NEWS AND VIEWS
Navigating the ERAD interaction network
Thibault Mayor
The endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, which orchestrates the degradation of ER
proteins by the proteasome, involves a plethora of proteins with diverse functions. Using a combination of proteomic and genetic
approaches, a recent study provides fresh insights into the organization of the mammalian ERAD interaction network and the
functions of its components.
Proteins targeted to the ER during translation have it tough. It is not uncommon for the
folding process to go awry; if left unchecked,
it can have profound implications on cellular
homeostasis. Indeed, several human diseases
are at least partially caused by malfunction of
the cellular machinery responsible for removing these misfolded proteins. Fortunately, our
cells normally have a robust mechanism for
dealing with this. ERAD is the major pathway
for ridding the cell of misfolded ER proteins,
and functions by targeting them to the 26S proteasome for degradation.
A central feature of ERAD is the coordination
of processes that occur in two separate cellular
compartments. Recognition and tagging of substrates occurs in the ER, whereas components
of the ubiquitin-proteasome system are located
in the cytosol. To accomplish such coordination, ERAD proteins are organized into distinct
functional modules that mediate recognition of
ERAD substrates, membrane dislocation and
extraction, ubiquitylation, and finally delivery
to the proteasome1. In particular, exactly how
the substrates are retrotranslocated from the ER
membrane remains elusive. Uncovering novel
ERAD components will help to further define
mechanisms that choreograph this complex
process. On page 93 of this issue, Christianson
et al. have used a combinatorial approach to
assemble an extensive protein interaction network map of the mammalian ERAD, revealing
new components and the preferred proteolytic
‘routes’ of several model substrates2.
Thibault Mayor is at the Department of Biochemistry
and Molecular Biology, Centre for High-Throughput
Biology, University of British Columbia, Vancouver,
V6T 1Z4 British Columbia, Canada
e-mail: [email protected]
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A key step in the ERAD pathway is substrate
recognition; this involves distinguishing bona
fide aberrant proteins that should be degraded
from those that are not fully folded but still
on the correct folding pathway. To complicate matters further, the many different ER
proteins are folded and processed at highly
variable rates. Failure to properly distinguish
between transiently and terminally misfolded
proteins can have dramatic consequences.
For instance, in cystic fibrosis, the misfolding
of a small region of the mutated cystic fibrosis transmembrane conductance regulator
(CFTR) protein leads to the ERAD-mediated
degradation of an otherwise functional ionchannel transporter 3.
Earlier work, primarily in budding yeast,
showed that at least one level of substrate
recognition was achieved by the ubiquitin
ligases that participate in ERAD. For instance,
yeast proteins containing intra-membrane or
ER-luminal lesions (ERAD-M and ERAD-L,
respectively) are targeted by the Hrd1 ubiquitin ligase, whereas ER substrates with misfolded cytosolic domains (ERAD-C) are
targeted by the Doa10 ligase4,5. In mammalian
cells, as many as 16 ubiquitin ligases are so far
implicated in ERAD, including Hrd1, gp78
(also called AMFR) and TEB4 (orthologous
to Doa10)6. A major challenge is to delineate
further how metazoan cells have expanded the
ERAD network to adapt to increasing cellular
complexity.
To address this issue, Christianson et al.
first performed a series of mass spectrometry
analyses of proteins that co-precipitated with
25 baits corresponding to different ERAD
components in human tissue culture cells.
They identified over 170 non-redundant
high-confidence interacting proteins, including 71 proteins not previously associated with
ERAD. Encouragingly, many of these potential
ERAD genes were upregulated by the unfolded
protein response, further suggesting a role in
protein quality control. Other interesting discoveries include: FAM8A1, which has been
identified as a potential regulator of Hrd1;
UBAC2, a ubiquitin-binding protein associated with gp78; and the uncharacterized
ER-membrane complex (EMC), which may be
involved in membrane dislocation. Although
the authors integrated many ERAD components into their workflow, several known effectors, including ubiquitin ligases such as TEB4,
were not identified. One potential explanation
is that in HEK293S cells (the cell type used for
this analysis) Hrd1 and gp78 are the two main
ubiquitin ligases that participate in ERAD.
Additional studies will no doubt complete this
first draft of the mammalian ERAD interaction map and determine whether other components exist in specific tissues, or at much
lower levels.
Using hierarchical clustering to regroup
proteins in the same complex or sub-complex,
the authors arranged the interaction network
around the Hrd1 and gp78 ubiquitin ligases.
Unsurprisingly, components of a given ERAD
module are topologically closely related. The
module that mediates substrate recognition
of ER luminal proteins (including OS-9 and
XTP3-B) was uniquely connected to Hrd1
and its established cofactor SEL1L. However,
other functional modules are highly interconnected, indicating that there are no clear ‘barriers’ separating these functional entities. For
instance, both the Hrd1 and gp78 modules
are connected to the same group of proteins
NATURE CELL BIOLOGY VOLUME 14 | NUMBER 1 | JANUARY 2012
© 2012 Macmillan Publishers Limited. All rights reserved
NEWS AND VIEWS
a
RNAi & Co-IP
Raw network
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Refined network
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A
A
?
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C
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B
C
B
B
RNAi
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B
RNAi
c
b
Sub-modular complexes
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Substrate-specific complexes
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Figure 1 Mapping the ERAD interaction network. (a) Schematic representation of the approach taken
to simplify the ERAD interaction network. Co-immunoprecipitation experiments combined with RNAi
help to distinguish the components of a protein complex that bind directly to the assessed protein
(green). (b) Schematic representation of sub-modular complexes. Only specific subunits (green) of
a given multi-protein complex are required for the degradation of a particular substrate (indicated
by light or dark grey), while the integrity of the complex is intact. (c) Schematic representation of
substrate-specific complexes. In this case, subunits make up different protein complexes that process
distinct substrates (indicated by light or dark grey).
that are involved in substrate dislocation and
extraction (for example, Derlin2 and UBDX8).
This may indicate that a similar mechanism
is used to retrotranslocate substrates of both
ubiquitin ligases across the ER membrane.
Small variations in specific interacting partners may allow optimization of the process for
different subsets of targets.
One inherent hurdle for parsing protein
interaction networks is their complexity,
which is exacerbated by proteomic analysis
that often leads to high interconnectivity
between components of a protein complex
and their interacting partners. So does each
connection between nodes represent direct
physical interaction between those proteins,
or is the interaction an indirect consequence
of the co-immunoprecitation? The authors
found an elegant solution to this problem by
using RNA interference (RNAi) analysis with
co-precipitation experiments and applying it
to the ERAD interaction network (Fig. 1a). By
doing so, they independently validated their
initial observations and simplified the interaction map by removing indirect interactions.
More specifically, they determined whether
co-precipitation was dependent (or not) on
closely interconnected proteins that were
downregulated by RNAi. Thus, the authors
could clarify the relationships between several components in the ERAD interaction
map. The outcome is a simplified network
that is almost as accessible as any metro map
of a large city.
Another major challenge is to define the
functional relevance of particular nodes in
the ERAD interaction network. To begin
addressing this issue, the authors targeted
specific ERAD proteins by RNAi and assessed
the impact on the degradation of five model
substrates. In this experiment, accumulation
of a green fluorescent protein (GFP)-fused
substrate in the absence of a specific ERAD
component suggests the assessed protein is
required for degradation of the substratec.
Effectively, this assay confirmed that many
identified components are involved in the
ERAD pathway. After assessing the outcome
for over 50 ERAD components, the authors
overlayed the results on the interaction map to
highlight which ‘routes’ are used by each individual substrate. One should remain cautious
NATURE CELL BIOLOGY VOLUME 14 | NUMBER 1 | JANUARY 2012
© 2012 Macmillan Publishers Limited. All rights reserved
about the interpretation of these results, as
only a few substrates were assessed and there
were possibly off-target effects. Nonetheless,
the data confirms previous work showing
that the mammalian Hrd1–SEL1L module
targets soluble ERAD-L, but not necessarily
ERAD-M, substrates7. Additionally, degradation of the two non-glycosylated luminal substrates is more reliant on HERP (ref. 8) and
additional components (for example, FAM8A1
and VCIP135) that may also be required for
their effective processing.
Intriguingly, in several cases, there was no
correlation between the requirement of a particular ERAD component for the degradation
of a model substrate and its direct neighbour
in the interaction network, even for members
of the same functional module. For instance,
downregulation of UBAC2 (associated with the
gp78 ubiquitin ligase) displays a substrate pattern similar to what is seen following deletion
of Hrd1, but not gp78. One possibility is that
within a protein complex, only specific components (that is, a sub-module) may be required
for the degradation of a particular substrate
or for regulating a different ERAD complex
(Fig. 1b). In this case, the integrity of the various
proteins complexes is maintained. Alternatively,
plasticity in the interaction network may allow
the formation of a specific protein complex for
the processing of a given substrate (Fig. 1c).
This may be the case for proteins associated
with the p97 AAA+ ATPase, which are involved
in the extraction of substrates from the ER
membrane. Future studies will be required to
determine the nature of this adaptive network
and which other proteolytic routes are followed
by additional ERAD substrates.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
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