Biochimica et Biophysica Acta 1833 (2013) 2392–2402

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Biochimica et Biophysica Acta

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Review

Co-translational targeting and translocation of proteins to the

endoplasmic reticulum☆
Yvonne Nyathi 1, Barrie M. Wilkinson, Martin R. Pool ⁎
Faculty of Life Sciences, University of Manchester, Oxford Road, M13 9PT, UK

a r t i c l e i n f o a b s t r a c t

Article history: Co-translational protein targeting to the endoplasmic reticulum (ER), represents an evolutionary-conserved
Received 11 December 2012 mechanism to target proteins into the secretory pathway. In this targeting pathway proteins possessing signal
Received in revised form 18 February 2013 sequences are recognised at the ribosome by the signal recognition particle while they are still undergoing syn-
Accepted 19 February 2013
thesis. This triggers their delivery to the ER protein translocation channel, where they are directly translocated
Available online 26 February 2013
into the ER. Here we review the current understanding of this translocation pathway and how molecular details
Keywords:
obtained in the related bacterial system have provided insight into the mechanism of targeting and translocation.
Protein translocation This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.
Protein targeting © 2013 Published by Elsevier B.V.
Protein biogenesis
Ribosome
Signal recognition particle
Translocon

1. Introduction The co-translational pathway utilizes the signal recognition particle

Proteins destined to enter the secretory pathway typically possess
N-terminal hydrophobic signal sequences, which direct them to the
endoplasmic reticulum (ER) membrane, where they are translocated
into the lumen of ER. Once inside the ER lumen they can traverse the
secretory pathway by vesicular transport, hence transport across the
ER membrane is the only membrane translocation event these proteins
require and represents the commitment step in entry to the secretory
pathway. There are several distinct pathways, which target secretory
protein to the ER. In this review, we focus on the universally conserved
co-translational translocation pathway, which also integrates mem-
brane proteins into lipid bilayer [1,2]. Alternative ER targeting path-
ways, which are not directly coupled to translation, are covered
elsewhere in this issue (Johnson et al, in this issue).
(SRP) to deliver secretory proteins to the ER membrane while they are
still being synthesized by ribosomes [1] (Fig. 1). SRP delivers the na-
scent secretory protein together with the associated ribosome to the
ER protein translocon (Sec61 complex) via the interaction with its
cognate receptor (SR) an ER resident membrane protein. When the
ribosome nascent chain complex (RNC) engages the Sec61 complex
protein synthesis continues, enabling the nascent chain to be directly
conveyed into the lumen of the ER where it can then fold to its final con-
formation. During translocation enzymes such as oligosaccharyl trans-
ferase (OST) and signal peptidase (SPase) can associate with the
translocon and either N-glycosylate or cleave the signal peptide from
the translocating nascent chain, respectively.
Bacteria possess a highly related targeting pathway that translo-
cates bacterial secretory proteins across the plasma-membrane into
the periplasm.

2. Structure and organisation of SRP

Abbreviations: BiP, binding immunoglobulin protein; tRNA, transfer RNA; RNC,
ribosome nascent chain complex; ER, endoplasmic reticulum; GppNHp, 5′-guanylyl
imidodiphosphate; OST, oligosaccharyl transferase; RAMP4, ribosome-associated mem-
brane protein 4; RNC, ribosome-nascent chain complex; rRNA, ribosomal RNA; SPase,
signal peptidase; SR, signal recognition particle receptor; SRP, signal recognition particle;
TM, transmembrane helix; TRAM, translocating chain-associated membrane protein;
TRAP, translocon-associated protein complex
☆ This article is part of a Special Issue entitled: Functional and structural diversity of
endoplasmic reticulum.
⁎ Corresponding author. Tel.: +44 161 275 5392.
E-mail address: martin.r.pool@manchester.ac.uk (M.R. Pool).
1
These authors contributed equally.
The signal recognition particle (SRP) is a ribonucleoprotein
complex found in all domains of life that couples the synthesis of
membrane and secretory proteins to their targeting [2,3]. Despite
this widespread conservation in function, the complexity of SRP
has increased considerably during evolution [2,4]. Mammalian
SRP is composed of a 7SL RNA of ~300 nt (which folds into a roughly
Y-shaped double-stranded secondary structure) in complex with six
protein subunits SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72 [5]
(Fig. 2A). These components can be divided into two structural domains;
the Alu and S domains, as revealed by micrococcal nuclease treatment [6].

0167-4889/$ – see front matter © 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.bbamcr.2013.02.021
 Y. Nyathi et al. / Biochimica et Biophysica Acta 1833 (2013) 2392–2402 2393

The S-domain, consists of the forked region of 7SL RNA together with
SRP19, SRP54, SRP68 and SRP72, while the Alu domain is made up helices
3–5 of the 7SL RNA and the SRP9–SRP14 heterodimers (Fig. 2A and B).
The composition of fungal SRP is similar to that of mammalian SRP
but with notable differences (Fig. 2A). For instance, yeast SRP differs
from its mammalian counterpart owing to the functional replacement
of SRP9/14 by an SRP14 homodimer and the presence of a yeast-
specific protein, SRP21 which is structurally related to SRP9 [7,8]. In addi-
tion, the yeast homologue of SRP19 (Sec65) is a much larger protein [9].
Moreover, the yeast SRP RNA frequently possesses additional insertion
elements of unknown function [8,9].
Bacterial SRP is much simpler than that of eukaryotes, consisting of
an SRP54 homologue, referred to as Ffh (fifty four homologue) in com-
plex with a much smaller 4.5S RNA that contains the most conserved
domain IV (helix 8) of the 7S RNA (reviewed in [3,10], Fig. 2A). It is
intriguing that despite the complexity of the mammalian SRP, the min-
imal bacterial SRP in concert with bacterial SR (FtsY), can still promote
targeting of mammalian proteins to the ER [11]. This demonstrates that
the conserved SRP54 and domain IV (helix 8) of the SRP RNA comprise
the functional core of SRP. It also suggests that the basic mechanism
of co-translational targeting is conserved from bacteria to mammals.
Indeed, much of the current understanding on the structure and mech-
anism of co-translational translocation has been obtained from studies
in bacteria.
3. Structure of SRP54
SRP54 is a key component in co-translational protein targeting and it
is conserved in all SRPs [2]. It plays a role in the interaction of SRP with
the ribosome, signal sequence recognition as well as GTP-dependent in-
teraction of SRP with SR, which together determine proper transfer of
the ribosome-nascent chain complex (RNC) to the translocon [12–14].
The functions of SRP54 are brought about by three domains; a C-
terminal α-helical methionine-rich (M) domain which binds the signal
sequence, an N-terminal four-helix bundle (N) domain, and a GTPase
(G) domain. The N and the G domains are closely packed together and
interact with the SR and are often collectively referred to as the NG
domain [15,16]. The M and NG domains are connected by a highly con-
served and flexible linker, which allows SRP54 to undergo substantial
structural rearrangements during the targeting cycle and permits
communication between the two domains [17,18]. The M domain
consists of four amphipathic helices organised around a small hydro-
phobic core (Fig. 2C). The hydrophobic core is a highly ordered region
with a hydrophobic groove implicated in signal sequence binding and
an arginine-rich motif required for binding to SRP RNA [17,19,20]. Before
the M-domain of SRP54 can bind to the SRP RNA, SRP19 must first bind to
the RNA via helices 6 and 8, inducing conformational rearrangements
in helix 8 that allow SRP54 binding [21].
Compared to prokaryotic Ffh, the C terminus of eukaryotic SRP54
extends beyond helix αM4 in the M domain by as many as ~ 100 res-
idues [22]. This methionine rich C-terminal extension is thought to be
involved in signal sequence binding as it is in close proximity to the
hydrophobic groove of the M domain. Moreover, deletion of a portion
of the C-terminus from mammalian SRP54 abolished cross-linking to
signal sequences in vitro [23].
4. SRP signal sequences
SRP signal sequences are highly divergent in length, shape and
amino acid composition [24–26]. Early studies identified a core of
8–12 hydrophobic amino acids that preferentially form an α-helix
as the major determinant that mediates signal sequence recognition by
SRP [24,25,27]. Although, it was generally agreed that a threshold level
of hydrophobicity is a key determinant in allowing a signal sequence to
specify the SRP pathway [28], recent work suggests that though impor-
tant, hydrophobicity is not a sufficient indicator for SRP-dependence
[29]. This idea is supported by the fact that the difference between
the hydrophobicity of SRP-dependent versus SRP-independent signal
sequences is relatively small [29]. In addition, some signal sequences
with hydrophobicity above the apparent ‘threshold’ fail to engage SRP
[29]. It has long been postulated that additional molecular features
of the signal sequence such as helical propensity [24], conformation/
orientation of the signal sequence at the ribosomal surface [30], the pres-
ence of N-terminal basic residues [31] and more recently the length of the

Fig. 1. The co-translation protein targeting pathway. 1. SRP binds the signal sequence (green) as it emerges from the ribosome forming the RNC–SRP complex. 2. The RNC–SRP com-
plex docks with the ER membrane by binding to the cognate SRP receptor (SR, consisting of α and β subunits). 3. Subsequently the RNC is then transferred from SR–SRP to the Sec61
translocon resulting in intercalation of the signal sequence in the translocon pore and its opening. 4. Translocation of the nascent chain proceeds through the pore and SRP also
disengages from SR. 5. Concomitant with translocation the signal peptidase (SPase) and oligosaccharyl transferase (OST) enzyme complexes are recruited to the translocon to
cleave the signal peptide and to add N-linked glycans to the nascent chain respectively. Termination of protein synthesis releases the nascent chain from the ribosome, it then com-
pletes its translocation and folds in the ER lumen.
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Fig. 2. Signal recognition particle and signal sequence recognition. A. Schematic representation of SRP organization in mammals, yeast and E. coli. SRP RNA helices are numbered
according to Larson and Zweib [137]. B. Structure of the mammalian SRP derived from a Cryo-EM reconstruction with X-ray structures of individual components fitted (PDB ID:
1RHY). No structure is available for SRP68/72 hence their approximate position is deduced as elucidated from RNA foot-printing experiments and unassigned density in
Cryo-EM maps. C. Structure of the SRP54 M-domain from Sulpholobus sulfataricus SRP54 (blue) with (bottom, PDB ID: 3KL4) and without (top, PDB ID: 1QZW) a signal sequence
bound (red) showing the NG-domain (green), M-domain (blue), and side chains of hydrophobic residues that line the signal sequence binding groove are shown in yellow. D. Struc-
ture of the Methanococcus jannaschii SRP with (PDB ID: 3NDB) and without (PDB ID: 2V3C) signal sequence showing the NG-domain (green), M-domain (blue), SRP RNA (grey), and
signal sequence (red). Note the large rotation of the NG domain upon signal sequence binding which brings the G domain close to the RNA tetraloop. E. Interaction of mammalian
SRP with wheat ribosome bearing a signal sequence. SRP54 and the S-domain are located adjacent to the polypeptide exit site on the 60S subunit. SRP54 contacts ribosomal proteins
L23a and L35. The Alu-domain is positioned at the subunit interface close to the GTPase associated centre at the elongation factor-binding site.

polypeptide chain [32] could affect signal sequence recognition. It is also
possible that other properties that are as yet to be identified [33] could
also play a role in specifying SRP dependence. Further work is necessary
in order to define all the molecular features of the signal sequence and
analyse their respective contributions in specifying SRP dependence.
5. Formation of the SRP–RNC targeting complex
SRP recognises the signal sequence while the nascent chain is still
associated with the ribosome. This interaction precludes the exposure
of the hydrophobic signal sequences to the bulk cytosol thereby

Fig. 3. A. Domain organization of SRP receptor and comparison to SRP54. Note the homology of the N and G (GTPase domains) between SRP54 and SRα. SRX (SRβ-interacting domain), mem-
brane binding A-domain of FtsY (A). B. Structure of the pseudo homo-dimer formed between the NG domains of SRP54 (Ffh) and SRα (FtsY) from Thermus aquaticus. Note that the two bound
GppNHp nucleotides directly contribute to the binding interface. C. GTPase cycle of SRP54 and SRα. The interaction of SRP and SR involves a series of kinetically controlled steps. 1: The RNC with
a signal sequence is bound by SRP. 2. SRP and SR first associate to form an early intermediate targeting complex, which is stabilized by the RNC. 3. The early complex subsequently rearranges to a
tightly bound closed state. 4. Interaction of SR with the translocon allows the SRP–SR complex to rearrange to the activated state, which drives the handover of cargo from the SRP to the
translocon. Note: transition to the closed and activated states collectively weakens the affinity of SRP for the RNC and promotes handover of the RNC to the translocon. 5. GTP hydrolysis drives
the disassembly and recycling of SRP and SR (T-GTP bound, D-GDP bound). D. Bacterial SRP–SR complex formation leads to a large conformational rearrangement about the SRP54 linker region,
such that the NG domains are brought close to a distal region of the SRP RNA. The GTPase pocket is approached by cytosine 83, providing a mechanism to induce hydrolysis. Right, note, in
mammalian SRP RNA the region where cytosine 83 is located overlaps the SRP68/72 binding site. E. Model for the structural rearrangements of the SRP.RNC complex upon interaction with
SR. The NG domain of SRP54 is moved away from L23a/L35, such that this site is now available for translocon binding and subsequent signal sequence handover.
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preventing their aggregation. SRP binds to ribosomes with low affinity
independent of whether or not a signal sequence is present. SRP binds
to non-translating ribosomes with equilibrium dissociation constant
(Kd) values of 70–80 nM, and to ribosomes translating cytosolic pro-
teins, with a Kd of 8 nM. However, its affinity for ribosomes is dramati-
cally increased when a signal sequence emerges from the ribosome exit
tunnel (Kd ~ 0.21 nM for a strong signal sequence [34–36]). Thus, SRP
is thought to sample many RNCs for the presence of a signal se-
quence by establishing a low affinity (salt sensitive) interaction with
the ribosomes and to then bind with higher affinity (salt resistant
interaction) once a signal sequence has been recognised [34,36,37].
Cross-linking and cryo-EM analyses indicate that during the sam-
pling mode, SRP has several contact points on the ribosome [38–40].
SRP54 is close to the exit site, where the polypeptide chain emerges
from the ribosome enabling the M-domain to contact the signal se-
quence and also contact elements of the 25S rRNA (Escherichia coli
23S rRNA) (Fig. 2E). The NG-domain of SRP54 (or Ffh) interacts with
ribosomal proteins L23a (L23 in E. coli) and to a lesser extent its neigh-
bour L35 (L29 in E. coli) [39,41]. Many nascent chain-associating factors
including trigger factor, N-acetyl transferases and the translocon also
interact with the ribosome via L23a/L35 (L23/L29 in E. coli) leading
to its description as a universal adaptor site [42].
The importance of the ribosome in driving the recruitment of SRP is
demonstrated by the fact that isolated signal peptides bind SRP weakly,
with Kd values in the micromolar range [43]. This observation suggests
that the ribosome acts synergistically with the signal sequence to drive
conformational changes which allow high affinity SRP binding to the
signal sequence [44]. Some of these different affinities of SRP mentioned
above were measured before the signal sequence had emerged from the
ribosome exit tunnel [34,35], suggesting that the ribosome exit tunnel
senses the different nascent chains and undergoes conformational
changes which are sequence specific. Indeed, it was recently shown that
a signal sequence within the ribosome exit tunnel enhances the binding
of SRP to the RNC [35,36,45]. Moreover, the presence of an alpha helical
motif in the distal part of the tunnel reduced the recruitment of trigger
factor to the ribosomes in E. coli [46]. Interestingly, the presence of a
signal anchor or transmembrane domain in the tunnel also appears to
have effects on downstream processes in the co-translocation pathway
including recruitment of a regulatory protein RAMP4 to the Sec61
translocon [47] and the opening and closing of the Sec61 translocon
[48]. Together, these observations suggest that sequence or structural
features of the nascent polypeptide inside the ribosome exit tunnel pro-
vide signals that can be sensed and transmitted to the ribosome exit
site leading to recruitment of different cytosolic factors to the ribosome.
6. Interaction of SRP54 with the signal sequence
Cross-linking and structural studies have revealed that the
M-domain of SRP54 is responsible for recognition of the signal sequence
[12,23,49,50]. The crystal structure of the M domain from Thermus
aquaticus Ffh [19] shows that the M domain forms a flexible hydrophobic
groove lined with many methionine residues, which are thought to facil-
itate the accommodation of a diverse array of signal sequences which are
mainly α-helical (Fig. 2C). Moreover, the ribosome-bound conformation
of SRP reveals a strikingly different conformation of the SRP core when
compared to the compact free state. SRP54 adopts an “open conforma-
tion” that would facilitate the accommodation of the signal sequence
within the hydrophobic groove of the M domain [44] (Fig. 2C).
Recently, Janda and coworkers determined the crystal structure
of a fusion of SRP54 (Ffh) from Sulfolobus solfataricus and the signal
anchor sequence of dipeptidyl aminopeptidase B (a characterized
SRP-dependent substrate from yeast) separated by a flexible linker.
This fusion protein oligomerised in solution through the interaction
between SRP54 and signal sequences of different polypeptide chains.
This structure (Fig. 2C) confirmed that the signal sequence binds to the
hydrophobic groove in an α-helical conformation [17]. It also revealed a
conformational adaptation of the signal sequence that occurs upon its
binding into the groove. This induced fit mechanism is likely to be a
general feature which confers sufficient plasticity of the groove to ac-
commodate a diverse array of signal sequences [17,51].
Another, recent structure of the SRP core bound to the signal sequence
at 3 Å resolution [18] identified conformational changes occurring to
the NG domain, which are coupled to signal sequence recognition. It
was shown that upon signal sequence binding, massive rearrangements
in the NG and M domain occur including formation of a GM-linker helix
and a 180° flip of the NG domain; structural changes which are likely to
promote receptor engagement [18] (Fig. 2D). These rearrangements of
the NG domains were not observed in the Janda et al. structure [17],
which lacked the SRP RNA, consistent with a role of the SRP RNA in
promoting receptor engagement.
7. SRP receptor
Once SRP and the ribosome-nascent chain (SRP–RNC) complex
has formed, the next stage in the targeting reaction is the interaction
with SRP receptor (SR) on the ER membrane. SR is composed of two
subunits: SRα, a 70 kDa alpha subunit, which is a peripheral ER
membrane protein that is tightly bound to SRβ, a 30 kDa integral
membrane protein possessing a single trans-membrane domain em-
bedded in the ER membrane. SRα is composed of two domains: an
N-terminal SRX domain with a longin domain fold that forms the in-
teraction interface with SRα [52,53] and a C-terminal region which
has high homology to the NG domain of SRP54, including a function-
al GTPase domain [22,51] (Fig. 3). SRα also possesses a GTPase do-
main [54], which interacts with the SRX domain only when SRα is
in the GTP bound state [52,55], suggesting that the GTP cycle of
SRβ controls the association and dissociation of the heterodimeric
SR [52]. However, the functional role and the precise events trigger-
ing GTP hydrolysis of SRβ remain to be elucidated. The bacterial SRP
receptor, FtsY has high sequence similarity to SRα but lacks the SRX
domain as well as the SRβ subunit.
SRα forms the major interaction interface with SRP and is specifically
able to interact with the NG domain of SRP54. Progress towards under-
standing the interaction between the SRP receptor and SRP in detail
comes from studies with the bacterial system. The two NG domains of
SRP54 and SRα can form a pseudo homo-dimer with interactions be-
tween both the N domains and the two G-domains (Fig. 3B) [56,57].
The interface of the two G-domains involves the nucleotide binding
pockets forming a single central catalytic cavity with the nucleotides
themselves contributing to the dimerization interface; they are arranged
in an antiparallel fashion such that γ-phosphate of one nucleotide inter-
acts with the ribose 3′ hydroxyl group from the opposing nucleotide,
such that tight complex formation only occurs when both proteins have
bound GTP.
8. The SRP GTPase cycle
In their dissociated state, both the SRP54 and SRα GTPase do-
mains bind nucleotide with relatively low affinity (μM range), par-
ticularly when compared to small GTPases such as Ras and Rho. This
is rationalised by the presence of a unique insertion-box domain
(IBD) inserted into the G-domain [15,16,58]. Elegant experiments
where the nucleotide specificity of SRα was altered to favour
xanthosine triphosphate (XTP) demonstrated that targeting to the
ER translocon requires nucleotide tri-phosphate binding by both
SRα and SRP54 [59,60], in accordance with the structural informa-
tion demonstrating that both nucleotides contribute to the binding
interface of SRP54 and SRα [56,57].
Detailed kinetic analysis of the targeting reaction has to date only
been performed for the bacterial system. However, the conserved
nature of the SRP GTPases and the fact that bacterial SRP and SR
can function in the context of the eukaryotic system in vitro [11]
 Y. Nyathi et al. / Biochimica et Biophysica Acta 1833 (2013) 2392–2402
suggest that it is highly likely that the mechanistic details uncovered
are relevant to ER targeting. Such kinetic analyses indicates a model
whereby Ffh and FtsY initially assemble with each other in an open
conformation to form an early intermediate, which subsequently
rearranges to a stable ‘closed’ conformation dependent upon GTP
binding to both GTPases [61]. Ffh binds to the RNC bearing a signal
sequence and then interacts with its receptor, FtsY resulting in the
formation of a transient early intermediate which is stabilized by
the RNC complex. In this early intermediate, Ffh and FtsY associate
primarily via electrostatic interactions between their N-domains
[62]. This is followed by extensive rearrangements in both GTPases
resulting in the formation of a range of contacts between the
NG-domains and direct interactions between the two GTP molecules
across the dimer interface, thereby forming the GTP-stabilized closed
complex [57,63,62]. Cooperative rearrangement of the (IBD) loops
in both proteins positions multiple catalytic residues adjacent to
the bound GTP, resulting in reciprocal GTPase activation, and subse-
quent hydrolysis which drives signal sequence release and complex
disassembly [62,61,64] (Fig. 3C).
SRP–RNC complexes dramatically accelerate formation of the
early intermediate providing a mechanism to strongly favour binding
of SRP receptor to SRP that is ribosome associated (i.e. likely to be en-
gaged with substrate) over free SRP [61,64,65]. This is rationalised by
the helix 8 tetraloop of SRP RNA which kinetically accelerates the
SRP.SR receptor interaction in response to signal sequence binding,
by promoting structural rearrangements which, as described above
(Fig. 2D), reposition the NG domain of SRP close to helix 8 tetraloop
where it can interact with the receptor [18,43,66]. This ensures
rapid delivery of the RNC complex to the membrane and avoids futile
interactions between the free SRP and SR.
Once the early SRP–SR complex has formed it needs to rearrange
to a closed and activated state in order to transfer the RNC complex
to the translocation machinery. In bacteria, anionic phospholipids
facilitate the rearrangement of SR into the closed conformation, thereby
accelerating the formation of the stable SRP–SR complex when SR is
membrane-bound [67]. However the region of FtsY that interacts with
lipids is not present in SRα and is replaced by the SRX and SRβ domains,
hence it is not clear if lipids play a similar role in eukaryotes. The confor-
mational rearrangements of the SRP–SR complex from the early to the
closed and activated states result in a reduced affinity (∼400 fold less)
of SRP for the RNC complex [64]; a step that facilitates handover of
the RNC complex to the translocon. It was shown that mutations in
the GTPases that block the transition from the closed to the activated
state result in the formation of a stable RNC–SRP–SR complex but
block the engagement of the RNC with the translocon [40,68]. Thus,
emphasising the importance of timing of GTP hydrolysis, as the SRP
must transfer the RNC to the translocon before GTP hydrolysis drives
the irreversible disassembly of the SRP–SR complex. Interestingly, the
RNC selectively stabilises the SRP–SR complex in the open conformation
and delays its subsequent rearrangements to the closed state, which ac-
tivates GTP hydrolysis [64]. This ‘pausing’ effect, provides the targeting
complex with an important time window to search for the membrane
localized translocon and prevents premature GTP hydrolysis that
would lead to an abortive targeting reaction [64,68].
Initial experiments in the eukaryotic system with non-
hydrolyzable nucleotide analogues such as GppNHp suggested that
hydrolysis is dispensable for release of the signal sequence, but need-
ed for disassembly of the SRP.SR complex [69,70]. More recent exper-
iments conducted with point mutations of SRP54 and SRα, that
prevent activation of the hydrolysis reaction and thereby blocking
the release of the signal sequence from SRP54, indicated, that nucleo-
tide hydrolysis and signal sequence release are coupled and likely to
be tightly regulated [68]. However, the initial observations with
GppNHp are still rationalised, as in the presence of the non-
hydrolyzable analogue the conformational changes that activate hy-
drolysis still occur. This indicates that conformational changes that
2397
activate hydrolysis are necessary for signal sequence release and
hydrolysis is required for the dissociation of the SRP–SR complex
[68]. Indeed, the activation reaction is accompanied by a reduction
in the affinity of SRP for the RNC which favours release of the signal
sequence [68].
Recent structures of the bacterial SRP–SR complex indicate an addi-
tional interaction of SR with the SRP RNA, suggesting two steps in
the interaction of SR with SRP. Initial dimerization involving the NG
domains occurs adjacent to the helix 8 tetra-loop of the SRP RNA (as
described above). Subsequent reorganisation of the complex with
respect to the SRP RNA then occurs, with a large conformational re-
arrangement around the linker such that the M-domain remains close
to the tetraloop but the NG-domains translocate to a distal site on the
SRP RNA [71]. The NG domain is repositioned such that the G-domain
is very close to the RNA and a single nucleotide is able to approach
the catalytic centre of the GTPase, where it could thereby induce activa-
tion [71] (Fig. 3D). Hence this rearrangement allows regulation of the
activation step thereby controlling when the signal sequence is re-
leased. Indeed, a very recent study indicates that the translocation chan-
nel can trigger the repositioning of the NG domains in the SRP.SR
complex to the distal site on the RNA, thereby providing a mechanism
to co-ordinate GTP hydrolysis and signal sequence release with the
availability of the Sec61/SecYEG complex [72].
The additional ‘distal’ contact region of SR in the bacterial SRP RNA
is well conserved in bacteria but not in the eukaryotic SRP RNA. Fur-
thermore, SRP68 and 72 are also present in this region (Fig. 3D), so it
remains to be seen whether this step occurs in an identical fashion in
the eukaryotic SRP and if so, whether SRP68/72 are involved.
9. Kinetics of SRP.SR interaction and signal sequence proof-reading
In addition to the recognition of the signal sequence by SRP54, the
kinetics of SRP.SR complex formation also provides additional proof-
reading of signal sequences [65]. Recent studies indicate that bona
fide targeting signals promote more rapid and stable SR.SRP dimeriza-
tion whereas non-secretory chains are more likely to lead to non-
productive dimerization [65]. Secondly, proof-reading also occurs at
the GTP hydrolysis step, as a signal sequence delays hydrolysis facilitat-
ing efficient transfer of the signal sequence to the translocon, whereas
non-secretory chains are more likely to promote rapid hydrolysis lead-
ing to their rejection. Hence, in addition to signal sequence selection
by SRP at the ribosome, these further steps contribute to overall specific-
ity and minimize the delivery of non-secretory nascent chains to the
translocon. This is highly reminiscent the recognition of cognate tRNAs
by EF-Tu and the ribosome, which also includes a series of steps which
are kinetically sensitive to substrate specificity and together lead to a
high degree of accuracy [73].
10. Elongation arrest
A unique feature of the eukaryotic SRP is that, unlike its bacterial
counterpart, it is capable of causing a transient arrest or slowing down
of the polypeptide chain elongation upon binding to the signal sequence
as it emerges from the ribosome [74]. Biochemical characterisation of SRP
revealed that the Alu domain is responsible for this elongation arrest ac-
tivity [11,75,76]. SRP14 has been directly implicated in elongation arrest;
SRP particles containing a C-terminal truncation of SRP14 can still be
assembled but fail to retard translation [7,77].
Cryo-electron microscopy analysis of mammalian SRP bound to
artificially-arrested ribosomes showed that mammalian SRP forms an
elongated, kinked structure, with its Alu domain reaching into the elon-
gation factor binding site of the ribosome (Fig. 2E) [39]. Furthermore,
SRP was found to interact with the ribosome at the step of the eEF-2
catalysed translocation of tRNA from the A to the P-site in yeast and
mammals [37,78]. Thus, the Alu domain might delay the elongation
cycle by preventing eEF2 binding. Cross-linking studies revealed that
2398 Y. Nyathi et al. / Biochimica et Biophysica Acta 1833 (2013) 2392–2402
the interaction of the Alu domain with the ribosome is dynamic and
changes upon signal sequence recognition [79].
Abolishing elongation arrest function of SRP in yeast and mam-
mals compromises the translocation efficiency in vitro and in vivo
resulting in severe defects in protein targeting and growth [7,78].
This led to this hypothesis that elongation arrest provides a crucial
time window that allows the targeting complex to find and engage
the translocon before the nascent chain loses translocation compe-
tence. Indeed, SRP fails to efficiently target nascent chain which ex-
ceeds a critical length [34,80]. Recent studies suggest that the rate
limiting step in SRP dependent targeting is the SR. Mutants of SRP,
which are compromised in elongation arrest activity lead to translo-
cation defects, and can be rescued by elevating the levels of SR [78].
Thus, SRP ensures that the nascent chains remain translocation com-
petent during the time window dictated by the intracellular concen-
tration of SR [78].
11. The Sec61 translocon
The Sec61 complex is a protein-conducting channel that mediates
the translocation of proteins either across or integration into the ER
membrane. It has high homology to the bacterial SecY complex, which
translocates proteins across the prokaryotic plasma-membrane [81].
This channel is a heterotrimer consisting of two essential and highly
conserved subunits named α, and γ and a less well-conserved and
non-essential β subunit. It has been shown by photocross-linking
experiments that the α-subunit of the Sec61 complex surrounds a
polypeptide during transfer across the membrane and thus forms
the pore [82]. The Sec61/SecY complex was shown to be an essential
membrane component for protein translocation after reconstitution
into proteoliposomes [83,84]. In addition, electrophysiology measure-
ments [85] and the analysis of the lifetimes of fluorescent probes incor-
porated into a translocating polypeptide chain have shown that the
channel has an aqueous interior [86].
Key features of the translocon have been identified from the crys-
tal structure of an archaeal SecY complex (Fig. 4) [87]. The α-subunit
consists of two halves, TM domains 1–5 and 6–10 arranged around a
central pore. The cytosolic loop between TM domains 5 and 6 at the
back of the α-subunit is proposed to serve as a hinge, allowing the
translocon to open at the front to the lipid bilayer via a lateral gate
formed by TM domains 2b and 7. The γ-subunit has a long TM domain
traversing the bilayer diagonally in contact with both halves of the
α-subunit at the back. The β-subunit however makes relatively few
contacts with the α-subunit and its role in translocon function is
less clear. In a side view the channel has an hourglass-shape and a
constriction halfway across the membrane formed by a ring of six
bulky hydrophobic residues with their side chains projected radially
inward. The luminal side of the channel is occluded by a plug formed
by a sub-domain of the α-subunit TM2 domain (TM2a) connected to
the channel by unstructured loops (Fig. 4A and B). Subsequent analysis
of bacterial SecY complexes has revealed a very similar architecture as
the archaeal complex [88,89]. Despite the lack of a crystal structure
for a eukaryotic Sec61 complex, sequence conservation and structures
derived from cryo-EM [90,91] also indicate a similar architecture and
hence a conserved mode of action.
The central constriction in the translocon pore has a diameter of
approx 6.0 Å, leading to the suggestion that it serves to seal the
inactive translocon and form a gasket around the translocating poly-
peptide [87]. This is supported by the efficient crosslinking of a
translocating substrate to the amino acid residues at the constriction
[92]. Electrophysiology and molecular dynamic simulation experi-
ments indicate that the pore could widen to a diameter of ~ 20 Å
via movement of TM domains harbouring the pore ring amino acids
[93,94]. Widening of the pore from the 6.0 Å closed state is required
to accommodate an unfolded protein or a single alpha helical domain
during translocation.
12. Ribosome interaction and translocon channel opening
It is envisaged that channel opening during co-translational trans-
location is most likely triggered by the binding of the ribosome to the
translocon causing a partial opening of the lateral gate and displace-
ment of the plug domain (Fig. 4C). This would allow translocation
to proceed with the insertion of the signal sequence tail first (type II
topology) into the pore with the following C-terminal flanking seg-
ment positioned in the pore forming a hairpin-loop conformation
with the N-terminus located at the cytosolic face of the translocon
[95]. In the next step, the hydrophobic portion of the signal sequence
becomes intercalated into the lateral gate. Data obtained from photo
cross-linking experiments indicate that the hydrophobic part of the
signal sequence is next to TM domains 2b and 7 and also to phospho-
lipid, consistent with an interface between the channel and lipid [96].
Signal sequence binding would enhance the opening of the lateral
gate by separating TM domains 2b and 7, thereby destabilising plug
interactions leading to the opening of the translocon at the luminal
face. The γ-subunit and specifically its diagonal TM domain at the
back of the channel are required at this final stage of translocon opening
[97]. As the polypeptide moves through the pore, the channel is now
stabilized in the open state. This model for channel opening is support-
ed by the deregulation of the translocon by mutations that destabilise
the closed state or lock the plug in the open state [98–102]. The signal
sequence remains stationary, but at some stage during translocation is
cleaved by signal peptidase (Fig. 4D). The plug domain can then move
back into the centre of the channel only when both the signal peptides
have exited the lateral gate and polypeptide translocation is complete.
In eukaryotes, the signal peptide can be further processed by cleavage
in the hydrophobic core by signal peptide peptidase [103]. Despite the
widespread use of the hairpin-loop model to describe signal sequence
insertion into the translocon pore, recent experiments have led to the
development of an alternative model [104]. In this model initial insertion
occurs in a head first (type I) topology coincident with tight association
between the ribosome and the translocon. The following C-terminal
flanking residues in this case are retained near the ribosome exit tunnel
in the ribosome–translocon complex. As translation proceeds there is a
rapid inversion from type I to type II topology and translocation of
C-terminal flanking residues. A single translocon pore is however too
small to entirely accommodate the inversion of a rigid helix leading to
the suggestion that this occurs partially outside the translocon and may
require the function of Sec61 complex accessory proteins [104].
The hypothesis outlined above whereby structural elements of SecY/
Sec61α are key to the regulation of translocon gating is supported by
extensive genetic and biochemical data. However, another body of evi-
dence suggests that the gating of the channel in the ER membrane is
carried out by the ER luminal Hsp70 chaperone, BiP [105,106]. Data
indicating that it may bind to a luminal loop of Sec61α between TM do-
mains 7 and 8, suggests that it may promote lateral gate closing via
proximity to TM7. Two Hsp40-type co-chaperones have been identi-
fied in the ER membrane with a J-domain located in the ER lumen
able to bind BiP and stimulate its ATPase activity. The first, Sec63p
is found stably associated with Sec61 complexes in both yeast and
mammals [107–109]. In yeast it has been shown that both Sec63p
and its Hsp70 partner, Kar2p (a homologue of BiP), are both essential
for co-translational translocation strongly suggestive of a fundamental
role in translocon function [110]. Similarly, Sec63 has also been shown
to have an important role in co-translational ER protein translocation
in mammalian cells [111]. The second Hsp40-type co-chaperone, ERj1,
identified in mammalian ER membranes binds 80S ribosomes at the
universal adaptor site(s) of the exit tunnel and recruits BiP to translating
ribosomes [112]. It has been postulated that the ribosome can bind ERj1
or Sec63 and the Sec61 complex simultaneously to recruit BiP to gate
the luminal face of the translocon [113]. This would enable BiP to main-
tain the ER permeability barrier by binding the nascent chain undergoing
transfer to the ER lumen and the Sec61 complex simultaneously. The
 Y. Nyathi et al. / Biochimica et Biophysica Acta 1833 (2013) 2392–2402 2399

Fig. 4. A. Structure of the SecYEβ translocon from the archaea Methanococcus jannaschii (PDB ID: 1RH5), viewed from the side (left) and the cytoplasmic face (right). SecY, the
homologue of Sec61α has 10 TM helices (numbered 1–10) and SecE is the Sec61γ homologue. B. Sec61α TM2a forms a plug on the luminal face, which occludes the channel
here in its closed state. Sec61α TM helices 2b and 7 form a ‘lateral gate’ which enables the translocon to open up exposing the central aqueous channel to the core of the lipid
bilayer. C. Binding of the ribosome-nascent chain complex to the translocon is proposed to lead to displacement of the plug domain and binding of the signal sequence (SS) at
the lateral gate in a hairpin-loop conformation. D. At a later translocation intermediate, the signal sequence has been processed by signal peptidase and has fully exited through
the lateral gate. This gate closes and translocation of the nascent polypeptide chain proceeds through the central aqueous pore.

permeability barrier may also be maintained by the binding of calmodulin
to the cytosolic face of Sec61α jointly with an 80S ribosome to limit leak-
age of Ca2+ ions through the active translocon [106,114].
13. Organisation of the translocation channel
Electron microscopy imaging has shown that a single copy of the
Sec61 complex is bound to a non-translating ribosome with the ribo-
some tunnel aligned with the translocon pore [91] an arrangement
which is also observed with translating ribosomes [90,115]. These
findings are consistent with the model for translocation previously
described in which a single copy of the Sec61/SecY complex forms
the translocon pore. Analysis of SecY stoichiometry during translocation
in vitro [116] and in vivo [117] is also strongly suggestive of this conclu-
sion. However, oligomeric forms of Sec61 have been detected by a num-
ber of methods in intact membranes including electron microscopy [118],
fluorescence energy transfer [119], crosslinking [120] and native gel anal-
ysis [121]. Several studies for instance implicate a dimer of SecY
complexes in a back-to-back conformation in SecA-mediated trans-
location [120,122,123] whereby SecA binding pushes the polypeptide
through one copy with additional stabilisation provided by binding to
the non-translocating partner. During co-translational translocation the
ribosome–translocon interaction may be similarly stabilized by binding
to additional Sec61 complexes. Whilst a single SecY/Sec61 complex may
be sufficient to form a translocation pore and to bind the ribosome, func-
tional oligomeric forms suggest a flexible and dynamic translocon.
The oligomerisation of Sec61 complexes may be stimulated by
accessory factors and may also serve in the recruitment of complexes
and factors associated with the biogenesis of secretory and membrane
proteins. In native yeast membranes, the oligomerisation of Sec61α
complexes requires stable interaction with Sec63p [121]. In addition,
signal peptidase and the OST complex which catalyses the addition
of N-linked glycans to polypeptide chains act co-translationally, sug-
gesting that a close association with the translocon is essential. The
translocating-chain associated membrane protein (TRAM) has also
been shown to stimulate the translocation of some secretory polypep-
tides [124]. Furthermore the Sec63/Sec62 complex [109], the TRAP com-
plex [125], RAMP4 [47], and luminal chaperones [105] may interact with
the Sec61 complex depending on the class of substrate. The binding of
such factors may play roles in providing a chaperone-like role particularly
for TM helices during membrane protein integration into the lipid bilay-
er and may be important in regulating channel oligomerisation.
14. Membrane protein biogenesis
In addition to providing a pore through the ER membrane, the Sec61
complex also functions to allow TM helices of membrane proteins to
become incorporated into the hydrophobic environment of the lipid
bilayer, and thus plays a vital role in the folding of such proteins. A
detailed overview of this aspect of translocon function is beyond the
scope of this review and readers are directed to [126]. TM segments
adopt an α-helical confirmation before emerging from the ribosome
[127]. When a TM helix is presented to the translocon during transloca-
tion, the process is interrupted to enable helix recognition and transfer
to the lipid bilayer. Integration of a simple type II (Ncyt-Clum) membrane
protein resembles the translocation of a secreted protein [128] with the
TM helix intercalating with the lateral gate TM2b and TM7 domains
allowing TM helix exit from the translocon and integration into the
2400 Y. Nyathi et al. / Biochimica et Biophysica Acta 1833 (2013) 2392–2402
lipid bilayer. The thermodynamic properties of a TM helix itself are a
key factor driving membrane integration [129,130]. Furthermore, it
has been shown that when a TM helix emerging from the ribosome
exit site engages with the translocon it is subjected to a biphasic force
[131]. This force is exerted firstly as the TM helix reaches the translocon,
but has not yet fully emerged from the ribosome and secondly when
the helix has completely entered the translocon pore and is partitioning
into the lipid bilayer [131].
The biogenesis of polytopic membrane proteins (PMPs) adds
considerable levels of complexity to translocon function. Alternating
Nlum-Ccyt (type I) and Ncyt-Clum (type 1I) orientated helices have to
be assembled and the intervening loops have to be located on the cor-
rect side of the membrane. This requires the interaction of the nascent
polypeptide with the ribosome, translocon and associated proteins in
a manner dependent on the structural features of the nascent chain. A
model has been developed in which each TM helix recognised by the ri-
bosome exit tunnel enables ribosome binding to co-ordinate alternate
cycles of translocon opening and gating by BiP/translocon J-protein
during the biogenesis of a PMP [132]. In addition, crosslinking studies
have shown that up to four helices of aquaporin [133] and three
helices of opsin [134] can contact Sec61α at a time suggesting that
the translocon has sufficient flexibility to allow multiple TM helices
to occupy the channel. The retention of TM helices has been confirmed
using FRET analysis suggesting that release into the lipid bilayer is not
immediate, but instead is dependent on translation termination or the
arrival of the next TM helix in the channel [135]. Some TM helices in
PMPs have insufficient hydrophobicity to integrate into membranes
themselves, and it has been found that the integration of such TM heli-
ces is driven by neighbouring helices possessing greater hydrophobicity
and a strong oriental preference [136]. The ability of the translocon to
retain TM helices and facilitate the integration of TM helices of insuffi-
cient hydrophobicity is likely to be a key factor in ensuring the correct
topogenesis of PMPs.
15. Concluding remarks
Considerable strides have been made in understanding the mecha-
nism of the ER co-translational translocation pathway, with significant
progress having been made in elucidating the sequence of interactions
that occur between the ribosome, SRP, SR and the Sec61 translocon at a
molecular level. A series of signal sequence recognition and kinetic
proof reading steps combine to ensure that co-translational translocation
occurs efficiently and with high fidelity. However, despite substantial
progress, much remains to be learned. For example, the role of the GTPase
activity of SRβ is poorly understood. It may only bind GTP during SR bio-
genesis, promoting stable recruitment of SRα, with GTP hydrolysis then
occurring to disassociate the SR subunits perhaps to down-regulate SR
activity. There is a paucity of data concerning the role of the SRP68/72
subunits of SRP, although it has been suggested to relay substrate binding
by the S-domain to the Alu domain of SRP to trigger elongation arrest
[39], and may also be important for SR binding and/or activation [80].
The transfer of the signal sequence from SRP54 to Sec61 is also poorly un-
derstood. At present, it is not possible to trap intermediates at this step,
which has limited our understanding of this key transition event. In
addition, the subunit stoichiometry of the translocon has been a topic of
intense debate and as discussed above it appears that under certain
conditions a single Sec61 heterotrimer is sufficient for translocation, yet
evidence suggests that many translocon complexes exist as dimers.
Hence if only one complex is required for translocation, what is the pur-
pose of such dimers? A host of accessory factors are recruited to the
translocon such as TRAM, TRAP, RAMP4 and the non-catalytic subunits
of the SPase and OST, yet the role these proteins play is currently unclear.
Moreover, the role of the ribosome in regulating the co-translational
translocation process and the recruitment of the translocation machinery,
and other protein biogenesis factors at the ER membrane awaits further
investigation. Hence, there is clearly still a great deal to understand and
discover in the field of co-translational translocation.
Acknowledgements
Work in our laboratory is funded by the Biology and Biotechnolo-
gy Research Council (BBSRC).
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