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

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:
Centro di Ricerca per la Viticoltura, Via
Casoni, 13/A, 31058 Susegana (TV), Italy
Fax: +39-0438-738058
Tel: +39-0438-73264
E-mail:

(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
M
r
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, 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-

itin protease activity [14,15], and in the ER-resident
ubiquitin ligase M
r
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].
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 the cofactors that direct Cdc48p to these
functions [3,21]. These functions and binding part-
ners are well conserved in mammals, consistent with
the fundamental importance of 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 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.
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

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
35
S-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.
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

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

(Fig. 2A,B). Alanine substitution at other sites had no
detectable effect, suggesting that Arg10, Arg13 and
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).
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
interaction was, instead, weakened by mutating the
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
In order to confirm a similar dependence on the VBM
for interaction between intact proteins, we then
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

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

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.
Extrapolating these findings to physiological protein
levels is complex. For example, it is possible that high
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 various VBM-containing proteins 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 it is clear that an
intact VBM is required for high-affinity binding inside
cells.
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
(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

cells, but these data are consistent with Hrd1 and
Ube4b also binding in a VBM-dependent manner
inside cells.
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.
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 1211
Mapping the Ube4b interaction domain in VCP
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 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

CDEF
GH I J
KL MN
OP QR
B
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 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

IBMPFD mutations. In view of the highly basic nature
of the VBM, we also tested whether the acidic
sequence EDEEE(192–196) of VCP is required for
binding. Individual point mutations in this VCP
sequence did not alter binding of Wld
S
(Fig. 4B).
Mutually exclusive binding of VBM-containing
proteins to VCP
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 Wld
S
, which contains
the N-terminal region of Ube4b [35] to allow maximal
blocking of the VBM-binding sites on VCP, we found
an inverse relationship between the amount of Wld
S
in
the input and the amount of VCP that Hrd1 could pull
down (Fig. 5). Half-maximal inhibition corresponded
to a Wld

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

range of organisms (Fig. 6 and Tables S1 and S2).
AB
Fig. 4. The VCP N-domain precipitates Wld
S
independently of pathogenic mutations (A)
Western blot (top) showing Wld
S
(and prote-
olytic fragments of Wld
S
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 Wld
S
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 Wld
S
was loaded with the input Wld
S
. MM,
marker lane.
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 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.

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. Intriguingly, however, the fact that
evolution has established more than one way for
these proteins to interact shows how important it is
that they do so.
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 are E3 ligases ubiquitinating ERAD
substrates [17,23]. Haploinsufficiency for Ube4b, an E4
Fig. 5. Hrd1 and Wld
S
bind VCP in a mutually exclusive manner. (A) Fast Blue-stained SDS ⁄ PAGE gel (below) showing the effect of preincu-
bating various quantities of Wld
S
bacterial extract with a bacterial extract containing recombinant VCP for 30 min at 4 ° 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 Wld
S
and Hrd1 to VCP is direct [8,24]. Western blots below show VCP and Wld
S
input. Densitometry values were
zeroed for the background. Note the decreasing interaction of VCP with GST–Hrd1(VBM) when it is preincubated with more Wld
S
. (B) Com-
parative histogram of VCP bound to GST–Hrd1(VBM); 750 lL of Wld
S

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-

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.

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

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 sites in the
hexamer, including unoccupied sites. This allows a
single cofactor molecule to determine the role of the
VCP complex, contributing to functional diversity. The
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].
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 Wld
S
, 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, the strong conservation of VBMs
among four mammalian proteins involved in ERAD
contrasts strikingly with the complete absence in
S. cerevisiae homologues, and poor conservation in
other invertebrates. The docking site on VCP also
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).
Evolutionary divergence in VCP binding during ERAD G. Morreale et al.
1216 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS
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
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

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 °C
to D
600 nm
= 1. Expression was induced by addition of
1mm isopropyl-thio-b-d-galactoside and further shaking at
30 °C for 12 h.
In vitro transcription and translation
Radioactive recombinant VCP was produced using the
pGBKT7 construct [8]. pGBKT7 was in vitro transcribed
and translated, incorporating [
35
S]methionine, using the
TNT T7 Reticulocyte Lysate Coupled Transcription ⁄
Translation kit from Promega (Promega Ltd, Southampton,
UK).
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 °C, and unbound protein was washed out using NaCl ⁄ P
i
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.
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

Ltd., Gillingham, UK) for 2 h at 4 °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.).
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-
ish peroxidase-conjugated secondary antibodies [either
sheep anti-(mouse IgG) (1 : 3000; GE Healthcare Ltd.) or
anti-rabbit IgG (1 : 3000; GE Healthcare Ltd.) were per-
formed in NaCl ⁄ P
i
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.
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 1217
Cell culture and transfection
Plasmid DNA was isolated using the endonuclease-free
plasmid kit (Qiagen, Crawley, UK). DNA was transfected
using LipofectAMINE2000 (Invitrogen) into ‘TV’ PC12
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
stably transfected with a tet-off inducible C-terminal

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.
Acknowledgements

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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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.

Table S2. Details of the VBM or putative VBMs in
ERAD proteins in a range of invertebrates.
Table S3. Full list of primers and templates used in
construct generation.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Evolutionary divergence in VCP binding during ERAD G. Morreale et al.
1220 FEBS Journal 276 (2009) 1208–1220 ª 2009 The Authors Journal compilation ª 2009 FEBS