Báo cáo khoa học: Evolutionary divergence of valosin-containing protein ⁄cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteins

Evolutionary divergence of valosin-containing protein ⁄cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteins Giacomo Morreale, Laura Conforti, John Coadwell, Anna L. Wilbrey and Michael P. Coleman

Laboratory of Molecular Signalling, The Babraham Institute, Cambridge, UK

Keywords endoplasmic reticulum-associated degradation; Hrd1; Ube4b; ubiquitin ligase; valosin-containing protein

Correspondence G. Morreale, The Babraham Institute, B501, Babraham Research Campus, Babraham, Cambridge CB22 3AT, UK Fax: +44 1223 496348 Tel: +44 1223 496251 E-mail: giacomo.morreale@bbsrc.ac.uk Centro di Ricerca per la Viticoltura, Via Casoni, 13/A, 31058 Susegana (TV), Italy Fax: +39-0438-738058 Tel: +39-0438-73264 E-mail: giacomo.morreale@entecra.it

(Received 21 August 2008, revised 9 December 2008, accepted 16 December 2008)

doi:10.1111/j.1742-4658.2008.06858.x

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell- autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor bind- ing would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b)–ubiquitin fusion degra- dation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1)–synoviolin and ataxin 3, and a related sequence in Mr 78 000 glycoprotein–Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in con- trast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP–Cdc48p. However, the differing molecular architecture of VCP–Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms.

Abbreviations Atx-3, ataxin 3; Cdc48p, cell division cycle protein 48; DAPI, 4¢,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; gp78, Mr 78 000 glycoprotein; GST, glutathione S-transferase; Hrd1, hydroxymethylglutaryl reductase degradation protein 1; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; OTUD7a, OUT domain-containing protein 7; SMURF, Smad ubiquitination regulatory factor; Ube4b, ubiquitin ligase E4b; Ufd, ubiquitin fusion degradation protein; VBM, valosin-containing protein binding motif; VCP, valosin-containing protein; WldS, slow Wallerian degeneration protein.

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is an AAA- Valosin-containing protein (VCP ⁄ p97) ATPase associated with a variety of cellular activities, most especially endoplasmic reticulum (ER)-associated degradation (ERAD) [1], and its functional diversity derives partly from its ability to bind a wide range of protein cofactors [2]. Some bind directly to VCP in a mutually exclusive manner, targeting VCP to a particu- lar function. For example, binding to the ubiquitin

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Evolutionary divergence in VCP binding during ERAD

fusion degradation protein (Ufd) 1–Npl4 dimer targets VCP to a function in ERAD, whereas binding to p47, which competes with Ufd1–Npl4, targets VCP to a role in homotypic membrane fusion [3,4]. In other cases, the function of the binding interaction is not fully understood, but there are further examples of mutual exclusivity [5–7]. Thus, the principle that cofac- tor binding determines functional specificity of VCP may be more wide-ranging, perhaps targeting VCP to different branches of the ubiquitin proteasome system according to which ligase it binds [5].

We hypothesized that additional ubiquitin-metaboliz- ing proteins would bind VCP through a similar VBM, and our search identified a functional VBM close to the C-terminus of the E3 ligase Hrd1, a protein involved in retrotranslocation during ERAD [23–25] and in turnover of the important disease-related proteins p53, expanded polyglutamine and Pael receptor [26–28]. Binding of Ube4b, Hrd1 and Atx-3 requires the sequence RXXR within a predicted a-helix. However, neighbouring amino acids also influence binding, and a similar motif required for VCP binding in gp78 toler- ates substitution of these two arginines. We map the site of binding of Ube4b to the N-domain of VCP and show that it competes for this site with Hrd1. Finally, we investigate the evolutionary divergence of the VBM and discuss its consequences for mechanism.

Results

Identification of the VBM in Hrd1 and its refinement in gp78

We recently reported that VCP binds directly to the N-terminal 16 amino acids of ubiquitination fac- tor E4b (Ube4b) [8], a protein involved in multiubiqui- tination and ERAD [9–12]. Similar arginine-rich sequences were subsequently identified in the polyglu- tamine protein ataxin 3 (Atx-3) [13], which has ubiqu- itin protease activity [14,15], and in the ER-resident ubiquitin ligase Mr 78 000 glycoprotein (gp78) (also known as autocrine motility factor receptor) [5], a key regulator of retrotranslocation during ER-associated degradation [16,17]. These sequences were respectively termed VCP-binding motif (VBM) and VCP-inter- acting motif [5,13].

the cofactors

importance of

VCP is a highly conserved protein whose functions have been extensively explored in invertebrate homo- logues. In particular, studies of cell division cycle protein 48 (Cdc48p) in Saccharomyces cerevisiae have uncovered roles in many cellular processes, including membrane fusion [18], ERAD [19] and spindle disas- sembly [20], and have played a key role in identify- ing that direct Cdc48p to these functions [3,21]. These functions and binding part- ners are well conserved in mammals, consistent with the fundamental these processes for cell survival. More specifically, S. cerevisiae Cdc48p interacts with Ufd2p, the homologue of Ube4b [11], so S. cerevisiae is used as a model for invstigating the role of VCP and its associated ubiquitin ligases during ERAD. However, despite the fundamental

Our search for additional mammalian ubiquitin ligases that contain a sequence similar to the VBM of Ube4b led us to Hrd1–synoviolin, which binds VCP through its C-terminal cytosolic region (amino acids 236–626) [24]. Within this region of mouse and human Hrd1, we identified four consecutive arginines close to the C-ter- minus at positions 599–602 [23]. Using the glutathione S-transferase (GST)-fused sequence DAAELRRRRL- QKLESPVAH, we then showed that this sequence is sufficient to pull down 35S-labeled VCP expressed by in vitro transcription and translation (Fig. 1A, lane 5). Using a similar approach, we also confirmed an earlier report that the Atx-3-derived peptide MTSEELRKR- REAYFEK binds VCP [13] (Fig. 1A, lane 4). We also identified arginine-rich motifs in three further ubiquitin- metabolizing proteins, the zinc finger deubiquitinating protein OUT domain-containing protein 7 (OTUD7a) [29], and the ligases Smad ubiquitination regulatory fac- tor (SMURF)1 and SMURF2, which are identical to each other in this region [30,31]. Neither of these sequences bound VCP in the GST pulldown assay (Fig. 1A, lanes 1 and 2), indicating both the importance of neighbouring amino acids and the specificity of the binding to Hrd1, Ube4b and Atx-3 VBM sequences.

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Another RING finger E3 ligase involved in ERAD, gp78, is homologous to Hrd1 [23]. The homology is mostly in the N-terminal regions of these two proteins, but the C-terminal 12 amino acids of gp78 show 50% identity to the sequence around the Hrd1 VBM, and VCP binding has been mapped successively to the last 49 and 30 amino acids of gp78 [5,17]. Therefore, we importance of ERAD to cell survival, and despite good conservation of the proteins and their binding partners, differences in binding sites have begun to emerge. For example, S. cerevisiae Ufd2p uses a C-terminal sequence to bind Cdc48p [12], in contrast to the N-terminal sequence used by mammalian Ube4b [8]. Mammalian VCP and S. cerevisiae Cdc48p are also not functionally inter- changeable, despite their strong homology [22]. Thus, to know how well the mechanism is conserved, it is important to understand fully the differences in how VCP–Cdc48p interacts with Ube4b–Ufd2p and with other VBM-containing proteins.

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Evolutionary divergence in VCP binding during ERAD

Fig. 1. An arginine-rich VBM common to several ERAD proteins. (A) Left: table show- ing GST-fused peptides tested for their abil- ity to pull down VCP. Each peptide is fused to pGEX vector-encoded amino acids at both the N-terminus and C-terminus. Right: western blot (top) and SDS ⁄ PAGE (bottom) showing precipitated VCP and GST peptides. All except the OTUD7a- and SMURF1 ⁄ 2-derived peptides efficiently precipitated VCP. Lane 8 refines our earlier mapping of the VBM in Ube4b [8] to amino acids 9–16. (B) Refining the VCP-binding sequence of gp78 using similar methods. The table (left) shows N-terminal and C-terminal deletions within the GST-fused peptide used in (A), and the western blot (right) shows that neither of the two arginine-rich sequences alone is sufficient to bind VCP in this case.

Leu14 are important for contacting the binding site on VCP. Similar mutation analysis in Atx-3 and Hrd1 revealed analogous binding requirements. The equivalent arginines were required for strong binding in both proteins, and in Hrd1 a leucine equivalent to Ube4b Leu14 was also required (Fig. 2B).

used the GST pulldown assay to test VCP binding of the peptide MLAAAAERRLQRQRTT, which spans this region of homology to the Hrd1 VBM. Surpris- ingly, in view of the ability of homologous sequences in Ube4b, Hrd1 and Atx-3 to bind VCP, this region of gp78 was not sufficient for binding. Instead, we found that gp78 has a bipartite VCP-binding sequence, requiring also a slightly more N-terminal arginine-rich sequence (Fig. 1B). Both arginine-rich sequences in gp78, including the VBM-like sequence, are necessary for binding, but neither is sufficient.

Mutational analysis of VBM in Ube4b, Hrd1, Atx-3 and gp78

In gp78, although the analogous sequence is neces- sary for VCP binding (Fig. 1B), mutation of amino acids aligning with Ube4b Arg10 and Arg13 (Fig. 2A) did not disrupt VCP binding (Fig. 2B). The gp78–VCP instead, weakened by mutating the interaction was, leucine that lies between them, and more severely affected by alanine substitution at Arg626 (Fig. 2C, indicated as position b). Thus, the VBM consensus sequence RXXR is necessary for VCP binding in Ube4b, Atx-3 and Hrd1, but the influence of neigh- bouring amino acids is indicated both by the require- ment for an additional leucine in Ube4b and Hrd1 and by the lack of VCP binding in OTUD7a and SMURF1 ⁄ 2.

VBM dependence for VCP binding in intact proteins in vitro and in cells

secondary altering the

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In order to confirm a similar dependence on the VBM then interaction between intact proteins, we for repeated the VCP pulldown experiment using full- length Atx-3 and R282A Atx-3 (Fig. 3A). As in the peptide experiments, there was a clear dependence on the VBM. A VCP concentration of approximately 8 ngÆlL)1 was sufficient to be pulled down by immobi- lized wild-type Atx-3. This is significantly lower than We then refined the sequence requirements for VCP binding in the homologous motifs of Ube4b, Hrd1, Atx-3 and gp78. First, we extended our previous dele- tion analysis of Ube4b [8] to show that amino acids 9–16 are necessary and sufficient to bind VCP, whereas amino acids 1–8 were dispensable as long as other amino acids supplied by the GST vector took their place (Fig. 1A, lane 8), possibly to maintain the appro- priate secondary structure. Complete removal of amino acids 1–8 should disrupt a predicted a-helix spanning amino acids 5–17 and may therefore alter binding indi- rectly (data not shown). We then showed that alanine substitution at Arg10 or Arg13, or at Leu14, disrupts or severely weakens binding of VCP in this assay with- structure predicted out (Fig. 2A,B). Alanine substitution at other sites had no detectable effect, suggesting that Arg10, Arg13 and

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Evolutionary divergence in VCP binding during ERAD

various VBM-containing proteins

is clear it

Fig. 2. Mutational analysis of VBMs in Ube4b, Hrd1, Atx-3 and gp78. (A) Table showing alignment of the VBM-containing peptides that were sufficient to bind VCP in Fig. 1. Amino acids 9–16 of Ube4b, which were shown to bind VCP in Fig. 1, are underlined, and the motif RXXR within this sequence is aligned with an equiva- lent motif in the other three peptides, with these two arginines also underlined. Amino acids counting from the first of these arginines are assigned positions e, f, g, h, i (bottom row), and the more N-terminal arginine-rich stretch in gp78, shown to be required for binding (Fig. 1B), is assigned positions a, b and c, as shown. (B, C) Amino acids e–i in each protein (B), and a, b and c in gp78 (C), were then mutated to alanine in turn, and the capacity for VCP binding was retested and compared to that of the nonmutated pep- tide (NM). Western blots of pulldown material indicate VCP binding. Arginines at positions e and h are essential for binding in Ube4b, Hrd1 and Atx-3, but in gp78, position b is the only individually essential arginine.

concentrations of VCP in the vicinity of the ER might still drive weak binding of mutated VBM-containing proteins. Competitive or cooperative binding between the introduces another variable (see below). Thus, we tested whether VBM-dependent complexes do form in living cells. Transiently transfected FLAG-tagged wild-type Atx-3 was able to coimmunoprecipitate VCP from HeLa cells, whereas R282A Atx-3, which lacks a functional VBM, could not (Fig. 3B). It is not feasible to do this experiment with endogenous protein, as even a knockin mouse may not survive, due to ER stress, but if R282A Atx-3 does not form stable complexes with VCP even when it is overexpressed, it is even less likely to bind strongly at endogenous protein levels. We cannot rule out the possibility that weak complexes formed inside cells fall apart during the coimmuno- precipitation experiment, but that an intact VBM is required for high-affinity binding inside cells.

these two proteins

[24] makes it difficult binding by

the average VCP concentration inside a cell, indicating should also bind in vivo. that Together with our earlier report that the removal of the N-terminal, VBM-containing 16 amino acids of Ube4b blocks binding of the otherwise intact protein [8], this indicates similarities between the VCP-binding sites of intact ERAD proteins and those indicated by the peptide experiments above.

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Extrapolating these findings to physiological protein levels is complex. For example, it is possible that high We then showed that both Hrd1 and Ube4b colocal- ize with VCP in transfected cells in a VBM-dependent manner. Binding of overexpressed Hrd1 to VCP has been shown to cause both proteins to accumulate in cytoplasmic aggregates [25]. We confirmed this prop- erty in our study, but when we disrupted the VBM of Hrd1 with an R599A mutation, this aggregation no longer occurred, consistent with a model in which Arg599 is a critical mediator of VCP binding (Fig. 3C–J). Mutant Hrd1 assumed a more reticular pattern, possibly reflecting binding to other ER pro- teins. VCP can also be partially redistributed by trans- fection with the slow Wallerian degeneration protein (WldS), this time into discrete intranuclear foci [8]. Although this is not the normal distribution of VCP, these foci do provide a site for specific colocalization studies, at least for proteins such as Ube4b, which enter the nucleus. Therefore, we transfected HeLa cells with WldS to determine whether Ube4b colocalizes with VCP in these foci in a VBM-dependent manner. FLAG-tagged Ube4b colocalized in most cells, but this was never seen with the R10A mutant Ube4b (Fig. 3L–S). These experiments have some unavoidable limitations. Both rely on mislocalized VCP, and the fact that Hrd1 is a multispanning ER membrane protein that also interacts with other VCP-binding to confirm ER proteins direct coimmunoprecipitation. Thus, the coimmunoprecipitation of VCP with Atx-3 remains the best evidence for VBM-dependent binding in cells, but these data are consistent with Hrd1 and Ube4b also binding in a VBM-dependent manner inside cells.

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A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

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Q

R

Fig. 3. (A) Coomassie-stained gel showing pulldown of VCP using full-length recombi- nant Atx-3 fused to GST and a VCP concen- tration of 8 ngÆlL)1. This interaction is prevented by the mutation of Arg282. (B) Western blots showing coimmunoprecipita- tion of VCP with FLAG-tagged Atx-3 trans- fected into HeLa cells. Again, the interaction is blocked by mutation of Arg282, confirm- ing dependence on the VBM for binding in cells. (C–J) Hrd1 redistributes VCP in a VBM-dependent manner. FLAG-tagged Hrd1 (C–F) or R599A mutant Hrd1 (G–J) was tran- siently transfected into the PC12 subline TV, which expresses EGFP-tagged VCP (R599 corresponds to position e in Fig. 2A). Over- expressed wild-type Hrd1 aggregates together with VCP, reflecting a binding inter- action as previously reported [25]. In con- trast, the R599A mutant does not redistribute VCP, and itself assumes a more reticular pattern, consistent with the R599A mutant failing to bind VCP. (L–S) HeLa cells were transfected with WldS to partially redistribute VCP into intranuclear foci as pre- viously reported [8], so that these foci could be used for specific colocalization studies. Faint FLAG-tagged Ube4b signal colocalized with VCP in these foci (arrows, N), whereas the R10A mutant (R) did not [this can be better seen in Fig. S3, where parts (L)–(S) are all equally enhanced by adjusting levels in PHOTOSHOP].

identical to the Ube4b VBM, Mapping the Ube4b interaction domain in VCP

of WldS, is the only VCP-binding site in this protein (Fig. S1). We then found that GST-fused VCP1–199 was sufficient to pull down WldS, indicating that the N-terminal Ube4b- (and WldS)-binding site of VCP also resides within this region (Fig. 4A). Thus, there are differences between mammals and S. cerevisiae in the sequences mediating binding both on the Ube4b–Ufd2p side [8] and on the VCP–Cdc48p side.

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The binding sites of gp78, Atx-3, Hrd1 have all been mapped to the N-domain of VCP (amino acids 1–199) [13,25,32,33]. As the VBM of Ube4b is very similar to those of the above proteins, we hypothesized that Ube4b should also bind to the N-domain. However, this conflicts with data from S. cerevesiae, where the N-domain of Cdc48p is neither necessary nor suffi- cient, and instead the D1D2 domain binds Ufd2p [34]. Therefore, we tested directly whether the N-domain of mammalian VCP is sufficient to bind the VBM of Ube4b, here represented by WldS, a fusion protein that shares its N-terminal 70 amino acids with Ube4b [35]. First, we confirmed that the N-terminal 16 amino acids We then investigated whether the binding of each of these VBM-containing proteins to the N-domain of VCP is disrupted in disease. Several mutations within the N-domain of VCP (positions 95, 155 and 191) cause inclusion body myopathy associated with Paget dementia disease frontotemporal bone and of

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Evolutionary divergence in VCP binding during ERAD

A

B

Fig. 4. The VCP N-domain precipitates WldS independently of pathogenic mutations (A) Western blot (top) showing WldS (and prote- olytic fragments of WldS recognized by the same specific antibody) precipitated by GST- fused VCP1–199 (SDS ⁄ PAGE, bottom). (B) The same VCP fragment was able to precip- itate WldS even when pathogenic VCP mutations were incorporated (top panel and lane 1 of bottom panel) or when the highly acidic sequence at amino acids 191–196 was mutated (bottom panel). Controls shown on the top panel are valid for the top and bottom panels (different gels from the same experiment). The lane marked WldS was loaded with the input WldS. MM, marker lane.

blocking of the VBM-binding sites on VCP, we found an inverse relationship between the amount of WldS in the input and the amount of VCP that Hrd1 could pull down (Fig. 5). Half-maximal inhibition corresponded to a WldS ⁄ VCP polypeptide ratio of approximately 2.4 (approximately 72 lg of VCP and 75 lg of WldS, with molecular masses of 97 and 43 kDa respectively). Thus, Hrd1 is excluded from binding VCP by increas- ing amounts of the Ube4b-derived VBM. Precisely how closely this models protein concentrations in the vicinity of the ER is unknown, but as most of these proteins are abundant at the ER, there is likely to be significant competition between the various VBM sequences for binding the VCP N-domain. the VBM, we also tested whether

Evolutionary conservation of VBM-containing proteins (IBMPFD) [36,37]. Not only does the binding site of each VBM-containing protein map to amino acids 1– 199, but that of Atx-3 has been mapped even closer to the IBMPFD mutations at amino acids 143–199 [38]. Therefore, we tested whether disruption of VBM bind- ing could be part of the pathogenic mechanism. GST constructs fused to VCP1–199 containing the point mutations R155C, R155H, R155P, R159H, R159T and R191Q were able to pull down bacterially expressed, His-tagged WldS (Fig. 4B). Together with a previous report that mutation of Arg93 or Arg155 does not block binding of Atx-3 [39], this suggests that binding of VBM-containing proteins is unaltered by the IBMPFD mutations. In view of the highly basic nature the acidic of sequence EDEEE(192–196) of VCP is required for binding. in this VCP Individual point mutations sequence did not alter binding of WldS (Fig. 4B).

Mutually exclusive binding of VBM-containing proteins to VCP

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As discussed above, several proteins bind the N-domain of VCP in a mutually exclusive manner, including the VBM-containing proteins Atx-3 and Ube4b [13]. Knowing which proteins compete with one another for binding is an essential step towards under- standing how VCP interacts with other proteins inside a cell. Thus, having identified Hrd1 as a new VBM- containing protein (Fig. 1), and having shown that Ube4b binds the N-domain (Fig. 4), like Hrd1 [25], we then looked for competition between them for VCP binding. Preincubating VCP with WldS, which contains the N-terminal region of Ube4b [35] to allow maximal We previously reported that the VBM of Ube4b is located within an N-terminal extension that is absent in S. cerevisiae [8]. We now show that the VBMs of Hrd1 and gp78 are also missing from their common S. cere- visiae homologue, as we map them to sites that are C-terminal extensions in the mammalian proteins [23]. Ube4b also docks at a different site on VCP from where Ufd2p binds Cdc48p (Fig. 4), and Atx-3 apparently has no close S. cerevisiae homologue. These observations indicate that there is evolutionary divergence in the molecular architecture of VCP–Cdc48p-containing complexes in ERAD, despite conservation of the princi- ple of VCP–Cdc48p binding. To understand more about the evolutionary conservation of the VBM in each of these proteins, we looked for VBM-like sequences in a range of organisms (Fig. 6 and Tables S1 and S2).

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Evolutionary divergence in VCP binding during ERAD

Fig. 5. Hrd1 and WldS bind VCP in a mutually exclusive manner. (A) Fast Blue-stained SDS ⁄ PAGE gel (below) showing the effect of preincu- bating various quantities of WldS bacterial extract with a bacterial extract containing recombinant VCP for 30 min at 4 (cid:2)C before precipitating with GST–Hrd1(VBM) bound to glutathione resin. Note that other proteins present in the bacterial extracts help to block nonspecific protein interactions and binding of WldS and Hrd1 to VCP is direct [8,24]. Western blots below show VCP and WldS input. Densitometry values were zeroed for the background. Note the decreasing interaction of VCP with GST–Hrd1(VBM) when it is preincubated with more WldS. (B) Com- parative histogram of VCP bound to GST–Hrd1(VBM); 750 lL of WldS was taken to confer half-maximal inhibition of Hrd1–VCP binding, a point corresponding to a WldS ⁄ VCP polypeptide ratio of approximately 2 : 4 (see text). Data points are mean values ± standard error; n = 4. ***P < 0.0001.

Interestingly,

these interactions that mediate

and from those in mammals other from S. cerevisiae probably reflects yeasts [40]. Caenorhabditis elegans, for VCP binding indicates that

very well fact the

in contrast to our earlier report [8], the recent database submission CAC19740 indicates there is a VBM-like sequence in Ufd2p of that Schizosaccharomyces pombe (Table S2), whose func- tionality we confirmed experimentally (Fig. S2). This the difference from a major divergence of present-day common ancestor in contrast, has a putative VBM in Hrd1 but not in Ufd2p, whereas in nearly every vertebrate that we studied, there was a well-conserved putative VBM in four proteins (Table S1). Thus, VBMs in these all four ERAD proteins conserved are among vertebrates, but only sporadically present in invertebrates. vation of most of the proteins involved, and strong the similarities in the pattern of binding partners, are sequences significantly different in S. cerevisiae invertebrate in many homologues. Not only is the corresponding protein domain absent, but our characterization of essential amino acids the VBM does not appear elsewhere in these proteins. These differences in molecular structure of the VCP ERAD complexes indicate a divergence point in this basic cellular mechanism that was not evident from earlier data. that Intriguingly, however, evolution has established more than one way for these proteins to interact shows how important it is that they do so.

Discussion

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are E3 Our data indicate that molecular interactions govern- ing ERAD diverge significantly between vertebrates and many invertebrates, despite the essential nature of this cell-autonomous process. Despite good conser- We have mapped the VCP-binding activity of Hrd1 to a C-terminal VBM, refined the VBMs of gp78 and Ube4b [5,8], and confirmed a functional VBM in Atx-3 [13]. Each protein is important for ERAD. Hrd1 and gp78 ligases ubiquitinating ERAD substrates [17,23]. Haploinsufficiency for Ube4b, an E4

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Evolutionary divergence in VCP binding during ERAD

Fig. 6. Evolutionary alignment of VBM of Ube4b–Ufd2, Hrd1, Atx-3 and gp78 among several vertebrate and invertebrate species. VBM or putative VBM sequences are indicated in bold. Atx-3 alignment is not shown for some species, as they lack a homologue to this protein. For further details, see Tables S1 and S2.

The binding motif that we define in Ube4b, Hrd1 and Atx-3 is similar but not identical to the motif (L ⁄ I ⁄ V ⁄ Y)-R-(K ⁄ R ⁄ W)-(R ⁄ K ⁄ L)-R-X-X-(Y ⁄ F)-(F ⁄ K ⁄ L ⁄ Y) reported in Atx-3 [13]. We find more tolerance of alanine substitution around the essential arginines, perhaps because different methods were used for muta- tion analysis. Short synthetic peptides [13] may not preserve secondary structure as effectively as GST fusion proteins, which is important because each VBM lies within a predicted a-helix, in which the two con- served arginines would project positively charged side chains to the same face. Alternatively, dimerization of the GST fusion proteins may influence the strength of VCP binding.

Interestingly, the Hrd1, gp78 and Ube4b VBMs are located within C-terminal or N-terminal extensions missing from their S. cerevisiae homologues [8,23]. Atx-3 has no S. cerevisiae homologue. Thus, differ- ences in VCP–Cdc48 interaction between Ube4b and Ufd2p [8] can be generalized to a wider range of ERAD proteins. This provides a structural explanation for the controversy regarding whether Hrd1 binds Cdc48–VCP directly. Whereas the S. cerevisiae pro- teins interact via mutual binding partner, Ubx2 [41,42], the corresponding mammalian proteins clearly exhibit a direct interaction through a sequence that is lacking in S. cerevisiae. These data do not exclude an addi- tional, direct binding site in yeast, and Ubx domains may also play a role in coordinating ERAD machinery in mammals through initiating or strengthening these interactions [41,43,44]. Mammalian VBM-containing proteins may also be linked to VCP through mutual binding partners [24,25]. Thus, evolution has generated more than one way of recruiting VCP–Cdc48p to ubiquitin ligases, and some molecular details of ERAD may differ accordingly.

ligase, triggers ER stress and neurodegeneration in mice [10]. Atx-3 inhibits retrotranslocation, probably through deubiquitination [7]. Despite excellent conser- vation among vertebrates, each VBM is absent in S. cerevisiae, and Ube4b–Ufd2p binds to different sites on VCP–Cdc48p in mammals and S. cerevisiae.

[25].

Several proteins bind VCP in a mutually exclusive manner. These include Ufd1–Npl4 and p47 [4], Ufd1– Npl4, SVIP and p47 [6], Ufd1–Npl4 and gp78 [5], and Ufd1–Npl4 and Atx-3 [7], and there is evidence that such competition can be important in regulating ERAD [45]. We now extend this to Ube4b and Hrd1 (Fig. 5), consistent both with their homology and with their shared use of the VCP N-domain for docking (Fig. 4) Interestingly, gp78 requires both the N-domain and D1-domain [5], mirroring the bipartite VCP-binding sequence that we report. As these pro- teins bind differently to S. cerevisiae Cdc48p, differ- ences in competition for binding are one way in which the ERAD mechanism could differ.

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The sequence RXXR is almost invariant through- out these VCP-binding sequences. Human gp78 dif- in having a conservative lysine for arginine fers substitution [23], in requiring a second arginine-rich stretch for VCP binding, and in tolerating arginine to alanine mutations in the sequence RXXR. How- ever, we class gp78 as a variant VBM, as this region is still required for VCP binding (Fig. 1). Mutational analysis and comparison with other RXXR proteins indicates that neighbouring amino acids also influ- ence VCP binding. In a biological context, two models are compatible with mutually exclusive binding: ternary complex and negative cooperativity (Fig. 7). Hexameric VCP [46]

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stoichiometry of one Ufd1–Npl4 dimer per VCP hexamer supports this model [47], as does the failure or Ufd2 and Ufd3 to coimmunoprecipitate in S. cerevi- siae [34].

Fig. 7. Ternary complex (A) and negative cooperativity (B) models for binding between VCP hexamer and VBM-containing proteins. (A) In the ternary complex model, different VCP polypeptides in the com- plex are able to bind different VBM proteins simultaneously, because competition operates only at the level of each individual N-domain. Thus, VCP coordinates formation of a ternary complex in which VBM-containing protein 1 (e.g. Ube4b) and VBM-containing protein 2 (e.g. Hrd1) are brought into close proximity with one another. (B) In the negative cooperativity model, binding of one VBM-containing protein to one VCP polypeptide (left) closes off all sites in the VCP hexamer through conformational change. VCP can only bind VBM protein 2 when no other VBM protein is bound (right).

VCP extracts ubiquitinated proteins from the ER and chaperones them to the cytoplasm for protea- some-mediated degradation. The differences in molecu- lar interactions between S. cerevisiae and mammals help to explain differences in ERAD. For example, one key question is what recruits VCP to the ER. S. cerevisiae Ubx2 optimizes this process by binding both Hrd1p and Cdc48p [41,42], whereas mammalian gp78 and Hrd1 both bind VCP directly, recruiting it to the ER and influencing ERAD [17,24]. In both pro- teins, we now map VCP binding to the extreme C-ter- minus. In Hrd1, the two critical arginines are located 11 and 14 amino acids from the C-terminus. Interest- ingly, Atx-3 has an opposite effect on retrotransloca- tion, inhibiting it in a VCP-binding dependent manner [7]. Thus, competition for a VCP-binding site between Atx-3 and Hrd1–gp78 could regulate the retrotranslo- cation process.

The VBM joins a growing list of VCP-binding sequences [2]. The Ubx domain of p47 [48] also occurs in many other proteins [2,43,44,49,50], and Ufd1–Npl4 binds similarly, despite lacking homology [51]. The PUB domain [52] is structurally different from VBM and Ubx, and, unlike both, binds C-terminally in VCP [53]. Interestingly, PUB domains are often found in higher eukaryotes but are also absent in S. cerevisiae, similarly to the VBM [52]. Finally, Ufd2p binds Cdc48 directly, despite lacking the VBM of its mammalian homologue Ube4b, so an alternative binding sequence exists [11,12].

Intriguingly, although S. cerevisiae Cdc48p does not use a VBM to bind the corresponding Ufd2p, Cdc48p can still bind the mammalian VBM (Fig. S2). Thus, there is an evolutionary pressure to maintain the VBM-binding site in S. cerevisiae Cdc48p that may come from other, as yet unidentified, binding partners. The VBM of Ube4b is shared with WldS, a mutant, chimeric protein that uniquely delays axon degenera- tion and is a fusion of Ube4b sequence with the NAD+-synthesizing enzyme Nmnat1 [35,54]. Ube4b sequences are required for the full phenotype [55], pos- sibly by competing for VCP binding with wild-type Ube4b. Our data show that competition with other VBM-containing proteins is another possibility. In summary, sites

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could assume a central, organizing role in a ternary complex where different VCP polypeptides bind differ- ent cofactors. Cofactors compete for each individual site, but the six VCP subunits bring together various ERAD proteins. Substrates ubiquitinated by E3 ligases Hrd1 and gp78 could be passed efficiently to a nearby E4 (Ube4b) for further ubiquitination [9,11], and indi- rect binding to more ligases via Ubx domain proteins provides even more scope for the coordination of ubiq- uitination in this way [43,44]. VCP interacts with Hrd1, derlin and VIMP in a ternary complex, although multiple pairwise interactions complicate the analysis [24,25], and gp78 and PNGase also bind VCP as a ter- nary complex through their different binding sites [33]. In the negative cooperativity model, cofactor binding to a single N-domain closes off all in the including unoccupied sites. This allows a hexamer, single cofactor molecule to determine the role of the VCP complex, contributing to functional diversity. The the strong conservation of VBMs among four mammalian proteins involved in ERAD strikingly with the complete absence in contrasts S. cerevisiae homologues, and poor conservation in other invertebrates. The docking site on VCP also

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Binding assays

GST fusion proteins were purified and coupled to glutathi- one–Sepharose 4B according to the manufacturer’s protocol (GE Healthcare, Little Chalfont, UK). For further affinity experiments, these purified proteins were mixed with various amounts of bacterial protein extracts in a 1.5 mL tube at 4 (cid:2)C, and unbound protein was washed out using NaCl ⁄ Pi plus 0.01% Triton X-100. The glutathione–Sepharose 4B beads were analysed by SDS ⁄ PAGE, the gel was dried and, when radioactive recombinant VCP was used, it was exposed directly to autoradiography film overnight.

differs for at least one of these proteins. These differ- ences in the molecular architecture of VCP–Cdc48p complexes indicate divergence in ERAD mechanisms that is not apparent from previous data. Differences are likely in how proteins compete to bind VCP and in the relative orientation of proteins and their key func- tional domains within the complex. Future studies now need to address the extent of these structural differ- ences, the consequences for the mechanism, and when and why key steps in the evolution of ERAD took place.

Experimental procedures

Coimmunoprecipitation

for

used

search

blastp 2.2.10 was the motif to EIRRRRLARLA, using a local mouse database. The sig- nificance cutoff was set at 1000 to allow for the shortness of the search string. In the absence of a protein sequence, the existence of a homologous gene was inferred from ensembl where possible (Table S1). clustal-w [56] was used for multiple sequence alignment of selected proteins.

Bioinformatics methods

Plasmid constructs were prepared using standard recombi- nant techniques [57]. All VBM motif sequences tested were derived from murine sequences and cloned into pGEX5T1 via EcoRI–XhoI. WldS and R10A WldS were cloned into pET28a via BamHI–HindIII. Flagged Ube4b, flagged R10A Ube4b, flagged Hrd1 and flagged R599A Hrd1 were cloned into pHbApr-1 via HindIII–BamHI. A list of templates, primers and plasmids used for this work is available in Table S3.

Constructs

Flagged Atx-3 and flagged R282A Atx-3 expression vectors were generated using standard cloning procedures, and veri- fied by restriction enzyme analysis and DNA sequencing. The coding regions of Atx-3 and R282A Atx-3 were PCR- amplified using primers harbouring appropriate restriction enzyme sites and FLAG-expressing sequences, with Pfu Polymerase (Promega Ltd.), and ligated into pCDNA3.1 (Invitrogen, Paisley, UK). HeLa cells were transfected with either flagged Atx-3 or flagged R282A Atx-3 expression vectors. After 24 h, cells were washed with NaCl ⁄ Pi, and harvested by adding lysis buffer [20 mm Tris, pH 7.5, 137 mm NaCl, 1 mm EGTA, 1% (v ⁄ v) Triton X-100, 10% (v ⁄ v) glycerol, and 1.5 mm MgCl2, supplemented with Complete Mini protease inhibitor cocktail tablets (Roche Diagnostics, Lewes, UK) and scraping cells after 20 min of incubation on ice. Lysates were subsequently collected and cleared by centrifugation by centrifugation for 30 min at 14 000 g at 4 (cid:2)C. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Hemel Hempstead, UK). FLAG-tagged proteins were immunoprecipitated from equal amounts of total protein by incubating with EZview Red ANTI-FLAG M2 affinity gel (Sigma-Aldrich Ltd., Gillingham, UK) for 2 h at 4 (cid:2)C. The beads were washed three times with lysis buffer, and analysed by SDS ⁄ PAGE followed by western blotting using VCP antibody (BD Biosciences, Oxford, UK) and monoclonal antibody against FLAG (M2) (Sigma-Aldrich Ltd.).

Expression of GST fusion proteins and other recombinant proteins

Transformed Escherichia coli BL21 cells were cultured in liquid LB medium (pGEX vectors with 50 lgÆmL)1 ampicil- lin and pET vectors with 25 lgÆmL)1 kanamycin) at 37 (cid:2)C to D600 nm = 1. Expression was induced by addition of 1 mm isopropyl-thio-b-d-galactoside and further shaking at 30 (cid:2)C for 12 h.

Western blotting

Proteins were separated by SDS ⁄ PAGE and semidry blot- ted onto nitrocellulose (Bio-Rad). Blocking, washing and incubation with primary antibodies and suitable horserad- [either ish peroxidase-conjugated secondary antibodies sheep anti-(mouse IgG) (1 : 3000; GE Healthcare Ltd.) or anti-rabbit IgG (1 : 3000; GE Healthcare Ltd.) were per- formed in NaCl ⁄ Pi plus 0.02% Tween-20 and 5% low-fat milk. Proteins were visualized using the ECL detection kit (GE Healthcare Ltd.) according to the manufacturer’s instructions.

Radioactive recombinant VCP was produced using the pGBKT7 construct [8]. pGBKT7 was in vitro transcribed incorporating [35S]methionine, using the and translated, TNT T7 Reticulocyte Lysate Coupled Transcription ⁄ Translation kit from Promega (Promega Ltd, Southampton, UK).

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In vitro transcription and translation

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containing protein (p97) is a regulator of endoplasmic reticulum stress and of the degradation of N-end rule and ubiquitin-fusion degradation pathway substrates in mammalian cells. Mol Biol Cell 17, 4606–4618.

2 Dreveny I, Pye VE, Beuron F, Briggs LC, Isaacson RL,

Plasmid DNA was isolated using the endonuclease-free plasmid kit (Qiagen, Crawley, UK). DNA was transfected into ‘TV’ PC12 using LipofectAMINE2000 (Invitrogen) cells immediately prior to differentiation by culturing in 100 ngÆlL)1 nerve growth factor on a type IV collagen substrate (Sigma-Aldrich Ltd.). The ‘TV’ PC12 subline is inducible C-terminal stably transfected with a tet-off enhanced green fluorescent protein (EGFP)-tagged VCP construct [58], and was grown in 1.0 lgÆmL)1 doxycycline (Sigma-Aldrich Ltd.), which was removed to induce VCP–EGFP expression. Protein location was observed 1–5 days after transfection.

Matthews SJ, McKeown C, Yuan X, Zhang X & Freemont PS (2004) p97 and close encounters of every kind: a brief review. Biochem Soc Trans 32, 715–720. 3 Kondo H, Rabouille C, Newman R, Levine TP, Pappin D, Freemont P & Warren G (1997) p47 is a cofactor for p97-mediated membrane fusion. Nature 388, 75–78. 4 Meyer HH, Shorter JG, Seemann J, Pappin D & War- ren G (2000) A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J 19, 2181–2192.

Cell culture and transfection

5 Ballar P, Shen Y, Yang H & Fang S (2006) The role of a novel p97 ⁄ valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degrada- tion. J Biol Chem 281, 35359–35368.

6 Nagahama M, Suzuki M, Hamada Y, Hatsuzawa K, Tani K, Yamamoto A & Tagaya M (2003) SVIP is a novel VCP ⁄ p97-interacting protein whose expression causes cell vacuolation. Mol Biol Cell 14, 262–273.

7 Zhong X & Pittman RN (2006) Ataxin-3 binds

VCP ⁄ p97 and regulates retrotranslocation of ERAD substrates. Hum Mol Genet 15, 2409–2420.

Cultured cells were fixed for 30 min in 4% paraformalde- hyde, permeabilized with Triton X-100 (0.1%, 5 min), blocked with 5% normal goat serum (Sigma-Aldrich Ltd.), and incubated with M2 antibody (Sigma-Aldrich Ltd.) and secondary antibody AlexaFluor568-conjugated anti-mouse IgG (Invitrogen; 1 : 200), with multiple washes in NaCl ⁄ Pi between each stage. Slides were mounted in Vectashield plus 4¢,6-diamidino-2-phenylindole (DAPI) (Vector Labora- tories Ltd, Peterborough, UK), and images were taken on a Zeiss LSM 510 META confocal system (LSM Software Release 3.2) coupled to a Zeiss Axiovert 200 microscope.

Immunocytochemistry

8 Laser H, Conforti L, Morreale G, Mack TG, Heyer M, Haley JE, Wishart TM, Beirowski B, Walker SA, Haase G et al. (2006) The slow Wallerian degeneration protein, WldS, binds directly to VCP ⁄ p97 and partially redistributes it within the nucleus. Mol Biol Cell 17, 1075–1084.

9 Hatakeyama S, Yada M, Matsumoto M, Ishida N & Nakayama KI (2001) U-Box proteins as a new family of ubiquitin-protein ligases. J Biol Chem 276, 32111– 33120.

10 Kaneko-Oshikawa C, Nakagawa T, Yamada M,

A Bio-Rad gel scanner (Gel Doc 2000) and densitometer with image j (NIH, Bethesda, MD, USA) was utilized to quantify the protein band intensity of stained SDS ⁄ PAGE gels. spss 15.0 software (SPSS Inc., Chicago, IL, USA) was used to analyse intensity measurements and calculate means and standard errors. Data were statistically evaluated by a univariate anova method. P < 0.05 was considered to be statistically significant.

Densitometry and statistical analysis

Acknowledgements

Yoshikawa H, Matsumoto M, Yada M, Hatakeyama S, Nakayama K & Nakayama KI (2005) Mammalian E4 is required for cardiac development and maintenance of the nervous system. Mol Cell Biol 25, 10953–10964. 11 Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU & Jentsch S (1999) A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644.

12 Richly H, Rape M, Braun S, Rumpf S, Hoege C &

Jentsch S (2005) A series of ubiquitin binding factors connects CDC48 ⁄ p97 to substrate multiubiquitylation and proteasomal targeting. Cell 120, 73–84. 13 Boeddrich A, Bo¨ ddrich A, Gaumer S, Haacke A,

We thank A. Segonds-Pichon for statistical advice, C. Wiggins and B. Gilley for experimental advice, A. Kakizuka (Kyoto University) for the ‘TV’ PC12 cell line, J. Mullally (Emory University) for the Cdc48- expressing plasmid, and T. Sommer (Max Delbru¨ ck Centre, Berlin) for antibody against cdc48. This work was funded by the BBSRC.

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list of primers and templates used in The following supplementary material is available: Fig. S1. SDS ⁄ PAGE showing pulldowns from wild- type mouse brain homogenate. Fig. S2. Western blotting for Cdc48 and VCP after pulldown of recombinant proteins and proteins from HeLa cell extract. Fig. S3. Parts (L)–(S) of Fig. 3 to demonstrate Ube4b intrunuclear puncta more clearly. Table S1. Details of the VBM or putative VBMs in ERAD proteins in a range of vertebrates. Table S2. Details of the VBM or putative VBMs in ERAD proteins in a range of invertebrates. Table S3. Full construct generation. This supplementary material can be found in the online version of this article.

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