2011 TA Insertion Into ER
2011 TA Insertion Into ER
2011 TA Insertion Into ER
Chaperones All biological membranes contain a structurally diverse compartments of the secretory and endocytic pathways,
A large group of proteins that assortment of integral membrane proteins, which col and both nuclear envelope membranes9.
facilitate the folding, assembly, lectively constitute ~30% of the cellular proteome 1,2. Insertion into the ER membrane can occur either
transport and degradation of These proteins impart essential functionality to the lipid co-translationally or post-translationally, each of which
non-native polypeptides by
minimizing inappropriate
bilayer to allow a range of cellular activities, including offers distinct advantages10,11. In the co-translational
interactions. transmembrane communication, transport and mem pathway, all of the steps from initial protein recognition
brane morphogenesis. The selective and asymmetric to final insertion into the membrane occur during pro
insertion of membrane proteins is therefore an evolu tein synthesis. By contrast, targeting and insertion via
tionarily ancient problem that was solved by the earliest post-translational pathways occur after complete syn
life forms. thesis of the membrane protein substrate. Thus, the ribo
The shared feature of all integral membrane pro some is a major functional component during all steps
teins is the highly hydrophobic transmembrane domain of co-translational insertion, whereas its role in post-
(TMD), which in the final structure resides within the translational pathways is limited to the very earliest steps.
lipid bilayer 3. Thus, a critical obstacle in membrane Although the co-translational pathway was discovered
protein insertion is the movement of these TMDs from over 30 years ago12–14 and has been extensively studied
the aqueous cytosol, where they are synthesized, into the in many systems15–19, the post-translational insertion
lipid bilayer, where they are energetically most stable4. pathway has only recently come into focus. Similarly to
*Medical Research Council This process necessitates selective TMD recognition, the post-translational translocation of soluble proteins
(MRC) Laboratory of shielding of the TMD from the aqueous cytosol, target into various organelles20–24, the basic paradigm of post-
Molecular Biology,
Hills Road, Cambridge,
ing to the membrane surface and integration of the translational ER membrane protein insertion involves
CB2 0QH, UK. TMD into the lipid bilayer in the correct orientation. cytosolic chaperones (mediating recognition and shield
‡
Department of Biochemistry All membrane protein insertion pathways must solve ing) that interact with a specific ER‑localized receptor
and Molecular Biology, these four problems, each of which typically involves (mediating targeting and insertion). Here, we review
The University of Chicago,
specialized and highly regulated factors in the cytosol this post-translational pathway and discuss how the
Gordon Center for Integrative
Science, Room W238, and target membrane. problems of recognition, shielding, targeting and inser
Chicago, Illinois 60637, USA. In eukaryotes, membrane proteins synthesized tion are solved by its machinery. As we outline, there is
e-mails: on cytosolic ribosomes can be targeted to mitochon now sufficient information about each step to provide a
[email protected]; dria5, peroxisomes6, chloroplasts (in plants)7 and the plausible mechanistic framework for the whole pathway.
[email protected]
doi:10.1038/nrm3226
endoplasmic reticulum (ER)8. Among these, the ER These insights are starting to reveal common themes
Published online accommodates the largest number of proteins, encom that are likely to apply to all membrane protein insertion
16 November 2011 passing all membrane proteins of the plasma membrane, processes.
2CTVKCNN[ENQUGF
1RGP
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/I
s#&2
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/GODTCPG
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2
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αJGNKECN GXGPVU
Figure 2 | Nucleotide-dependent conformational changes in the Get3 homodimer. Each Get3 monomer comprises
two distinct regions: an α‑helical subdomain and an ATPase subdomain. In the presence0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
of ATP, the Get3 helical subdomains
become intimately associated, forming an extended composite hydrophobic groove (FIG. 3) that recognizes and binds to
the transmembrane domain (TMD) of a tail-anchored (TA) substrate. ATP hydrolysis, which occurs at some stage before the
release of the TA substrate at the endoplasmic reticulum (ER) membrane, produces an ADP-bound Get3 homodimer that is
partially closed. Following the release of the TA substrate and ADP, Get3 shifts back to an open conformation. Subsequently,
ATP binding allows recycling of Get3 back to the cytosol in a closed conformation. The insets show crystal structures of the
fungal Get3 homodimer in the nucleotide-free state (Protein Databank (PDB) ID 2WOO), the Mg2+–ADP-bound state
(PDB ID 3IQX) and the Mg2+–ADP–AlF4–-bound state (which seems to mimic the ATP-bound state; PDB ID 2WOJ).
Near-simultaneous reports of Get3 crystal struc recognition element in Ffh (the bacterial SRP54 homo
tures from multiple fungal species and in multiple states logue) is constructed from an α‑helical protein scaf
revealed that Get3 was a symmetric homodimer 69–73. fold that presents a large hydrophobic surface area for
Each Get3 monomer comprises a core ATPase domain substrate binding 74 (FIG. 3). Moreover, these scaffolds are
decorated with an α‑helical domain. The arrange highly dynamic. As two crystal structures of the SRP54–
ment of Get3 subunits depends on the nucleotide state, signal peptide complexes show, this flexibility can be
transitioning from a fully open state in the absence of leveraged to accommodate targeting signals of differ
nucleotide to a fully closed conformation in the presence ent lengths and sequence75,76. A similar mechanism is
of Mg 2+–ADP–AlF4– (which seems to mimic the ATP- probably at play in the case of Get3, although this awaits
bound state) and a partially closed state in the presence further structural analysis of Get3–TMD complexes.
of Mg 2+–ADP (FIG. 2). In contrast to the relatively rigid In addition to flexibility in accommodating different
conformation of the ATPase domain, the conformation sequences, substrate recognition by Get3 must also be
of the α‑helical domain is sensitive to nucleotide bind selective in at least two ways. First, the C‑terminal TMDs
ing. In the fully closed, ATP-bound state, the helical of TA proteins must be distinguished from the internal
subdomains are in direct contact and define a large, TMDs of co-translational substrates. Second, Get3 must
hydrophobic groove that spans both Get3 monomers71 avoid the TMDs of TA proteins destined for other orga
(FIGS 2,3). nelles (such as peroxisomes, mitochondria and chloro
Three lines of evidence illustrated that the helical plasts). As there is little difference between the TMDs
domains mediate recognition. First, the size, shape, of these different substrates77, a key issue is how the Get
hydrophobicity, flexibility and the ATP-dependent form pathway selects only the correct substrates for targeting.
ation of the composite groove argued for this being the One clue comes from the observation that, in the
site of TMD binding 69–72. Second, hydrogen exchange absence of SRP (or other competing factors), Get3 can
mass spectrometry (HX-MS) studies demonstrated bind substrates containing internal TMDs. Conversely,
protection of the α‑helical subdomains upon binding SRP cannot recognize a TMD it normally binds when that
to a TA substrate69. Third, perturbing the composite same TMD is near the C terminus. This suggests that,
hydrophobic groove by introducing negative charges or under physiological conditions, SRP binding to inter
disrupting dimerization reduced TA substrate binding nal TMDs is strongly favoured by its association with a
in vitro and led to growth defects in vivo71. translating ribosome. Hence, its very high local concen
The process of substrate recognition in the co- and tration near the ribosomal exit tunnel ensures that SRP
post-translational pathways shows important func will out-compete any other available binding proteins,
tional and mechanistic similarities. As with Get3, the such as Get3.
sequences inside the ribosome can influence its inter binding to Get3, was defective for TA protein insertion
action with the SEC61 translocon90–92. Alternatively, into the membrane95. These observations suggested that
the BAG6 complex might be recruited to ribosomes Get2 functions to recruit the Get3–TA protein targeting
by signals that are present in the mRNA that encodes complex to the membrane.
the TA protein. Indeed, studies indicate that cis-acting The structure of nucleotide-free Get3 bound to
sequences in the TMD-coding region of bacterial the Get1 cytosolic fragment revealed two Get1 frag
membrane protein mRNAs can direct these mRNAs ments bound to equivalent sites on opposite faces of
to the plasma membrane93,94. The initial capture step the open Get3 dimer 95,97 (FIG. 5). Strikingly, each Get1
by the BAG6 complex remains to be studied in mecha coiled-coil inserts itself between the two Get3 subunits
nistic detail, as does the apparent regulation of transla to completely disrupt the closed dimer interface. This
tional termination and the relationship between these observation immediately suggested that Get1 functions
two processes. to release substrate from Get3. Consistent with this,
functional analysis showed that Get1, but not Get2, pro
Targeting and release at the ER motes substrate release95,96. Moreover, Get1 was unable
After a TA protein is successfully loaded onto Get3 by to promote substrate release from an ATPase-deficient
the action of the TRC, the resulting Get3–TA protein Get3 variant (in which Asp57 was replaced with Asn),
complex must next be targeted to the ER. In budding suggesting that it functions on a Get3–TA substrate
yeast, genetic studies have indicated that Get1 and Get2, complex in which the ATP has already been hydrolysed.
both of which are multi-spanning ER membrane pro The crystal structures also provided key insights into
teins, are needed for targeting 65. Furthermore, their how targeting and substrate release are coordinated by
ability to form a complex with Get3 in the absence of the two receptor subunits. The Get1- and Get2‑binding
other factors61,66 suggested that these three proteins sites on Get3 are partially overlapping, and interaction
could be the minimal factors required for targeting, and analyses showed that the receptor subunits compete
possibly insertion into, the ER membrane. for binding to Get3 (REF. 95), which is consistent with a
Insight into the role of Get1 and Get2 came from sequential handover mechanism (FIG. 5). A complex of
recent reconstitution studies95,96. Genetic and bio Get3 bound simultaneously to portions of Get2 and Get1
chemical depletion and add-back experiments estab can be detected at high concentrations by NMR, and this
lished that Get1 and Get2 are each indispensable for may represent the transient intermediate during hand
Get3‑dependent insertion of the TA substrate into the over 97. Taken together, these studies suggest a model in
membrane95,96. Remarkably, efficient targeting and inser which Get2 recruits the Get3–TA substrate targeting
tion could be achieved in proteoliposomes containing complex, with Get3 in a closed dimer conformation, and
only recombinant Get1 and Get2 at physiological con subsequently transfers it to Get1, which drives substrate
centrations95. Thus, Get1 and Get2 are both necessary release by disrupting the composite hydrophobic groove
and sufficient for the membrane-associated events of TA and stabilizing the open state of Get3.
protein targeting and insertion. The stoichiometry of the Get1–Get2 receptor com
The availability of a simple reconstituted system plex remains to be established. The simplest possibility
using completely purified, recombinant components in view of the crystal structures and the symmetric Get3
permitted a detailed analysis of how these two mem dimer is that two Get1 and two Get2 subunits form a
brane proteins interact with, and regulate the func heterotetrameric assembly. The resulting high-avidity
tion of, Get3 (REF. 95). Interaction analysis illustrated interaction of two Get1 subunits with the Get3–TA
that Get1 and Get2 associate through their membrane substrate complex would facilitate substrate release at
domains and that they each interact with Get3 via their physiological concentrations. Nevertheless, a hetero
prominent (non-homologous) cytosolic domains95,96. dimeric receptor is also plausible, with Get2 binding
The unusual feature of a receptor that interacts with to one side of the Get3 heterodimer and Get1 binding to
Get3 in two different ways suggested that these two the other. Evidence that this mechanism could work is
interactions serve distinct functions: targeting of the provided by the finding that artificially heterodimerized
Get3–TA protein complex to the ER, followed by sub Get1–Get2 cytosolic domains can mediate substrate
strate release at the ER membrane. This indeed proved release in vitro96.
to be the case, as revealed by a combination of structural This critical step of releasing a substrate from its
and functional studies. tightly bound targeting factor at the correct place and
Insight into both steps was provided by structures of time is a problem faced by all targeting pathways. In the
the complexes that form between Get3 and the cytosolic TA protein pathway, substrate release is first obligatorily
Get1 and Get2 receptor fragments95,97 (FIG. 5). The struc ‘primed’ by nucleotide hydrolysis, whereas its actual
ture of the amino‑terminal end of the Get2 cytosolic release is promoted by the Get3–Get1 interaction95,96.
fragment in complex with Mg2+–ADP–AlF4–-bound Get3 This two-step mechanism is similar in concept, albeit
showed two Get2 fragments bound to equivalent sites on different in details, to the SRP-mediated co-translational
opposite faces of the closed Get3 dimer. Importantly, the insertion pathway. Here, a substrate bound to SRP54 is
Get3 hydrophobic groove was intact and accessible, with released in two successive steps, one involving nucleotide
the N‑terminal ends of Get2 tethered to the membrane binding and the other involving receptor interaction. The
by a long, flexible linker. A Get1–Get2 complex contain nucleotide-dependent step involves GTP binding to both
ing a structure-based mutation in Get2 that disrupts SRP and its receptor to allow targeting 98,99. The second
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)GV
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Figure 5 | Targeting and substrate release at the ER membrane. Nucleotide- (either ADP or ATP) and tail-anchored
(TA) substrate-bound Get3 is captured at the endoplasmic reticulum (ER) membrane0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
by the long, flexible amino termini
of Get2. The Get3–TA substrate complex, now in an ADP-bound, partially closed state, is transferred to Get1, which
wedges open the composite hydrophobic groove to promote TA substrate (and ADP) release. Finally, ATP re-binding
dissociates the stable Get1–Get3 ‘post-insertion’ complex to recycle Get3 back to the cytosol. Although depicted here
as a stable heterotetramer, the stoichiometry and subunit composition of the Get1–Get2 receptor complex is not known.
The insets show crystal structures of Mg2+–ADP–AlF4–-bound Get3 in complex with the cytosolic fragment of Get2
(left, Protein Databank (PDB) ID 3ZS9), and of nucleotide-free Get3 in complex with the cytosolic fragment of Get1
(right, PDB ID 3ZS8). C, carboxyl terminus.
step is a GTP-dependent interaction between SRP and a hydrophobic TMD to the membrane surface is insuf
the SRP receptor that results in structural rearrangements ficient for insertion, and that the SEC61 complex has a
that expose the M‑domain–signal sequence module to crucial role. On the basis of structural and functional
facilitate release to the translocon100. analysis, this crucial function is twofold102–104. First, SEC61
seems to directly recognize substrate TMDs via a spe
The elusive insertion step cific binding site within its membrane domain. Second,
Virtually nothing is known about how TA substrates are this binding site also serves as a ‘lateral gate’, which pro
inserted into the ER membrane. Following its release vides TMDs direct access to the lipid bilayer. Thus, the
from Get3, the substrate must avoid improper inter TMD takes a route through the centre of SEC61 to bypass
actions (in particular, aggregation) and insert into the phospholipid headgroups that otherwise preclude
the membrane bilayer in the correct orientation. On the facile access to the hydrophobic core of the membrane.
basis of the rigid interaction between Get1 and Get3, Applying these principles to the TA protein pathway,
the TMD is presumably released parallel to and abut it is attractive to speculate that Get1 and Get2, both of
ting the bilayer surface. From this position, insertion which are multi-spanning membrane proteins, interact
requires the hydrophobic TMD to cross the polar head directly with the TA protein’s TMD to chaperone it into
groups of the phospholipids and reach the hydrophobic the bilayer. Such a recognition event could provide an
membrane core. This could either occur ‘spontaneously’ additional layer of proofreading, as has been ascribed
(that is, without direct assistance from any factors) or the to the SEC61 complex 105–107. More importantly, it would
Get1–Get2 complex could chaperone the TA protein into explain how the TMD efficiently accesses the hydro
the membrane (FIG. 6). Although in vitro studies show that phobic core of the bilayer with minimal possibility of
less-hydrophobic TMDs can insert spontaneously into ‘off-pathway’ events such as aggregation or inappro
liposomes44,51, most TA proteins cannot. The mechanistic priate interactions. One speculative model for how
basis of this final step awaits additional studies, but some chaperoning could be achieved via coupled conforma
insight can be gleaned from experiments done in the tional changes in Get1–Get2–Get3 is depicted in FIG. 6.
co-translational system. Alternatively, Get1–Get2 could distort the lipid bilayer in
A type I membrane protein containing a single its vicinity to facilitate TMD insertion. The biochemical
N‑terminal TMD, a short luminal domain and an exten strategies used to study SEC61‑mediated insertion in
sive cytosolic C‑terminal domain can be efficiently tar the co-translational pathway 101,104,108–110 will clearly be
geted to the membrane surface via the SRP pathway, useful in determining the insertion mechanism of the
but subsequent insertion fails in the absence of the TA protein pathway. However, distinguishing between
SEC61 complex 101. This indicates that simply targeting what can happen in a simplified system and what does
C7PCUUKUVGF D%JCRGTQPGCUUKUVGF
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6#UWDUVTCVG
0 0
#&2 #&2 #&2 #&2
)GV
Figure 6 | Alternative models for the insertion of TA proteins into the ER membrane. a | After release from Get3,
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
the tail-anchored (TA) substrate might transiently associate with the membrane surface before inserting ‘spontaneously’
into the lipid bilayer. b | Alternatively, the transmembrane domains (TMDs) of Get1 and Get2 may interact directly with the
TA substrate to chaperone it into the bilayer. In the model shown here, binding of two Get1 subunits to the partially closed
targeting complex results in a ‘strained’ configuration of the Get1–Get2 receptor complex. This arrangement might
provide a hydrophobic gate through which the TMD could diffuse into the bilayer. ‘Wedging open’ the Get3 dimer for
substrate release would then allow the Get1–Get2 receptor complex to ‘relax’ back into a low-energy configuration that
is sealed-off from the cytosol. Thus, conformational changes in Get3 might be coupled to those of the receptor TMDs to
promote TA substrate integration. C, carboxyl terminus; ER, endoplasmic reticulum; N, amino terminus.
happen in a physiological context may be challenging, that distinct Get1–Get2–Get3 and Get3–Get4–Get5
as the role of Get1–Get2 in insertion might simply be to complexes can be isolated from fractionated yeast 66,
accelerate an already favourable reaction, and any con and provides an elegant mechanism for spatially
sequences may only be apparent in a highly crowded regulating Get3 activity in the cytosol and at the ER
context that reflects the in vivo situation. membrane.
to have been hydrolysed95,96. This is probably because BAG6, Sgt2, Get3, HSP70 and possibly others)? This crit
the binding site for Get1 is partially buried in the fully ical checkpoint determines the fate of the hydrophobic
closed ATP-bound state of Get3 (REF. 95). Get1 binding substrate: insertion into the ER membrane (via Get3),
causes Get3 to fully open, thereby favouring substrate insertion into the mitochondrial outer membrane
release. Once Get3 opens, the ADP is no longer trapped (by an unknown mechanism), or ubiquitylation (in a
and probably dissociates rapidly. The re-binding of ATP process involving BAG6) and proteasomal degradation for
to Get3 dissociates it from Get1. This effectively means misfolded membrane proteins. Defining the mechanism
that Get1 acts as a nucleotide exchange factor that dis of this process is therefore an important future goal.
places ADP (via opening of the Get3 dimer) and allows The biochemical and structural framework estab
its replacement with ATP. lished over the past few years also sets the stage for
Whereas most parts of the ATPase cycle have strong detailed mechanistic studies of events at the ER mem
experimental support, the timing of ATP hydrolysis brane. Determining the structure and organization of the
remains unclear. It must occur at a point after the sub Get1–Get2 receptor complex will be critical for under
strate binds to Get3 and before the interaction of this standing insertion. Is the receptor a stable heterodimeric,
protein with Get1. Although it is attractive to posit that heterotetrameric or higher order assembly? Or are there
hydrolysis stimulated by either substrate or Get2 inter temporal variations in its stoichiometry and subunit
action provides an additional checkpoint, this may not be composition? Similarly, the order and timing of Get3–
necessary. In fact, a slow rate of intrinsic hydrolysis might substrate interactions with the receptor have not been
provide a mechanism for kinetic proofreading. In this directly established. Most significantly, almost nothing is
model, loosely bound substrates would dissociate before known about how the TA substrate inserts into the bilayer
hydrolysis, thereby preventing their membrane-localized after its release from Get3. Does it insert spontaneously?
release by Get1. This might reduce inappropriate target Or is it actively chaperoned by the conserved TMDs of
ing of mitochondrial TA proteins (which generally have Get1 and Get2? Answers to these questions will require
TMDs of lower hydrophobicity than ER‑directed TA high-resolution structural analysis of Get1–Get2–Get3
proteins)47–49,77 to improve the overall fidelity of sorting. complexes trapped at different stages of this cycle. This
Similar kinetic proofreading mechanisms have been promises to be technically challenging, but the goal is in
uncovered in the SRP pathway and are thought to maxi reach now that robust expression systems for functional
mize the sorting efficiency of an otherwise promiscuous Get1 and Get2 proteins are in hand.
interaction between SRP and various proteins112. Finally, there may be much to learn from studying TA
protein biogenesis in other organisms. The recent discov
Future challenges and perspectives ery of a conserved Get3 orthologue in archaea indicates
Since the discovery of the TA protein insertion pathway in that the post-translational TA pathway is more broadly
2007, rapid advances have been made through the appli conserved than previously appreciated116,117. No obvious
cation of genetic, biochemical and structural approaches. sequence homologues have been identified for other
Over the past 5 years, all of the core components have been cytosolic components (for example, BAG6, Sgt2, Get4 or
identified, and a general mechanistic understanding of the Get5), but functional data are consistent with the pres
pathway from ribosome to the ER is now in hand. But the ence of an orthologous integral membrane receptor in
details of many key steps that take place in the cytosol archaea117. If such a receptor exists in archaea, it shares
and at the membrane remain a mystery. For example, how only limited sequence homology with the yeast recep
does the TRC selectively interact with cytosolic ribosomes tor subunits, Get1 and Get2. There are also intriguing
that contain a TMD in the exit channel? Structural studies evolutionary differences between the yeast and mam
are needed to identify the ribosomal binding site of the malian pathways. The absence of a BAG6 homologue in
TRC, and to determine how the TRC senses the presence yeast suggests increasing complexity in the pre-targeting
of a TMD in the exit channel. After this initial capture machinery of higher eukaryotes. And, whereas the human
step, what is the mechanism for TA substrate transfer Trp-rich basic (WRB) protein shows sequence and func
from the TRC to Get3? Although crystal structures exist tional homology to yeast Get1 (REF. 118), no obvious
for Get4 and portions of Get5 and Sgt2 (REFS 80,113–115), sequence homologue to Get2 has been found in higher
mechanistic insight awaits structural analysis of the intact eukaryotes. Thus, the identification and characterization
TRC. More broadly, how does the TRC sort different of components in the archaeal and higher eukaryotic
hydrophobic substrates between its multiple hydropho pathways promises new insight into the mechanism of
bic region-binding factors (including the chaperones TA protein targeting and insertion.
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