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
Valosin-containing protein (VCP ⁄ p97) is an AAA-
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
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: [email protected]
Centro di Ricerca per la Viticoltura, Via
Casoni, 13/A, 31058 Susegana (TV), Italy
Fax: +39-0438-738058
Tel: +39-0438-73264
E-mail: [email protected]

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, M
r
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;
Wld
S
, slow Wallerian degeneration protein.
1208 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 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-

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.
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
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

G. Morreale et al. Evolutionary divergence in VCP binding during ERAD
FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS 1209
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
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-
out altering the predicted secondary structure

8ngÆlL
)1
was sufficient to be pulled down by immobi-
lized wild-type Atx-3. This is significantly lower than
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.
Evolutionary divergence in VCP binding during ERAD G. Morreale et al.
1210 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS
the average VCP concentration inside a cell, indicating
that these two proteins should also bind in vivo.
Together with our earlier report that the removal of
the N-terminal, VBM-containing 16 amino acids of

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
(Wld
S
), 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 Wld
S
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
ER proteins [24] makes it difficult to confirm
direct binding by coimmunoprecipitation. Thus,
the coimmunoprecipitation of VCP with Atx-3 remains
the best evidence for VBM-dependent binding in

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 Wld
S
, a fusion protein that
shares its N-terminal 70 amino acids with Ube4b [35].
First, we confirmed that the N-terminal 16 amino acids
of Wld
S
, identical to the Ube4b VBM, is the only
VCP-binding site in this protein (Fig. S1). We then
found that GST-fused VCP1–199 was sufficient to pull
down Wld
S
, indicating that the N-terminal Ube4b-
(and Wld
S
)-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.
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
disease of bone and frontotemporal dementia
A

were transfected with Wld
S
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].
Evolutionary divergence in VCP binding during ERAD G. Morreale et al.
1212 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS
(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 Wld
S
(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

S
⁄ VCP polypeptide ratio of approximately 2.4
(approximately 72 lg of VCP and 75 lg of Wld
S
, 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.
Evolutionary conservation of VBM-containing
proteins
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

FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS 1213
Interestingly, in contrast to our earlier report [8],
the recent database submission CAC19740 indicates
that there is a VBM-like sequence in Ufd2p of
Schizosaccharomyces pombe (Table S2), whose func-
tionality we confirmed experimentally (Fig. S2). This
difference from S. cerevisiae probably reflects the
major divergence of present-day yeasts from a
common ancestor [40]. Caenorhabditis elegans,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
all four proteins (Table S1). Thus, VBMs in these
four ERAD proteins are very well conserved
among vertebrates, but only sporadically present in
invertebrates.
Discussion
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-
vation of most of the proteins involved, and strong
similarities in the pattern of binding partners, the
sequences that mediate these interactions are
significantly different from those in mammals in
S. cerevisiae and in many other invertebrate
homologues. Not only is the corresponding protein
domain absent, but our characterization of essential
amino acids for VCP binding indicates that the
VBM does not appear elsewhere in these proteins.

was taken to confer half-maximal inhibition of Hrd1–VCP binding, a
point corresponding to a Wld
S
⁄ VCP polypeptide ratio of approximately 2 : 4 (see text). Data points are mean values ± standard error; n =4.
***P < 0.0001.
Evolutionary divergence in VCP binding during ERAD G. Morreale et al.
1214 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS
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.
The sequence RXXR is almost invariant through-
out these VCP-binding sequences. Human gp78 dif-
fers in having a conservative lysine for arginine
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.
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-

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) [25]. 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.
In a biological context, two models are compatible
with mutually exclusive binding: ternary complex and
negative cooperativity (Fig. 7). Hexameric VCP [46]
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.
G. Morreale et al. Evolutionary divergence in VCP binding during ERAD
FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS 1215
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

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 Wld
S
, a mutant,
chimeric protein that uniquely delays axon degenera-
tion and is a fusion of Ube4b sequence with the
NAD
+

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
Bioinformatics methods
blastp 2.2.10 was used to search for the motif
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.
Constructs
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. Wld
S
and R10A Wld
S
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.
Expression of GST fusion proteins and other
recombinant proteins

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.
Coimmunoprecipitation
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 ⁄ P
i
, 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 MgCl
2
, 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 °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

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.
Immunocytochemistry
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 ⁄ P
i
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.
Densitometry and statistical analysis
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.
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Supporting information
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.