhoff, R.A. Nixon, M.J. Monteiro, Characterization of erasin (UBXD2): a new ER protein that promotes ERassociated protein degradation, J. Cell Sci. 119 (2006) 4011–4024.
[14] J.A. Johnston, C.L. Ward, R.R. Kopito, Aggresomes: a cellular response to misfolded proteins, J. Cell Biol. 143 (1998) 1883–1898. [15] M. Nagahama, T. Yamazoe, Y. Hara, K. Tani, A. Tsuji, M. Tagaya, The AAAATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1, Biochem. Biophys. Res. Commun. 346 (2006) 1075–1082. [16] L. Carim-Todd, M. Escarceller, X. Estivill, L. Sumoy, Identification and characterization of UBXD1, a novel UBX domain-containing gene on human chromosome 19p13, and its mouse ortholog, Biochim. Biophys. Acta 1517 (2001) 298–301. [17] M. Hirabayashi, K. Inoue, K. Tanaka, K. Nakadate, Y. Ohsawa, Y. Kamei, A.H. Popiel, A. Shinohara, A. Iwamatsu, Y. Kimura, Y. Uchiyama, S. Hori, A. Kakizuka, VCP in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration, Cell Death Differ. 8 (2001) 974– 984. [18] T. Suzuki, H. Park, E.A. Till, W.J. Lennarz, The PUB domain: a putative protein–protein interaction domain implicated in the ubiquitinproteasome pathway, Biochem. Biophys. Res. Commun. 287 (2001) 1083–1087. [19] G. Alexandru, J. Graumann, G.T. Smith, N. Kolawa, R. Fang, R.J. Deshaies, UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1a turnover, Cell 134 (2008) 804–816. [20] L. Madsen, K.M. Andersen, S. Prag, T. Moos, C.A. Semple, M. Seeger, R. Hartmann-Petersen, Ubxd1 is a novel co-factor of the human p97 ATPase, Int. J. Biochem. Cell Biol. 40 (2008) 2927–2942.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation Masami Nagahama a,*, Machi Ohnishi a, Yumiko Kawate b, Takayuki Matsui a, Hitomi Miyake a, Keizo Yuasa a, Katsuko Tani b, Mitsuo Tagaya b, Akihiko Tsuji a a b
Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
a r t i c l e
i n f o
Article history: Received 27 February 2009 Available online 9 March 2009
Keywords: AAA ATPase ERAD UBX domain VCP
a b s t r a c t AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1–Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domaincontaining proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Eukaryotes have an elaborate endoplasmic reticulum (ER) quality control system to ensure that misfolded or unassembled proteins are retained in the ER and then directed for degradation by the pathway called ER-associated degradation (ERAD) [1,2]. ERAD consists of multi-step processes involving misfolded protein recognition, polyubiquitination, dislocation into the cytosol, and proteasomal degradation. These steps appear to be tightly coupled. The cytoplasmic ATPase valosin containing protein (VCP; p97; and Cdc48 in yeast) is likely involved in the dislocation of the substrates using energy provided by ATP hydrolysis in association with other ERAD components such as ubiquitin ligases and a putative dislocation channel component Derlin-1. VCP is a member of the AAA (ATPase associated with various cellular activities) protein family, and is proposed to serve as a molecular chaperone in the ubiquitin signaling pathway, through which it participates in a wide range of cellular activities, including organelle membrane fusion, protein degradation, transcriptional activation, cell-cycle control, apoptosis, and DNA repair [3,4]. The functional diversity of VCP is partly explained by its interaction with a variety of cofactors or adaptors, which direct VCP into different cellular pathways. In the ERAD pathway, VCP forms a com* Corresponding author. Fax: +81 88 655 3161. E-mail address: [email protected] (M. Nagahama). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.012
plex with the Ufd1–Npl4 heterodimer. In addition, activities of VCP in ERAD are known to be regulated by several other cofactors. We previously identified the small VCP-interacting protein (SVIP) [5]. Overexpression of SVIP in cells forms large ER membrane-derived vacuoles. The VCP-binding sites of SVIP and ERAD E3 gp78 share significant sequence similarity, which are termed VCP-interacting motifs (VIM) [6]. By negatively regulating the assembly of the gp78–VCP–Derlin-1 complex, SVIP acts as an endogenous inhibitor of ERAD [7]. Roles of proteins containing an ubiquitin regulatory X (UBX) domain in ERAD have also been reported recently. The UBX domain has a tertiary structure similar to that of ubiquitin and is considered to be a general VCP-binding module [8]. In the yeast Saccharomyces cerevisiae, seven UBX domain-containing proteins have been identified and all the members were confirmed to bind Cdc48. In addition, most of them were shown to be involved in intracellular proteolysis [9,10]. In particular, the participation of a membrane-bound UBX protein, Ubx2, in ERAD has been studied in detail [11,12]. On the ER membrane, Ubx2 interacts with Cdc48 and membrane-integrated ubiquitin ligases via its UBX and UBA domains, respectively. Thereby, it spatially links those activities at the site of ERAD. In mammals, the involvement of erasin (UBXD2) in ERAD has been reported [13]. Erasin is an ER membrane-integrated protein, which binds VCP via its UBX domain. In addition, it is in a complex with gp78 and Derlin-1, and is required for the degradation of the model substrate CD3-d by ERAD.
304
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
Materials and methods Plasmid construction. To construct pFT-GFP-CFTR4F508 for stably expressing GFP-CFTR4F508 in the Flp-In T-REx system (Invitrogen, Carlsbad, CA), a cDNA fragment encoding GFP-CFTR4F508 [14] was inserted into pcDNA5/FRT/TO (Invitrogen). pFT103 was constructed by inserting a fragment encoding One-STrEP-tag (a modified Strep-tagII), which was excised from pEXPR-IBA103 (IBA, St. Louis, MO), into the multiple cloning site of pcDNA5/ FRT/TO. Full-length cDNA for rat and human UBXD1 was amplified respectively from a rat liver and a human kidney cDNA library (Clontech, Palo Alto, CA) by PCR. The rat UBXD1 cDNA was subcloned into pFLAG-CMV-6 (Sigma–Aldrich, St. Louis, MO) to express FLAG-tagged UBXD1, and into pcDNA5/FRT/TO (Invitrogen) to express UBXD1 in the doxycycline (a tetracycline derivative) inducible Flp-In T-REx system. The human UBXD1 cDNA was subcloned into pGEX-6P-1 (GE Healthcare, Piscataway, NJ) to express UBXD1 as a glutathione-S-transferase (GST)-fused protein, and into pFT103 to express strep-tagged UBXD1. Expression plasmids for the GST- or FLAG-tagged SVIP, Ufd1, and p47 were described previously [5]. Antibodies. Rabbit anti-VCP and anti-SVIP antibodies have been described previously [5]. The monoclonal anti-FLAG M2, anti-strep, and anti-GFP antibodies were purchased from Sigma–Aldrich, Qiagen (Chatworth, CA), and Covance (Berkeley, CA), respectively. The monoclonal anti-Ufd1 and anti-calnexin antibodies were purchased from BD Biosciences (San Jose, CA). The polyclonal anti-Derlin-1 and anti-VIMP antibodies were purchased from Sigma–Aldrich. To generate the polyclonal anti-UBXD1 antibody, rabbits were immunized with GST-fused full-length UBXD1 expressed in Escherichia coli. Antibodies reactive to GST in the antiserum were removed by passage through a column of GST-immobilized Sepharose beads and the anti-UBXD1 antibody were affinity purified using GST-UBXD1-immobilized beads. Cell culture, transfection, and immunofluorescence. HeLa cells were cultured in a-minimum essential medium supplemented with 10% fetal bovine serum. Human embryonic kidney 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Flp-In T-Rex-293 cells (Invitrogen) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 lg/ml Zeocin (InvivoGen, San Diego, CA), and 15 lg/ml blastcidin S (Kaken Pharma, Tokyo, Japan). Transfection was performed with LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. To generate stable cells exhibiting doxycycline-inducible expression of cDNAs, Flp-In T-Rex-293 cells were cotransfected with pFT-GFP-CFTR4F508 or pFT103-UBXD1 and the Flp recombinase plasmid pOG44. Stable integrants were selected by culture in hygromycin B (100 lg/ml) (Wako, Osaka, Japan) and blastcidin S (15 lg/ml) and maintained under the same conditions. Cells were induced for the cDNA expression with varying concentrations of doxycycline (Sigma–Aldrich) for 24 h before use in the experiments. Immunofluorescence staining of cells was performed as described previously [15]. RNA interference. Synthetic small interference RNA (siRNA) was purchased from B-Bridge International (Mountain View, CA). The siRNA sequences targeting UBXD1 and SVIP were siUBXD1–302, 5’-GGACCAACGUGGUAUCUGA-dTdT-3’; siUBXD1–1312, 5’-GAGAA GCUCUUGUGAAAUA-dTdT-3’; and siSVIP, 5’-GGTGGACTTAGG TGGACAG-dTdT-3’. The siRNA targeting luciferase (GL3) was used as a negative control. The siRNAs were transfected with LipofectAMINE 2000 as described by the manufacturer. Pull-down assays. For GST pull-down assays, cells expressing GST-fusion proteins were lysed for 20 min at 4 °C in lysis buffer [20 mM Tris–HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 5 mM MgCl2, 10 lg/ml leupeptin, 1 lg/ml pepstatin A,
and 1 mM phenylmethyl sulfonylfluoride (PMSF)]. The lysate was then centrifuged at 14,000 rpm for 20 min at 4 °C, and supernatant was incubated with glutathione–Sepharose 4B beads (GE Healthcare) for 2 h at 4 °C with gentle rotation. After incubation, the beads were collected by centrifugation, washed four times with wash buffer (lysis buffer containing 0.05% Triton X-100 instead of 0.5%), and then treated with SDS–PAGE sample buffer. Proteins in the precipitates were analyzed by SDS–PAGE and Western blotting with an enhanced chemiluminescence reagent (Pierce Chemical, Rockford, IL). For strep pull-down assay, lysate from cells transiently expressing strep-tagged UBXD1 was incubated with Strep-Tactin beads (IBA) for 2 h at 4 °C and processed as for GST pull-down analysis. For pull-down experiments with purified proteins, 2 lg of GST-fusion proteins was incubated with 6 lg of Histagged proteins in a buffer consisting of 20 mM HEPES–KOH (pH 7.4), 100 mM KCl, 0.5% Triton X-100, 1 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, and 5% glycerol and then pulled down with glutathione–Sepharose 4B. The precipitated materials were separated by SDS–PAGE and detected by Coomassie brilliant blue (CBB) staining. Results Identification of UBXD1 as a VCP-interacting protein To identify proteins that interact with SVIP, we conducted a yeast two-hybrid screen using a rat liver cDNA library and the full-length SVIP as bait. A positive clone encoded a UBX domaincontaining protein UBXD1. The UBXD1 gene was previously identified but its biochemical and cell biological study remains sparse [16]. To examine whether the interaction found by the yeast two-hybrid system exists in mammalian cells, GST-fusion proteins of SVIP, p47, Ufd1, and VCP were expressed in 293 cells, and their interaction with endogenous UBXD1 was tested by GST pull-down with glutathione beads. As shown in Fig. 1A, the endogenous UBXD1 did not coprecipitate with SVIP or other VCP adaptors, although it did coprecipitate with VCP. These results suggest that UBXD1 directly binds VCP rather than SVIP. To further confirm the above result, strep-tagged UBXD1 (UBXD1-str) was expressed in cells, and pull-down analysis using Strep-Tactin beads was conducted (Fig. 1B). The results revealed that UBXD1-str interacted with endogenous VCP but not with SVIP or Ufd1. Remarkably, UBXD1 also associated with Derlin-1, suggesting the potential involvement of UBXD1 in ERAD. We interpret the above results to indicate that UBXD1 directly interacts with VCP, and that the SVIP-UBXD1 interaction observed in the yeast two-hybrid system may be an indirect one, probably mediated by the yeast VCP ortholog Cdc48. To confirm the direct interaction of UBXD1 with VCP, we conducted in vitro binding assays using recombinant proteins. In the first experiment, recombinant His-tagged VCP was incubated with GST-fused UBXD1 or GST as a control, and a GST pull-down assay was performed using glutathione beads. The results showed that His-VCP coprecipitated with GST-UBXD1 but not with control GST (Fig. 1C). Next, we performed the reverse experiment using His-tagged UBXD1 and GST fusion proteins of VCP or its cofactors (Fig. 1D). The results showed that His-UBXD1 coprecipitated with GST-VCP, but not with GST-fusion proteins of the cofactors including SVIP. Thus, these in vitro binding assays indicate that UBXD1 directly interacts with VCP but not with its cofactors. Overexpression of UBXD1 inhibits interaction between VCP and Ufd1 We next asked whether UBXD1 could influence the interaction of VCP with other cofactors. To investigate this possibility,
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
305
Fig. 1. UBXD1 interacts with VCP and Derlin-1. (A) Human embryonic kidney 293 cells were transfected with indicated GST-fusion proteins. After 24 h, cell lysates were subjected to pull-down with glutathione beads. The precipitates (Pull-down) and 4% of the starting material (Input) were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Human embryonic kidney 293 cells were transfected with strep-tagged UBXD1 (UBXD1-str) or an empty vector as a control. After 24 h, cell lysates were subjected to pull-down with Strep-Tactin beads. (C) Recombinant GST-UBXD1 or GST as a control was incubated with His-VCP and subjected to pull-down with glutathione beads. The precipitated proteins were detected by SDS–PAGE followed by CBB staining. The amounts of 10% of the His-VCP added in the reaction mixtures are shown in Input. (D) Indicated recombinant GST-fusion proteins were incubated with His-UBXD1 and subjected to pull-down with glutathione beads. The reaction with GST-Ufd1 was added with His-tagged Npl4. The precipitated proteins were analyzed by SDS–PAGE followed by CBB staining. The amounts of 10% of the His-UBXD1 added in the reaction mixtures are shown in Input.
GST-fusion proteins of the various cofactors were expressed in cells in the absence or presence of FLAG-UBXD1. The cell lysates were then prepared and subjected to a GST pull-down assay to monitor the interaction of the GST-fused cofactors with endogenous VCP (Fig. 2A). In the absence of FLAG-UBXD1 expression, interactions of VCP with GST-SVIP, GST-Ufd1, and GST-p47 were observed with similar efficiency (Fig. 2A, lanes 8–10). Interestingly, in the presence of FLAG-UBXD1 expression as shown in the right panel, the interaction of VCP with GST-Ufd1 was dramatically inhibited (Fig. 2A, lane 19), whereas that with GST-SVIP or GST-p47 was not affected. These results suggest that UBXD1 specifically attenuates the interaction between VCP and Ufd1. FLAG-UBXD1 was also coprecipitated with GST-SVIP and GSTp47, which is probably mediated by VCP interactions (Fig. 2A, lanes 18 and 20).
The above results were further confirmed in cells where the expression of UBXD1 is under the control of a doxycycline-inducible promoter. The cells were transfected with the expression plasmid for GST-Ufd1 and then grown with different concentrations of doxycycline to modulate the level of UBXD1 expression. The cellular extracts were then subjected to a GST pull-down assay to monitor the interaction between VCP and GST-Ufd1. The results showed that the increase of UBXD1 expression decreased the VCP binding to GST-Ufd1 in a dose-dependent manner (Fig. 2B). UBXD1 is a cytosolic protein whose overexpression causes cell vacuolation To investigate the intracellular localization of UBXD1, 293 cells were processed for fractionation into cytosol and microsomes, and
306
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
Fig. 2. Overexpression of UBXD1 causes the dissociation of VCP and Ufd1. (A) Human embryonic kidney 293 cells transiently transfected as indicated were processed for GST pull-down analysis. The precipitates (Pull-down) and 4% of the starting material (Input) were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Doxycycline (Dox)-inducible 293 cells that stably express UBXD1 were transiently transfected with GST-UBXD1 and then treated with various concentrations of Dox to induce UBXD1 expression for 24 h. Extracts were prepared and subjected to GST pull-down analysis.
proteins were detected by Western blotting. The membrane-anchored protein SVIP and the membrane-spanning proteins calnexin and Delin-1 were detected only in the microsomal membranes. Endogenous UBXD1 was detected in both the cytosol and membrane fraction (Fig. 3A). Next, to examine how UBXD1 associates with the membrane, the membrane fraction was subjected to alkaline extraction with Na2CO3. As in the case of VCP, which is known to be peripherally associated with the microsomes, a significant amount of UBXD1 was released into the supernatant by the treatment, whereas the treatment had no effect on the membrane association of SVIP, calnexin, and Delin-1. These data suggest that, similar to VCP, UBXD1 is a cytosolic protein and a part of it peripherally associates with microsomal membranes. To confirm the intracellular localization of UBXD1, FLAG-tagged UBXD1 was expressed in cells and processed for immunofluorescence analysis (Fig. 3B). The results revealed that UBXD1 was distributed throughout the cytoplasm. Interestingly, we noticed that large, aberrant vacuoles formed in cells expressing FLAG-UBXD1. Similar vacuoles are observed in cells overexpressing SVIP or a dominant negative mutant of VCP, and they have been shown to be derived from the ER and might be caused by accumulation of misfolded proteins [5,17]. These observations suggest that UBXD1 may be involved in a quality control system of the ER by regulating the function of VCP.
UBXD1 is required for efficient degradation of CFTRDF508 by ERAD To assess the involvement of UBXD1 in ERAD, we established 293 cells that express a mutant form of CFTR (CFTR4F508), a widely used ERAD substrate, as a GFP-fusion protein. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of cycloheximide, a protein synthesis inhibitor. As shown in Fig. 4A, GFP-CFTR4F508 was rapidly degraded in cells transfected with the empty vector as a control (lanes 1–4). In contrast, overexpression of UBXD1 gave a significant increase of the steady-state level of the substrate and its stability during the cycloheximide chase (lanes 5–8), indicating that the degradation of GFPCFTR4F508 was inhibited in these cells. In a parallel experiment, overexpression of SVIP, which has been shown to function as an endogenous inhibitor of ERAD by using CD3-d as a substrate [17], increased the steady-state level and stability of GFP-CFTR4F508 (lanes 9–12). Furthermore, we investigated the requirement of UBXD1 in ERAD in GFP-CFTR4F508 expressing cells via RNA interference mediated silencing of UBXD1 expression. A significant reduction in the level of UBXD1 protein was achieved by transfection with two specific siRNAs (UBXD1–302 and UBXD1–1312) (Fig. 4B). The UBXD1 knockdown led to a marked increase of the steadystate level of GFP-CFTR4F508 (lanes 4 and 7). A cycloheximide
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
307
of GFP-CFTR4F508 (lanes 10–12). This is consistent with a previous report that SVIP knockdown enhances turnover of CD3-d [7]. These data suggest that, in contrast to the inhibitory role of SVIP in ERAD, UBXD1 is a regulatory component required for efficient progression of the ERAD pathway. Discussion
Fig. 3. (A) Subcellular fractionation shows cytosolic localization of UBXD1. Human embryonic kidney 293 cells were homogenized and the cytosol (Cyt) and microsomes (Mem) were isolated from the post-nuclear supernatant. For alkaline extraction, the microsomes were extracted with Na2CO3 (pH 12) or Tris–HCl (pH 7.4) as a control. The membranes (ppt) and supernatants (sup) fractions were resolved by SDS–PAGE and then analyzed by Western blotting. (B) Overexpression of UBXD1 causes cell vacuolation. The FLAG-tagged UBXD1 was expressed in HeLa cells. The cells were double immunostained with antibodies against FLAG and calnexin.
UBX domain-containing proteins constitute a large and widespread family of diverse VCP cofactors. They are thought to be involved in substrate recruitment to VCP or in the temporal and spatial regulation of its activity. The Saccharomyces cerevisiae genome encodes seven UBX domain-containing proteins, which have been shown to be required for sporulation or protein degradation [9,10]. In humans, 13 UBX domain-containing proteins have been identified, including erasin/UBXD2, which is involved in ERAD [13]. However, the functional correlations between yeast and human protein members are obscure. The primary structure of UBXD1 is characterized by two recognizable protein domains, UBX and PUB domains, both of which are considered to be involved in the ubiquitin-related pathways. The PUB domain was first identified in peptide N-glycanase, which cleaves N-glycan chains from misfolded glycoproteins during ERAD, and is thought to function as a protein-protein interaction domain in this process [18]. These structural features of UBXD1 suggest its involvement in ERAD via interaction with VCP and other ERAD-related proteins. UBXD1 is found in eukaryotes except yeast and shows a high degree of sequence conservation among species [16], suggesting that this protein participates in an important function specific for multicellular organisms. We showed that both overexpression and knockdown of UBXD1 in cells cause defects in the degradation of CFTR4F508 by ERAD (Fig. 4). These results suggest that UBXD1 is a regulatory component in the ERAD pathway, and that its appropriate expression level is critical for efficient substrate degradation. Consistent with these observations, we found that overexpression of UBXD1 induces specific dissociation of Ufd1, the adaptor for the ERAD pathway, from the VCP complex (Fig. 2). This finding suggests a regulatory role of UBXD1 in the VCP-Ufd1 interaction, although further evidences are required to support a physiological significance of this effect in the ERAD regulation. Recently, interactions of UBXD1 with VCP and other ERAD components such as Hrd1 and HERP were reported [19,20]; however, siRNA-mediated depletion of UBXD1 did not affect the ERAD of CD3-d [20]. This discrepancy may be due to the different experimental systems employed. UBXD1 may be involved in degradation of particular substrates or it may function in particular cellular environments. Acknowledgments
Fig. 4. (A) Effects of UBXD1 overexpression on ERAD. Doxycycline (Dox)-inducible 293 cells that stably express GFP-CFTR4F508 were transiently transfected with FLAG-tagged UBXD1, FLAG-tagged SVIP, or vector as a control. The cells were then treated with 0.1 lg/ml Dox for 24 h to induce GFP-CFTR4F508 expression. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of 50 lg/ml cycloheximide (CHX). The cell extracts were subjected to SDS– PAGE and then analyzed by Western blotting. (B) Effect of UBXD1 knockdown on ERAD. Dox-inducible 293 cells that stably express GFP-CFTR4F508 were transfected with 100 nM control siRNA or siRNAs directed against UBXD1 (siUBXD1–302 or siUBXD1–1312) or SVIP for 72 h, followed by induction with 0.1 lg/ml Dox to induce GFP-CFTR4F508 expression for 24 h. Disappearance of GFP-CFTR4F508 in the cells was monitored over time in the presence of 50 lg/ml CHX.
chase analysis showed that degradation of GFP-CFTR4F508 is remarkably delayed in UBXD1 knockdown cells (lanes 4–9). In a parallel experiment, SVIP knockdown led to enhanced degradation
We are grateful to Dr. G. Warren and Dr. R. Kopito for the kind gifts of the plasmids. This work was supported in part by Grantsin-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and by a grant from the Ichiro Kanehara Foundation. References [1] B. Meussr, C. Hirsch, E. Jarosch, T. Sommer, ERAD: the long road to destruction, Nat. Cell Biol. 7 (2005) 766–772. [2] K. Römisch, Endoplasmic reticulum associated degradation, Annu. Rev. Cell Dev. Biol. 21 (2005) 435–456. [3] P.G. Woodman, p97, a protein coping with multiple identities, J. Cell Sci. 116 (2003) 4283–4290. [4] Q. Wang, C. Song, C.C. Li, Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions, Struct. Biol. 146 (2004) 44–57.
308
M. Nagahama et al. / Biochemical and Biophysical Research Communications 382 (2009) 303–308
[5] M. Nagahama, M. Suzuki, Y. Hamada, K. Hatsuzawa, K. Tani, A. Yamamoto, M. Tagaya, SVIP is a novel VCP-interacting protein whose expression causes cell vacuolation, Mol. Biol. Cell 14 (2003) 262–273. [6] P. Ballar, Y. Shen, H. Yang, S. Fang, The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation, J. Biol. Chem. 281 (2006) 35359–35368. [7] P. Ballar, Y. Zhong, M. Nagahama, M. Tagaya, Y. Shen, S. Fang, Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation, J. Biol. Chem. 282 (2007) 33908–33914. [8] C. Schuberth, A. Buchberger, UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97, Cell. Mol. Life Sci. 65 (2008) 2360–2371. [9] A. Decottignies, A. Evain, M. Ghislain, Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation, Yeast 21 (2004) 127–139. [10] C. Schuberth, H. Richly, S. Rumpf, A. Buchberger, Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation, EMBO Rep. 5 (2004) 818–824. [11] O. Neuber, E. Jarosch, C. Volkwein, J. Walter, T. Sommer, Ubx2 links the Cdc48 complex to ER-associated protein degradation, Nat. Cell Biol. 7 (2005) 993–998. [12] C. Schuberth, A. Buchberger, Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation, Nat. Cell Biol. 7 (2005) 999–1006. [13] J. Liang, C. Yin, H. Doong, S. Fang, C. Peterhoff, R.A. Nixon, M.J. Monteiro, Characterization of erasin (UBXD2): a new ER protein that promotes ERassociated protein degradation, J. Cell Sci. 119 (2006) 4011–4024.
[14] J.A. Johnston, C.L. Ward, R.R. Kopito, Aggresomes: a cellular response to misfolded proteins, J. Cell Biol. 143 (1998) 1883–1898. [15] M. Nagahama, T. Yamazoe, Y. Hara, K. Tani, A. Tsuji, M. Tagaya, The AAAATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1, Biochem. Biophys. Res. Commun. 346 (2006) 1075–1082. [16] L. Carim-Todd, M. Escarceller, X. Estivill, L. Sumoy, Identification and characterization of UBXD1, a novel UBX domain-containing gene on human chromosome 19p13, and its mouse ortholog, Biochim. Biophys. Acta 1517 (2001) 298–301. [17] M. Hirabayashi, K. Inoue, K. Tanaka, K. Nakadate, Y. Ohsawa, Y. Kamei, A.H. Popiel, A. Shinohara, A. Iwamatsu, Y. Kimura, Y. Uchiyama, S. Hori, A. Kakizuka, VCP in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration, Cell Death Differ. 8 (2001) 974– 984. [18] T. Suzuki, H. Park, E.A. Till, W.J. Lennarz, The PUB domain: a putative protein–protein interaction domain implicated in the ubiquitinproteasome pathway, Biochem. Biophys. Res. Commun. 287 (2001) 1083–1087. [19] G. Alexandru, J. Graumann, G.T. Smith, N. Kolawa, R. Fang, R.J. Deshaies, UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1a turnover, Cell 134 (2008) 804–816. [20] L. Madsen, K.M. Andersen, S. Prag, T. Moos, C.A. Semple, M. Seeger, R. Hartmann-Petersen, Ubxd1 is a novel co-factor of the human p97 ATPase, Int. J. Biochem. Cell Biol. 40 (2008) 2927–2942.