REVIEW
published: 15 April 2021
doi: 10.3389/fmolb.2021.664241
The Dynamic SecYEG Translocon
Julia Oswald 1 , Robert Njenga 1,2 , Ana Natriashvili 1,2 , Pinku Sarmah 1,2 and
Hans-Georg Koch 1*
1
Institute for Biochemistry and Molecular Biology, Zentrum für Biochemie und Molekulare Medizin (ZMBZ), Faculty
of Medicine, Albert Ludwigs Universität Freiburg, Freiburg, Germany, 2 Faculty of Biology, Albert Ludwigs Universität
Freiburg, Freiburg, Germany
Edited by:
Kürşad Turgay,
Max Planck Unit for the Science
of Pathogens,
Max-Planck-Gesellschaft (MPG),
Germany
Reviewed by:
Damon Huber,
University of Birmingham,
United Kingdom
Anastassios Economou,
KU Leuven, Belgium
*Correspondence:
Hans-Georg Koch
Hans-Georg.Koch@
biochemie.uni-freiburg.de
Specialty section:
This article was submitted to
Protein Folding, Misfolding
and Degradation,
a section of the journal
Frontiers in Molecular Biosciences
Received: 04 February 2021
Accepted: 24 March 2021
Published: 15 April 2021
Citation:
Oswald J, Njenga R, Natriashvili A,
Sarmah P and Koch H-G (2021) The
Dynamic SecYEG Translocon.
Front. Mol. Biosci. 8:664241.
doi: 10.3389/fmolb.2021.664241
The spatial and temporal coordination of protein transport is an essential cornerstone of
the bacterial adaptation to different environmental conditions. By adjusting the protein
composition of extra-cytosolic compartments, like the inner and outer membranes or
the periplasmic space, protein transport mechanisms help shaping protein homeostasis
in response to various metabolic cues. The universally conserved SecYEG translocon
acts at the center of bacterial protein transport and mediates the translocation of
newly synthesized proteins into and across the cytoplasmic membrane. The ability of
the SecYEG translocon to transport an enormous variety of different substrates is in
part determined by its ability to interact with multiple targeting factors, chaperones
and accessory proteins. These interactions are crucial for the assisted passage of
newly synthesized proteins from the cytosol into the different bacterial compartments.
In this review, we summarize the current knowledge about SecYEG-mediated protein
transport, primarily in the model organism Escherichia coli, and describe the dynamic
interaction of the SecYEG translocon with its multiple partner proteins. We furthermore
highlight how protein transport is regulated and explore recent developments in using
the SecYEG translocon as an antimicrobial target.
Keywords: SecYEG translocon, protein transport, YidC, signal recognition particle, SecA, PpiD, FtsY, stress
response
INTRODUCTION
The dynamic control of protein synthesis, folding and degradation under different environmental
conditions is essential for maintaining a functional proteome in eu- and prokaryotic cells (Mogk
et al., 2011; Song et al., 2020). Protein trafficking pathways expand this proteostasis network and
target proteins into subcellular compartments with specific folding conditions (Figure 1; Kudva
et al., 2013; Tsirigotaki et al., 2017). Cell compartmentalization is a unifying principle in all cells
and diversifies their metabolic activity by generating membrane-bordered reaction chambers.
Prokaryotes lack the sophisticated intracellular organization that is usually observed in eukaryotes,
but still maintain distinct compartments like the cytosol, the inner membrane, the periplasm and
in Gram-negative bacteria also the outer membrane (Figure 1). Each extra-cytosolic compartment
contains a dedicated protein composition which can only be maintained due to the presence
of protein transport systems that export proteins out of the cytosol. The Gram-negative model
organism Escherichia coli synthesizes approx. 4.400 different proteins1 and contains a predicted
1https://www.ncbi.nlm.nih.gov/genome/
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FIGURE 1 | The proteostasis network in bacteria. For details see text. Secretion systems refer to the type I–IX protein secretion systems that have been identified in
bacteria, although some of these secretion systems are only found in some species (Christie, 2019). IM, inner membrane; PP, periplasm; OM, outer membrane.
total number of 3–4 × 106 proteins per cell, calculated based on
cell volume, average protein mass and average cellular protein
concentration (Milo, 2013). Ribosome profiling studies suggest
that roughly one third of these proteins, accounting to approx.
1.5 × 106 proteins per cell, execute their function outside of
the cytosol (Li et al., 2014). The STEPdb databank of subcellular
topologies of E. coli polypeptides2 lists approx. 1,000 different
inner membrane proteins, approx. 400 periplasmic proteins and
approx. 160 outer membrane proteins (OMPs) (Loos et al., 2019),
all of which have in common the requirement for dedicated
protein transport systems. N-terminal, cleavable signal sequences
in secretory proteins and non-cleavable signal anchor sequences
in inner membrane proteins provide the means to identify those
proteins that have to be exported (Pugsley, 1990; von Heijne,
1994; Hegde and Bernstein, 2006; Steinberg et al., 2018).
The majority of exported proteins engage the SecYEG
translocon, a universally conserved protein transport channel
2
that resides in the inner bacterial membrane and facilitates the
insertion of membrane proteins into the inner membrane as well
as the translocation of proteins across the inner membrane into
the periplasm (Figure 1; Kudva et al., 2013; Denks et al., 2014).
The heterotrimeric SecYEG translocon consists of SecY, SecE, and
SecG as core proteins, but constitutes only a passive and sealed
pore that connects the cytoplasm to the periplasm and the lipid
phase of the membrane. For being active in protein transport, the
SecYEG translocon depends on the coordinated interaction with
multiple partner proteins that select potential SecYEG substrates
(Lill et al., 1990; van der Does et al., 1996; Angelini et al.,
2005), provide the driving force for protein transport (Tsukazaki
et al., 2011; Knyazev et al., 2018), coordinate substrate release
from the SecYEG channel (Beck et al., 2001; Houben et al.,
2004; Sachelaru et al., 2017) and communicate with components
of the proteostasis network (Kihara et al., 1996; Schäfer et al.,
1999; Jauss et al., 2019). The SecYEG translocon also cooperates
with additional protein transport systems (Figure 1), like the
YidC insertase (Scotti et al., 2000; Sachelaru et al., 2015, 2017;
https://stepdb.eu/
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FIGURE 2 | SecA- and SRP-dependent protein targeting in bacteria. The SecA- and SRP-dependent protein targeting pathways constitute the two main protein
targeting pathways in bacteria and both can operate in a co- or post-translational mode. However, post-translational targeting of secretory proteins by SecA and
co-translational targeting of membrane proteins by SRP are the preferred modes. Substrates of the post-translational SecA pathway are kept in a translocation
competent state by chaperones, like the ribosome-bound TF or the cytosolic SecB. SecA serves as receptor for signal sequences (shown in red) of secretory
proteins and is bound to the SecYEG translocon, which serves as main protein transport channel in bacteria. Repetitive ATP hydrolysis cycles by SecA allows for the
translocation of the polypeptide across the SecY channel. SecA can also associate with the ribosome and target potential substrates co-translationally to the
SecYEG translocon. The subsequent ATP-dependent translocation likely occurs then post-translationally, i.e., after the substrate is released from the ribosome. SRP
binds with high affinity to translating ribosomes and traps the signal anchor sequence of a membrane protein when it emerges from the ribosomal peptide tunnel.
SRP then delivers the translating ribosome (ribosome-associated nascent chain, RNC) to the SRP receptor FtsY. FtsY serves as SecYEG-bound receptor for
nascent membrane proteins and engages similar binding sites as SecA on the SecYEG translocon. After SRP-FtsY contact, the translating ribosome docks onto the
SecYEG translocon and ongoing translation inserts the protein into the lipid phase. FtsY can also associate with the YidC insertase and SRP can deliver less
complex membrane proteins co-translationally to the YidC insertase for insertion. Small membrane proteins (<50 amino acids) and likely tail-anchored membrane
proteins are post-translationally bound by SRP and targeted to SecYEG or YidC only after they have been released from the ribosome. This post-translational
insertion by SRP is likely initiated by a so far largely uncharacterized mRNA-targeting step (Steinberg et al., 2020), which is not depicted in this cartoon.
protein targeting primarily acts on secretory proteins that contain
a cleavable N-terminal signal sequence and this pathway is
generally described as post-translational event (Figure 2). In
contrast, inner membrane proteins with non-cleavable signal
anchor sequences engage the signal recognition particle (SRP)dependent targeting pathway, which operates primarily cotranslationally and involves the ribosome-bound SRP (Pool et al.,
2002; Gu et al., 2003; Halic et al., 2004; Schaffitzel et al., 2006)
and the SecYEG-bound SRP receptor FtsY (Angelini et al.,
2005, 2006; Kuhn et al., 2015; Draycheva et al., 2016; Steinberg
et al., 2018; Figure 2). The SRP pathway can deliver membrane
proteins also to the YidC insertase (Welte et al., 2012; Dalbey
et al., 2017; McDowell et al., 2021), which can insert membrane
proteins independently of SecYEG but also cooperates with the
SecYEG translocon (Houben et al., 2000; Scotti et al., 2000;
Serek et al., 2004; du Plessis et al., 2006; Yuan et al., 2007;
Sachelaru et al., 2015, 2017; Dalbey et al., 2017). It is important to
emphasize that the classification into post-translational targeting
by SecA and co-translational targeting by SRP does not apply
Dalbey et al., 2017; Petriman et al., 2018), the Tat transport
machinery (Keller et al., 2012; Kudva et al., 2013; Tooke et al.,
2017) and the Bam complex (Wang et al., 2016; Alvira et al.,
2020), which inserts β-barrel proteins into the outer membrane.
Additional partner proteins of the SecYEG translocon have been
recently identified by proteomic approaches (Chorev et al., 2018;
Carlson et al., 2019; Jauss et al., 2019), further highlighting the
dynamic nature of the SecYEG translocon, which is probably
the basis for its ability to transport a large variety of highly
different substrates.
TARGETING THE SECYEG
TRANSLOCON
The selective recognition of SecYEG substrates is achieved by
two protein targeting systems that operate in parallel in bacterial
cells (Koch et al., 2003; Rapoport, 2007; Driessen and Nouwen,
2008; Kudva et al., 2013; Smets et al., 2019). SecA-dependent
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FIGURE 3 | Structures of SecA and SecA bound to the ribosome. (A) Structure of B. subtilis SecA (PDB 5EUL), showing its multiple domains. The two
nucleotide-binding domains NBD1 and NBD2 are shown in cyan and in olive, respectively. The peptide-binding domain (PBD) is shown in raspberry-red, the helical
wing domain (HWD) and the helical scaffold domain (HSD) in gray and the two-helix finger (2HF) in red. (B) Structure of E. coli SecA bound to a translating ribosome
(PDB 6S0K). The 50S ribosomal subunit is shown in light-blue and the nascent RodZ chain in green. Ribosomal proteins that are in contact with SecA [uL23 (blue),
uL29 (pink), and uL24 (green)] and the different domains of SecA are labeled and shown in the same color-code as in (A).
(Pohlschroder et al., 1997; Figure 3A). In E. coli it is present in
about 2,000–5,000 copies per cell (Kudva et al., 2013; Smets et al.,
2019) and therefore much more abundant than the SecYEG
complex, which exists in about 500 copies (Kudva et al., 2013).
SecA binds with high-affinity to the cytosolic loops of SecY
(Douville et al., 1995; Mori and Ito, 2006; Kuhn et al., 2011) and
to negatively charged phospholipids (Lill et al., 1990; Gold et al.,
2010; Koch et al., 2016, 2019). In addition, a fraction of SecA
is located in the cytosol (Chun and Randall, 1994; Hoffschulte
et al., 1994), where it can exist as dimer (Woodbury et al., 2002;
Banerjee et al., 2017a). The oligomeric state of membrane-bound
SecA is controversially discussed. Liposome studies indicate
that only the SecA monomer binds to phospholipids (Roussel
and White, 2020), but a SecA dimer is functional in protein
translocation (de Keyzer et al., 2005) and can function as receptor
for preproteins (Gouridis et al., 2013). It has been suggested
that one protomer is required for docking onto the SecYEG
to all substrates. A co-translational targeting by SecA has been
observed for the inner membrane protein RodZ, which contains
a large cytosolic domain preceding its single transmembrane
domain (Rawat et al., 2015; Wang et al., 2017), and for the
periplasmic maltose binding protein MBP (Huber et al., 2017).
This is in line with the ability of SecA to interact with translating
and non-translating ribosomes (Eisner et al., 2003; Karamyshev
and Johnson, 2005; Huber et al., 2011; Knupffer et al., 2019; Origi
et al., 2019; Wang S. et al., 2019). On the other hand, a posttranslational interaction of SRP has been shown for the small
bacterial membrane proteins YohP and YkgR (Steinberg et al.,
2020) and for the tail-anchored proteins DjlC, Flk, and SciP
(Pross et al., 2016; Peschke et al., 2018).
Targeting by SecA
The ATPase SecA is a multi-domain protein of 102 kDa
that is found exclusively in bacteria and chloroplasts
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with both the ribosome and with SecYEG or phospholipids
(Knupffer et al., 2019; Origi et al., 2019) and thus SecA binding
to ribosomes or to SecYEG appears to be mutual exclusive. This
suggests that co-translational targeting by SecA is followed by
a post-translational translocation across the SecYEG translocon.
This assumption is also in line with the observation that SecA
and ribosomes use almost identical binding sites on SecY (Prinz
et al., 2000; Mori and Ito, 2006; Kuhn et al., 2011; Banerjee
et al., 2017b) and that SecA and ribosomes compete for SecYEG
binding (Wu et al., 2012).
complex, while the second copy is involved in the downstream
translocation upon ATP-dependent dissociation of the dimer (Or
et al., 2002; Gouridis et al., 2013).
SecYEG-bound SecA primarily recognizes its substrates after
they have been released from the ribosome (Randall, 1983; Hartl
et al., 1990; Swidersky et al., 1990; Chun and Randall, 1994;
Fekkes et al., 1998). N-terminal signal sequences are bound via
a shallow groove within the preprotein-binding domain (PBD) of
SecA, also called preprotein cross-linking domain (PPXD) (Gelis
et al., 2007; Grady et al., 2012). The PBD domain is located close
to the two nucleotide binding domains (NBD1 and NBD2) and
dynamic movements within the PBD link substrate recognition
to ATP binding and hydrolysis (Karamanou et al., 2007; Gouridis
et al., 2013; Figure 3A). Although signal sequences are probably
the most important determinants for SecA-dependent targeting
(Hegde and Bernstein, 2006), additional sequences within the
mature domain of a secretory protein can also contribute to the
specificity of the targeting reaction (Chatzi et al., 2017). Binding
of SecA to sequences within the mature domain might be in
particular important for keeping substrates in a translocation
competent state, e.g., largely unfolded. Translocation competence
is furthermore supported by chaperones like Trigger factor (TF)
(Saio et al., 2014, 2018; Can et al., 2017; De Geyter et al., 2020)
or SecB (Bechtluft et al., 2010; Huang et al., 2016; Figure 2).
Due to its high affinity to ribosomes and its ability to bind to
the ribosomal protein uL23 (Kramer et al., 2002), TF is one of
the first contacts of the emerging nascent chain (Deuerling et al.,
1999; Bornemann et al., 2014). Different to SecA, TF does not
specifically bind to signal sequence-containing proteins but also
binds to cytosolic proteins, although β-barrel OMPs appear to
be the preferred target (Teter et al., 1999; Oh et al., 2011). It
has been shown that protein translocation of some substrates
is accelerated upon TF deletion and it was suggested that this
reflects prolonged contact between TF and these outer membrane
substrates (Lee and Bernstein, 2002). TF can also interact with
SecB and the SecYEG-bound SecA, which probably helps to
connect protein folding and protein transport (De Geyter et al.,
2020). SecB is present in proteobacteria only and like TF not
essential (Deuerling et al., 2003; Crane and Randall, 2017). It
forms a tetramer with surface-exposed hydrophobic areas, which
are involved in substrate binding (Knoblauch et al., 1999). SecB
binds only to a small number of secretory proteins and releases its
substrates upon binding to the C-terminus of SecA (Baars et al.,
2006; Crane et al., 2006; Castanie-Cornet et al., 2014).
In addition to this post-translational substrate recognition,
SecA can bind to its substrates also co-translationally (Eisner
et al., 2003; Karamyshev and Johnson, 2005; Huber et al., 2011,
2017; Figure 2). This was observed for secretory proteins, like
MBP (Chun and Randall, 1994; Huber et al., 2017), but also for
the membrane protein RodZ (Rawat et al., 2015; Wang et al.,
2017). SecA binds to the ribosome close to the ribosomal tunnel
exit, which is formed by the ribosomal proteins uL23, uL24,
and uL29 (Huber et al., 2011; Knupffer et al., 2019; Wang S.
et al., 2019; Figure 3B). This is also the binding site for SRP and
for many ribosome-associated chaperones and processing factors
(Kramer et al., 2002, 2009; Denks et al., 2017; Knupffer et al.,
2019). Importantly, it is the N-terminus of SecA that interacts
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Targeting by the SRP Pathway
The SRP pathway is a universally conserved targeting system that
bacteria primarily use for inner membrane proteins (Figure 2)
(Ulbrandt et al., 1997; de Gier et al., 1998; Valent et al., 1998;
Cristobal et al., 1999; Koch et al., 1999, 2003; Koch and Muller,
2000). In E. coli, SRP consists of the protein Ffh and the 4.5S
RNA (Figure 4A) and thus represents a basic version of the
eukaryotic SRP, which consists of six protein subunits bound to
the 7SL RNA (Koch et al., 2003). Still, the bacterial SRP and
its receptor FtsY are sufficient to support protein targeting to
mammalian endosomal membranes (Powers and Walter, 1997).
The SRP pathway in bacteria not only targets the SecYEG
translocon, but also the YidC insertase (Welte et al., 2012;
Petriman et al., 2018), which inserts less-complex membrane
proteins (Samuelson et al., 2000; Dalbey et al., 2017). Ffh and FtsY
share a homologous NG domain with highly similar architecture
and amino acid sequence (Freymann et al., 1997; Montoya
et al., 1997). The respective N-domains form a four-helix bundle
that is followed by the Ras-like GTPase domain (G-domain)
(Figure 4A). The NG-domain of Ffh is C-terminally continued
by the M-domain, which forms a flexible groove that is able to
accommodate signal anchor sequences of different lengths and
hydrophobicities. This flexibility explains why the bacterial SRP
recognizes the hydrophobic signal anchor sequences of basically
all inner membrane proteins and also the signal sequences of
some secretory proteins and amphipathic helices of integral and
membrane-associated proteins (Beha et al., 2003; Huber et al.,
2005; Maier et al., 2008; Lim et al., 2013; Schibich et al., 2016).
Substrate recognition by SRP is a multi-step process that is
initiated by SRP binding to the ribosome, where it contacts
primarily uL23, uL29, and the 23S rRNA close to the tunnel
exit (Halic et al., 2006a,b; Schaffitzel et al., 2006; Figure 4B).
SRP binds to vacant ribosomes with high affinity (K d 50–60 nM)
(Bornemann et al., 2008; Holtkamp et al., 2012) and the flexible
C-terminus of Ffh protrudes into the ribosomal tunnel where
it contacts the intra-tunnel loop of uL23 (Jomaa et al., 2016,
2017; Denks et al., 2017). This scanning mode allows SRP to
screen ribosomes for potential substrates. When translation is
initiated and the nascent chain reaches a length of approx. 25
amino acids, SRP is displaced from the intra-tunnel loop, which
now contacts the nascent chain (Denks et al., 2017). However,
SRP maintains contact to the surface-exposed domain of uL23
and this anticipatory or stand-by mode further increases the
affinity (K d 1 nM) and likely orients the M-domain for binding
to the signal anchor sequence. When the nascent chain reaches
a length of approx. 45–50 amino acids and the signal anchor
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FIGURE 4 | Structures of the SRP-FtsY-complex and the SRP-ribosome complex. (A) Structure of the E. coli SRP-FtsY complex (PDB 2XXA) (Ataide et al., 2011).
Ffh, the protein component of the bacterial SRP is shown in yellow and the 4.5S RNA in dark-blue. The domains of Ffh are indicated. The NG-domain of FtsY is
shown in green; the structure of the N-terminal A-domain of FtsY has not been solved yet and is shown as green box. (B) Structure of an SRP-RNC complex (PDB
5GAH). The 50S ribosomal subunit is shown in light-blue and the ribosomal proteins that provide the contact site for SRP are indicated, uL23 (blue), uL29 (pink),
uL24 (green), and bL32 (light-green). Ffh is shown in yellow and the 4.5S RNA in dark-blue. The nascent PhoA chain is shown in dark red.
sequence is exposed to the outside of the ribosome, SRP forms
a stable complex with the ribosome-associated nascent chain
(RNC) (K d ≤ 1 nM) (Holtkamp et al., 2012; Schibich et al., 2016;
Denks et al., 2017). The SRP-RNC complex is then targeted to the
SRP receptor FtsY. Although some initial studies proposed that
the SRP-RNC complex interacts with FtsY already in the cytosol
(Shan et al., 2007; Saraogi et al., 2014), FtsY in Gram-positive and
Gram-negative bacteria is almost exclusively membrane-bound
(Mircheva et al., 2009).
Membrane binding of FtsY is mediated by the A-domain,
which precedes the NG-domain (Figure 4A), and by a
membrane-targeting sequence at the interface of the A- and
NG-domains (de Leeuw et al., 2000; Parlitz et al., 2007; Weiche
et al., 2008; Braig et al., 2009; Erez et al., 2010; Kuhn et al.,
2011). The A-domain is highly variable in length and sequence
and so far no structural information is available, suggesting
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intrinsic flexibility (Montoya et al., 1997). The A-domain is not
essential for protein targeting in E. coli (Eitan and Bibi, 2004),
which is explained by the presence of additional binding sites
for SecY and phospholipids in the N-domain of FtsY (Parlitz
et al., 2007; Weiche et al., 2008; Braig et al., 2009; Erez et al.,
2010; Kuhn et al., 2011). However, the A-domain is important for
increasing the fidelity of the targeting reaction because it shields
the SRP binding site when FtsY is not in contact with the SecYEG
complex (Draycheva et al., 2016; Lakomek et al., 2016) and it
thus prevents futile SRP-FtsY interactions. Binding of SRP-RNCs
to the FtsY-SecYEG complex generates a transient quaternary
complex (Kuhn et al., 2015; Jomaa et al., 2017; Draycheva et al.,
2018; Figure 5). Subsequent movements of SRP expose the
SecY binding site on the ribosome (Halic et al., 2006b) and
simultaneous movements of FtsY expose the ribosome binding
site on SecY (Halic et al., 2006b; Kuhn et al., 2015). This then
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FIGURE 5 | Structure of the quaternary RNC-SRP-FtsY-SecYEG complex. Structure of the quaternary complex (PDB 5NCO), depicting an early state of
co-translational protein insertion. The subunits SecY, SecE and SecG of the SecYEG translocon are indicated by green, orange and blue color, respectively. The
color code of the FtsY-SRP complex is as in Figure 4 and the nascent PhoA is shown in dark-red. Please note that in this structure, the SecYEG translocon is only
tentatively fitted and would have to tilt by ∼20◦ to be accommodated within the membrane (Jomaa et al., 2017).
allows for the docking of the RNC onto the SecYEG translocon
and subsequent GTP hydrolysis by the FtsY-SRP complex (Egea
et al., 2004; Focia et al., 2004; Saraogi et al., 2014). GTP-hydrolysis
induces the dissociation of the FtsY-SRP complex and allows for
the next round of targeting (Egea et al., 2004; Shan et al., 2004;
Akopian et al., 2013a).
Importantly, the SecA and SRP pathways have several features
in common: (1) SecA and SRP engage the same docking site
on the ribosome and both protrude into the ribosomal tunnel
(Denks et al., 2017; Knupffer et al., 2019; Wang S. et al.,
2019). (2) FtsY and SecA are activated upon binding to anionic
phospholipids and SecY (Mircheva et al., 2009; Kuhn et al.,
2011; Stjepanovic et al., 2011; Draycheva et al., 2016; Koch
et al., 2016). (3) FtsY, SecA and the ribosome use largely
identical binding sites on SecY (Mori and Ito, 2006; Kuhn
et al., 2011, 2015). A computational approach for investigating
the early evolutionary history of protein transport systems
indicates that the SRP/FtsY targeting pathway is the most ancient
protein delivery system that probably even existed before the
last universal common ancestor (LUCA) (Harris and Goldman,
2021). As protein transport is faster than translation (Pugsley,
1990; Rodnina and Wintermeyer, 2016), the evolution of a
second targeting system in fast growing bacteria probably ensures
that secretory proteins are kept in a translocation-competent
state, when the limited number of SecYEG translocons are cotranslationally engaged by SRP-substrates.
Finally, translation-independent membrane localization of
some mRNAs encoding for membrane proteins has been
observed in bacteria (Nevo-Dinur et al., 2011; Kannaiah and
Amster-Choder, 2014; Kannaiah et al., 2019). One example is
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the small membrane protein YohP, which consists of just 27
amino acids and is predicted to be involved in the bacterial
stress response (Hemm et al., 2010). The yohP mRNA was found
to be almost exclusively membrane localized, but membrane
insertion of the YohP protein by either the SecYEG complex
or YidC still required SRP and FtsY (Steinberg et al., 2020).
SRP contacts YohP post-translationally both in vivo and in vitro
(Steinberg et al., 2020), questioning the paradigm that SRP
has to be ribosome-bound for substrate recognition. For small
membrane proteins, the post-translational recognition by SRP
can be easily explained by the fact that they are already released
from the peptidyl transferase domain of the ribosome before
they are sufficiently exposed on the ribosomal surface for cotranslational SRP recognition. Considering the rapidly increasing
number of small membrane proteins discovered in bacteria (Storz
et al., 2014; Weaver et al., 2019), the post-translational targeting
by SRP could be as abundant as the co-translational targeting
and might also be executed for C-tail anchored membrane
proteins in bacteria (Abell et al., 2004; Pross et al., 2016;
Peschke et al., 2018; Figure 2).
THE SECYEG COMPLEX IN THE
RESTING AND ACTIVE STATE
The first X-ray structure of the Sec translocon was obtained
for the homologous SecYEβ complex from the archaeon
Methanococcus janaschii and represented the resting state with
a sealed pore (Van den Berg et al., 2004). In this resting
conformation, which was later also obtained from other species
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FIGURE 6 | Structure of the SecYEG translocon in its resting state and active state. (A) Structure of T. thermophilus SecYEG in the resting state (PDB 5AWW) and
the active state (PDB 5CH4). SecY is shown in green, SecE in orange and SecG in blue. The SecY transmembrane domains that constitute the lateral gate are
shown in light green and the plug in magenta. The upper structures depict the front views of the SecYEG translocon and the lower structures the top view from the
cytosol, respectively. (B) Schematic front view and view from the cytosol of the SecYEG translocon.
SecY is rapidly degraded by the membrane protease FtsH in the
absence of SecE (Kihara et al., 1995; Lycklama a Nijeholt et al.,
2013). SecG, the third subunit of the bacterial SecYEG complex,
consists of two transmembrane domains, which are connected by
a cytosolic loop (Figure 6). SecG is not essential for cell viability,
but 1secG strains of E. coli exhibit protein transport defects
in vivo (Nishiyama et al., 1994, 1996).
Activation of the SecYEG channel and subsequent protein
transport requires opening of the lateral gate, expansion of the
pore ring and movement of the plug (Collinson et al., 2015;
Voorhees and Hegde, 2016b; Figure 6A). These movements
have been documented by additional structures and a wealth
of biochemical data. For the transport of secretory proteins,
the SecYEG channel is activated by SecA, which serves a
dual function: it acts as SecYEG bound receptor for proteins
with cleavable signal sequences and provides the energy for
translocation by multiple ATP-hydrolysis cycles (Douville et al.,
1995; Manting et al., 1997; Tomkiewicz et al., 2006; Alami et al.,
2007; Das and Oliver, 2011; Gold et al., 2013; Gouridis et al.,
2013). A first structure of a SecYEG-SecA complex (Zimmer
et al., 2008) revealed the insertion of the hairpin-like two-helix
finger (2HF) of SecA into the cytoplasmic vestibule of SecY and a
partial opening of the lateral gate. This opening is required for
intercalation of the signal sequence within the lateral gate (du
Plessis et al., 2009; Hizlan et al., 2012; Corey et al., 2016). This
is depicted in the structure of the SecYEG-SecA complex with
a covalently linked signal sequence (Li et al., 2016; Figure 7A).
(Li et al., 2007; Tsukazaki et al., 2008; Tanaka et al., 2015), SecY
is organized in two halves formed by transmembrane helices
(TMs) 1 to 5 and 6 to 10, respectively, which are connected by
a loop between TM5 and 6, termed the hinge (Figure 6). In this
clamshell-like structure, SecY forms two vestibules with a central
constriction, called the pore ring, in the middle. The pore ring is
formed by six bulky and hydrophobic isoleucine residues in E. coli
and is sealed on the periplasmic side by a short helix (TM2a;
the plug) (Figure 6B). The plug and the pore ring are important
for maintaining the membrane barrier in the resting state and
during translocation (Saparov et al., 2007; Park and Rapoport,
2011). This structural arrangement provided a first glimpse into
how the SecY channel is able to translocate proteins across the
membrane, but also to insert proteins into the membrane (Van
den Berg et al., 2004). At the front of SecY, TMs 2/3, and 7/8
constitute a flexible crevice, called the lateral gate that allows
access to the lipid phase (du Plessis et al., 2009; Hizlan et al., 2012;
Bischoff et al., 2014; Gogala et al., 2014; Figure 6A). Cytosolically
exposed loops of SecY provide the docking sites for SecA (Mori
and Ito, 2006; Das and Oliver, 2011; Kuhn et al., 2011), FtsY
(Angelini et al., 2005, 2006; Kuhn et al., 2011) and ribosomes
(Prinz et al., 2000; Frauenfeld et al., 2011; Kuhn et al., 2011).
Although these sites are not identical, they largely overlap (Kuhn
et al., 2011), which indicates that SecA, FtsY and ribosomes
compete for SecY binding (Wu et al., 2012; Kuhn et al., 2015).
The tilted TM3 of SecE further stabilizes the hinge at the back
of SecY and this appears to be crucial for its integrity because
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FIGURE 7 | Structure of the substrate-engaged SecA-SecYEG complex and model for SecA-dependent translocation across the SecYEG-translocon. (A) Structure
of the SecA-SecYEG complex from B. subtilis (PDB 5EUL). SecY and SecE are shown in green and orange, respectively, and the translocating peptide in yellow. The
different domains of SecA are indicated. 2HF corresponds to the two-helix finger. (B) Upon ATP binding to SecA, the 2-helix-finger (2HF) inserts into the SecY
channel and pushes the polypeptide into the channel. The signal sequence is depicted in red. For preventing back-sliding, the polypeptide binding domain (PBD) of
SecA rotates toward the nucleotide-binding domain (NBD2) and forms a clamp that traps the polypeptide. This step likely occurs before or simultaneously with
ATP-hydrolysis. Closing the clamp also leads to the retraction of the 2HF. After phosphate release, the clamp opens again and the polypeptide can slide deeper into
the channel but in principle also backward. In vivo, backsliding at this stage could be prevented by contacts of the polypeptide to periplasmic chaperones, like Skp
(Schäfer et al., 1999) or the PpiD/YfgM complex (Götzke et al., 2014; Jauss et al., 2019). In addition, the membrane potential is likely important for maintaining
directionality of translocation (Driessen and Nouwen, 2008; Knyazev et al., 2018). Figure was modified after (Catipovic et al., 2019).
This structure shows that the hydrophobic segment of the signal
sequence is located outside of the opened lateral gate. The
segment following this hydrophobic part is trapped between TM3
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and TM7 on the periplasmic part of the lateral gate and the signal
sequence cleavage site is located within the periplasmic vestibule.
Opening of the channel is further accompanied by movement
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Inner membrane proteins are targeted to the SecYEG
translocon co-translationally as RNCs by the SRP pathway
(Figure 2; Koch et al., 1999; Beck et al., 2000; Neumann-Haefelin
et al., 2000; Akopian et al., 2013b; Steinberg et al., 2018). The SRP
receptor FtsY docks onto the SecYEG translocon and engages
largely identical binding sites as SecA and the ribosome (Angelini
et al., 2005, 2006; Kuhn et al., 2011, 2015). FtsY and SecA have
comparable affinities for the SecYEG translocon and are present
in comparable copy numbers in E. coli (Douville et al., 1995;
Kudva et al., 2013; Kuhn et al., 2015) and it is currently unknown
how access of either FtsY or SecA to the SecYEG translocon is
regulated. Importantly, only SecY-bound FtsY exposes the SRP
binding site and is thus able to direct the SRP-RNC complex
to the SecYEG translocon (Mircheva et al., 2009; Draycheva
et al., 2016). Structural information on the isolated FtsY-SecYEG
complex is not available, but Cryo-EM structures of RNCs bound
to the Sec translocon in the presence and absence of SRP and
its receptor have been obtained from different species (Becker
et al., 2009; Frauenfeld et al., 2011; Bischoff et al., 2014; Gogala
et al., 2014; Voorhees et al., 2014; Jomaa et al., 2016, 2017;
Voorhees and Hegde, 2016a; Kater et al., 2019). Binding of a
non-translating ribosome to the Sec translocon, primarily via the
cytosolic loop C5, results in small rearrangements which slightly
open the cytosolic part of the lateral gate (Voorhees et al., 2014;
Figure 8). The structure of a quaternary ribosome-SRP-FtsYSecYEG complex revealed that FtsY aligns the ribosomal tunnel
exit with the SecYEG channel (Jomaa et al., 2017; Figures 5, 8).
The exposure of a short nascent membrane protein further
opens the lateral gate on the cytosolic side (Kater et al., 2019)
and full insertion of the signal anchor sequence leads to a
rotation of helices 2–5 and 10 and allows trapping of the signal
anchor sequence at the lateral gate (Voorhees and Hegde, 2016a;
Figure 8). Simultaneously, the plug is displaced from its position
at the pore ring and the channel is open to both the trans-side
and the lipid side of the membrane. TMs downstream of the
signal anchor sequence can exit the Sec translocon laterally one
by one or in pairs (Heinrich and Rapoport, 2003; Houben et al.,
2004; Sadlish et al., 2005). Lipid partitioning of TMs is largely
determined by their hydrophobicity (Hessa et al., 2007; White
and von Heijne, 2008) and moderately hydrophobic TMs possibly
require the interaction with a more hydrophobic second TM to
enter the lipid phase (Heinrich and Rapoport, 2003). These helixhelix interactions could occur within the Sec channel (Pitonzo
et al., 2009), at the channel-lipid interface (Sadlish et al., 2005;
Cross and High, 2009) or even before, at the end of the ribosomal
tunnel (Tu et al., 2014; Holtkamp et al., 2015; Nilsson et al.,
2015). Lateral release of transmembrane domains out of the SecY
channel is further facilitated by YidC (Beck et al., 2001; Houben
et al., 2002), which associates with the lateral gate of SecY to form
a tetrameric protein channel (Sachelaru et al., 2015, 2017).
Although there are some variations in the translocon
structure when activated by SecA or the ribosome, the step-wise
channel opening during post-translational translocation or cotranslational insertion appears to be a conserved feature of the
Sec translocon and is in line with multiple biochemical studies
(du Plessis et al., 2009; Bonardi et al., 2011; Hizlan et al., 2012;
Knyazev et al., 2013, 2014; Ge et al., 2014; Mercier et al., 2020).
of the plug to the back of the channel, where it resides close
to SecE, validating previous cross-linking studies (Harris and
Silhavy, 1999; Tam et al., 2005).
The activation of SecYEG by SecA initiates the step-wise
translocation of secretory proteins across the membrane. The
reconstituted SecYEG-SecA complex was shown to generate a
mechanical force of about 10pN (Robson et al., 2007; Gupta
et al., 2020). Consequentially, several models were proposed
on how the high conformational flexibility of SecA might be
used for the ATP-dependent and stepwise translocation of a
preprotein across the SecYEG channel (Erlandson et al., 2008a,b;
Kusters et al., 2011; Gouridis et al., 2013; Ernst et al., 2018; Fessl
et al., 2018; Corey et al., 2019; Komarudin and Driessen, 2019).
Central to most models is the 2HF-domain of SecA (Erlandson
et al., 2008a). The 2HF was shown to insert into the cytosolic
vestibule of SecY, where it resides in close proximity to the
preprotein (Zimmer et al., 2008). A highly conserved tyrosine
residue at the tip of the loop is essential for SecA function, but
immobilizing the 2HF on the SecYEG complex does not interfere
with translocation (Whitehouse et al., 2012), suggesting that
even restricted movements of the 2HF are sufficient to support
translocation. Latest data support a push-and-slide mechanism
of protein translocation that depends on a power stroke by SecA
(Catipovic et al., 2019; Catipovic and Rapoport, 2020). In this
model (Figure 7B), the 2HF moves toward the SecY channel
upon ATP binding, thereby pushing the polypeptide into the
channel. While the 2HF retracts during ATP hydrolysis from
the channel, movement of the polypeptide-binding domain of
SecA toward the nucleotide-binding domain generates a clamp
that fixes the polypeptide in the channel. Phosphate release from
SecA is suggested to open the clamp, which allows for some
passive sliding of the polypeptide until the next ATP binds
and the 2HF pushes the next segment of the polypeptide into
the channel. The observation that cross-linking the 2HF to the
cytosolic loop C4 of SecY does not impair protein translocation
(Whitehouse et al., 2012) is possibly explained by the inherent
flexibility of the large C4 loop which might still allow sufficient
movements of the 2HF.
The 2HF is also central to an alternative model for SecAdependent translocation, which suggests a Brownian ratchet
mechanism (Collinson, 2019). In this model, SecA regulates
channel opening via the 2HF, while substrate movement across
the channel occurs via Brownian movement (Allen et al., 2016,
2020). ATP hydrolysis by SecA is suggested to prevent partial
folding of substrates at the SecA-SecY interface, while the partial
folding on the periplasmic side would prevent back-sliding and
thus impose directionality to protein translocation (Fessl et al.,
2018; Corey et al., 2019).
In both models, substrate translocation is further stimulated
by the proton-motif-force (PMF), which probably adds to
vectorial translocation (Brundage et al., 1990; Nouwen et al.,
1996; Knyazev et al., 2018). Prior to completion of translocation,
the signal sequence is cleaved off by signal peptidase and the
mature domain is released into the periplasm (Josefsson and
Randall, 1981a,b; Paetzel et al., 2002). This latter step is likely
supported by periplasmic chaperones (Schäfer et al., 1999; Furst
et al., 2018; Chum et al., 2019; Mas et al., 2019) (see below).
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FIGURE 8 | Model of membrane protein insertion via the SecYEG translocon. In the resting state of the SecYEG-translocon, the lateral gate, composed of
transmembrane domains (TMs) 2/3 on one side (orange) and TMs 7/8 (blue) on the other side, is closed. Binding of the translating ribosome to the cytosolically
exposed loop connecting TM 6 and 7 of SecY (C5-loop, not shown), causes the lateral gate to slightly open, which is then primed for the approaching nascent
chain. The emerging nascent membrane protein (red) disrupts contacts between TM 2 and TM 7 on the cytosolic side of the membrane further, while TM 7 moves
closer toward TM 3 on the periplasmic side. This creates a V-shaped crevice during the early state of insertion. This state is likely further stabilized by the two
N-terminal TMs of SecE (not shown). Ongoing chain elongation positions the hydrophobic core of the signal peptide (red zylinder) at the lateral gate, where it
occupies approx. the same position as TM 2 in the resting SecYEG channel, before it is released into the membrane.
with SecA (Zimmer et al., 2008) or YidC (Boy and Koch,
2009; Sachelaru et al., 2017), and as heterohexameric complexes
with SecDFYajC (Duong and Wickner, 1997) or FtsY-SRPRNCs (Jomaa et al., 2017). Finally, a heteroheptameric SecYEGSecDFYajC-YidC complex was characterized and referred to as
Holo-translocon (HTL) (Schulze et al., 2014; Komar et al., 2016).
Several additional partner proteins have been identified, like the
YfgM-PpiD chaperone complex (Antonoaea et al., 2008; Götzke
et al., 2014; Sachelaru et al., 2014; Furst et al., 2018; Jauss et al.,
2019), or the cytosolic protein Syd, which is suggested to serve
together with the protease FtsH in quality control of the Sec
translocon (Akiyama et al., 1996; Dalal et al., 2009; Table 1 and
Figure 9). Non-proteinaceous partners are equally important
for SecYEG function, like anionic phospholipids and cardiolipin
(Prabudiansyah et al., 2015; Collinson, 2019; Bogdanov et al.,
2020; Ryabichko et al., 2020) or the glycolipid MPiase, which was
shown to support protein transport via the SecYEG translocon
(Moser et al., 2013; Nishiyama and Shimamoto, 2014). The highly
dynamic equilibrium between different SecYEG assemblies likely
allows the SecYEG complex to adapt to a wide variety of different
substrates and to different physiological conditions.
It is, however, currently unknown how channel opening and
transport across the SecYEG translocon works for membrane
proteins that are co-translationally targeted by SecA, like RodZ
(Rawat et al., 2015; Wang et al., 2017; Figure 3). A simultaneous
binding of SecA and the ribosome to SecY appears unlikely,
considering that both engage overlapping binding sites on SecY
(Kuhn et al., 2011). One possibility is that SecA starts inserting
RodZ only after it is released from the ribosome. In this
case, only targeting would occur co-translationally, while the
actual insertion would be post-translationally. A similar situation
is encountered during co-translational insertion of membrane
proteins with large periplasmic loops, because their translocation
requires SecA (Neumann-Haefelin et al., 2000; Deitermann
et al., 2005). How the access of SecA to these loops during
co-translational insertion is coordinated is currently unknown.
Finally, how the SecYEG translocon handles small membrane
proteins that are post-translationally targeted by SRP (Steinberg
et al., 2020; Figure 2), i.e., when neither the ribosome nor SecA
are involved, requires further analyses.
THE SECYEG INTERACTION NETWORK
YidC
The Sec translocon in bacteria and eukaryotes is organized as a
highly modular protein complex and multiple different entities
have been structurally and biochemically characterized (Zimmer
et al., 2008; Boy and Koch, 2009; Frauenfeld et al., 2011; Denks
et al., 2014; Komar et al., 2016; Kater et al., 2019). The E. coli
SecYEG translocon was found to exist as a functional monomer
(Menetret et al., 2007; Kedrov et al., 2011; Park and Rapoport,
2012) and as a dimer stabilized by cardiolipin (Gold et al., 2010).
SecYEG was furthermore found in heterotetrameric complexes
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YidC is an inner membrane protein with six TMs in E. coli and a
Nin -Cin -topology (Figure 10A). It belongs to a conserved group
of proteins with homologues in mitochondria, chloroplasts, the
endoplasmic reticulum and archaea (Borowska et al., 2015;
Anghel et al., 2017; Kuhn and Kiefer, 2017; McDowell et al.,
2021). Although YidC can act as SecYEG-independent insertase
for some membrane proteins (Samuelson et al., 2000; Luirink
et al., 2001; Serek et al., 2004; Welte et al., 2012), it also associates
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TABLE 1 | Interaction partners of the SecYEG translocon.
Protein/protein complex Function
Method of identification
References
Outer membrane
BAM complex
Folding and insertion of OMPs into the
OM
Site-directed cross-linking, EM,
pull-down, protein modeling
Wang et al., 2016; Alvira et al., 2020; Jin, 2020
Skp
Periplasmic chaperone
Cross-linking, translocation
intermediates
Schäfer et al., 1999; Jauss et al., 2019
Inner membrane
F1 F0 -ATPase
ATP synthesis coupled proton transport Native MS, peptidiscs
Tat complex
Twin-arginine translocation system
In vitro transport studies, qAP/MS
Chorev et al., 2018; Young et al., 2020
Keller et al., 2012; Tooke et al., 2017; Jauss et al., 2019
YajG
Putative lipoprotein
SEC-PCP-SILAC
Carlson et al., 2019
YibN
Putative sulfurtransferase
qAP/MS; SEC-PCP-SILAC
Carlson et al., 2019; Jauss et al., 2019
YicN
Unknown function
qAP/MS; His-tagged peptidiscs
Carlson et al., 2019; Jauss et al., 2019
YidD
Putative membrane protein insertion
efficiency factor
In vitro transport/cross-linking
Yu et al., 2011
FtsH
Protease, regulated by the FtsH
inhibitor YccA
Co-purification; qAP/MS
Akiyama et al., 1996; van Stelten et al., 2009; Jauss
et al., 2019
Hsp70/Hsp40 chaperone
qAP/MS
Stenberg et al., 2005; Wickstrom et al., 2011b;
Castanie-Cornet et al., 2014; Jauss et al., 2019
Cytoplasm
DnaK/DnaJ
GroEL
Hsp60 chaperone
Suppressor screen
Danese et al., 1995; Castanie-Cornet et al., 2014
Syd
Membrane associated regulator of
SecY function
Suppressor screen, MS
Shimoike et al., 1995; Matsuo et al., 1998; Dalal et al.,
2009; Zhang et al., 2012
Listed are potential interactors of the SecYEG translocon, their localization in the cell, their putative function based on gene ontology (GO) assignments (http:
//geneontology.org/) and the method that was used for identifying these interactors. Only those interacting proteins are listed for which a detailed characterization of
the interaction with the SecYEG complex is still missing. qAP/MS, quantitative affinity purification-mass spectrometry; EM, electron microscopy; SEC-PCP-SILAC, size
exclusion chromatography-protein correlation profiling-Stable isotope labeling of amino acids in cell culture.
with the SecYEG complex (Scotti et al., 2000; Nouwen and
Driessen, 2002; Li et al., 2013; Sachelaru et al., 2015, 2017).
The conserved TMs 2 to 6 of YidC are organized as a
globular helix bundle that forms a hydrophilic groove within the
membrane, while the structure and position of TM1 is unknown
(Kumazaki et al., 2014a,b; Figure 10A). The hydrophilic groove
is blocked on the periplasmic side of the membrane by the
short amphipathic EH1 helix, which is oriented in parallel to
the membrane surface. The EH1 helix is part of the large P1loop that connects TM1 and TM2 on the periplasmic side (Saaf
et al., 1998; Oliver and Paetzel, 2008; Ravaud et al., 2008). On
the cytosolic side of TM2, the C1-loop forms a helical coiled-coil
domain that is essential for YidC function (Geng et al., 2015).
The hydrophilic groove likely faces the TM domains of SecY
and cross-link data demonstrate that TM1, TM3 and TM5 of
YidC are in close contact to the lateral gate of SecY (Sachelaru
et al., 2015; Petriman et al., 2018). YidC can even enter the
SecY channel (Sachelaru et al., 2017) and this is achieved via
the flexible TM1 and the P1-loop that reaches deep into the
periplasmic cavity of SecY, where it makes contact to the plug
domain of SecY (Jauss et al., 2019). TM1 was also found in contact
with SecG, supporting its intrinsic flexibility (Petriman et al.,
2018). Further contacts between YidC and SecY were observed
for the C1-loop, while the P1-loop also contacts SecG, SecE and
SecF. The C1-loop also provides the docking site for FtsY and is
essential for the insertase function of YidC (Geng et al., 2015),
but SecY-YidC contacts are maintained even in the absence of
the C1-loop (Petriman et al., 2018). Crystallization and molecular
dynamics simulations demonstrate that the C2 loop linking TM4
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and TM5 is highly flexible (Tanaka et al., 2018). Together with the
C-terminus of YidC, the C2-loop provides the ribosome binding
site of YidC (Geng et al., 2015) and shields the hydrophilic
groove on the cytosolic side (Tanaka et al., 2018). The intimate
contact between the hydrophilic groove of YidC and the lateral
gate of SecY provides further support for the concept that TMs
leaving the SecY channel are first bound by YidC before they are
released into the lipid phase (Beck et al., 2001; Houben et al.,
2002). TMs exit the SecY channel in most cases sequentially
(Serdiuk et al., 2019) and the hydrophilic groove of YidC probably
reduces the hydrophobicity of the adjacent lateral gate of SecY
and therefore further stimulates the release of the TMs into the
inner membrane by a greasy slide. The amphipathic helix EH1
could act as a mechanical switch, tilting TM3 and supporting
substrate release (Dalbey et al., 2017; He et al., 2020).
The SecDFYajC Complex
The inner membrane proteins SecD, SecF and YajC form a stable
complex (Pogliano and Beckwith, 1994a,b) and were shown to
interact with SecYEG and YidC (Duong and Wickner, 1997;
Nouwen and Driessen, 2002). Depletion of SecDF causes cold
sensitivity and the accumulation of precursor proteins in the
cytosol, supporting their role in stimulating protein translocation
across the membrane (Pogliano and Beckwith, 1994a). SecD
mutants also lead to elevated levels of SecA (Rollo and Oliver,
1988), which is a typical sign of impaired protein translocation
(Ito et al., 2018).
The crystal structure of the SecDF complex shows 12 TMs, six
each in SecD and SecF, and three periplasmic domains, termed
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FIGURE 9 | Schematic view on the protein interaction network of the E. coli SecYEG complex. Interactions within the inner membrane are shown in blue boxes,
those that take place at the cytosolic phase of the inner membrane in orange boxes, those at the periplasmic side of the inner membrane in a green box, and those
with the outer membrane are boxed in yellow. For details see text.
P1-head, P1-base and P4 (Tsukazaki et al., 2011; Figure 10B).
The P1-head can undergo a large rotation, resulting in two
distinct conformations, the F- and I-form. An amphiphilic cavity
within the P1-head was proposed to bind precursor proteins
(Furukawa et al., 2017, 2018). As protein translocation is strongly
dependent on the PMF (Driessen and Wickner, 1991; Mori
and Ito, 2003; Corey et al., 2018; Knyazev et al., 2018), PMFdriven conformational changes of the P1-head could help to
pull substrates out of the SecYEG channel (Tsukazaki et al.,
2011; Tsukazaki, 2018). This is in line with the assumption
that the SecDF complex is necessary at a later stage of protein
translocation (Pogliano and Beckwith, 1994a; Tsukazaki, 2018).
The predicted low abundance of the SecDFYajC complex in E. coli
(Pogliano and Beckwith, 1994a,b) suggests that such a pulling
is only required for particular substrates or that other proteins
execute a similar function, e.g., the YfgM-PpiD complex that
also associates with the SecYEG translocon (Götzke et al., 2014;
Sachelaru et al., 2014; Jauss et al., 2019).
SecF interacts with the P1-loop of YidC and the nonconserved residues 215–265 in the P1-loop are sufficient for SecF
interaction (Xie et al., 2006), but these residues are not required
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for YidC function (Jiang et al., 2003). The phenotype of a secDF
depletion strain can be rescued by YidC-overproduction, further
supporting a cooperation between SecDF and YidC (Nouwen
and Driessen, 2002; Li et al., 2013). The SecDF complex likely
stabilizes the SecYEG-YidC interaction (Nouwen and Driessen,
2002; Tsukazaki, 2018), although the SecYEG-YidC interaction
is also observed in the absence of the SecDFYajC complex (Boy
and Koch, 2009; Sachelaru et al., 2015). Finally, SecDF might
also play a role in efficient maturation and folding of OMPs
(Alvira et al., 2020) and it was proposed that SecDF is part of
an inter-membrane trafficking machinery that connects transport
processes across the inner membrane with those at the outer
membrane (Alvira et al., 2020) (see below).
The Holo-Translocon
The existence of a HTL was first shown after co-expression and
purification of its seven constituents (Schulze et al., 2014). The
HTL comprises the core SecYEG translocon and its ancillary
subunits SecDFYajC and YidC, forming a heteroheptameric
complex (Schulze et al., 2014; Botte et al., 2016; Komar et al.,
2016). The periplasmic domains of SecDF and YidC are localized
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FIGURE 10 | Structures of YidC, SecDF and a model of the holo-translocon. (A) Structure of YidC from E. coli (PDB 6AL2). The conserved transmembrane domains
(TMs) 2 to 6 of YidC are indicated (TM2, light blue; TM3, yellow; TM4, orange; TM5, light pink; TM6, red), while the structure of TM1 is still unknown. The short
amphipathic helix EH1 is depicted in dark blue, the periplasmic loop P1 in dark green and the cytoplasmic loop C1 in light green. (B) Structure of the SecDF complex
from Thermus thermophilus (PDB 5YHF). SecDF consists of 12 TMs, six each in SecD (TM1-6, pink) and SecF (TM7-12, green), and three periplasmic domains,
termed P1-head (dark blue), P1-base (light blue) and P4 (yellow). (C) Modell of the holo-translocon based on the cryo-EM structure from E. coli (PDB 5MG3). SecY is
shown in green, SecE in orange and SecG in blue, its ancillary subunits SecD in pink, SecF in green and YidC in yellow.
of theoretically possible HTLs, but not the real number in the
E. coli membrane.
on top of the SecY channel and are suggested to interact with
emerging substrates, potentially preventing their backsliding
(Botte et al., 2016; Figure 10C). The seven subunits of the HTL
are arranged around a central lipid-filled chamber, which might
provide a flexible and protected environment for TMs to fold, to
acquire their final topology and to assemble (Goder et al., 1999;
Dowhan et al., 2019; Martin et al., 2019). The presence of the
lipid chamber could also promote the assembly of membrane
protein complexes, a function that was assigned to YidC when
in complex with SecYEG (Wagner et al., 2008). This concept
would attribute the HTL a particular role during membrane
protein insertion and indeed in vitro studies showed that the
HTL was more efficient in protein insertion and less effective
in SecA-dependent protein secretion than the SecYEG complex
(Schulze et al., 2014). However, in these studies the HTL also
increased the insertion of proteins that were classified as SecYindependent, like the phage protein Pf3 or subunit c of the
F1 F0 ATPase (Serek et al., 2004; van der Laan et al., 2004). The
abundance of the HTL in the E. coli membrane is not entirely
clear. Initial estimations suggested that the SecDF complex is
present in only 30–100 copies per cell and thus about 10 times
less abundant than SecYEG (Pogliano and Beckwith, 1994a,b). In
contrast, ribosome profiling data indicated a 4:1 SecYEG:SecDF
ratio (Li et al., 2014) and a recent proteomics study even proposed
a 1:1 ratio (Schmidt et al., 2016). Considering that the HTL is only
one of several SecYEG assemblies, it is important to emphasize
that these absolute numbers would only predict the number
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The Interaction of the SecYEG Complex
With Periplasmic Chaperones and the
Outer Membrane
The interaction of the SecYEG complex with periplasmic
chaperones was first shown for Skp and it was suggested that Skp
could facilitate substrate release from the SecY channel (Schäfer
et al., 1999; Harms et al., 2001; Figure 9). A similar function
was also proposed for the membrane-anchored periplasmic
chaperone PpiD, which was found to contact a secretory protein
exiting SecY (Antonoaea et al., 2008). PpiD forms a complex
with YfgM, which contains like PpiD a single TM and a large
periplasmic domain (Maddalo et al., 2011; Götzke et al., 2014).
YfgM was also found as contact partner of SecYEG and the
PpiD-YfgM complex was suggested to mediate substrate transfer
from the SecYEG complex to other periplasmic chaperones, like
SurA, Skp, or DegP (Götzke et al., 2014; Furst et al., 2018).
PpiD contacts the lateral gate of SecY (Sachelaru et al., 2014)
and its periplasmic domain deeply inserts into the periplasmic
cavity of the SecY channel (Jauss et al., 2019). When the plug
domain of SecY is deleted, the interaction between SecYEG and
PpiD is enhanced both at the lateral gate as well as in the
channel interior which suggests that channel opening controls
the SecY-PpiD contact. These SecY-PpiD contacts as revealed
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loops of SecDF, YidC, and the BAM complex (Alvira et al., 2020).
The periplasmic domain of SecD has multiple contact sites with
BamBCD, while YidC interacts with BamABCD. Furthermore,
there might be a potential interaction between YajC and BAM
(Carlson et al., 2019). In contrast, the SecYEG complex alone
is not able to directly interact with the BAM complex, probably
due to the lack of large periplasmic domains. The HTL-BAM
complex is further stabilized by cardiolipin (Alvira et al., 2020),
which was already shown to be important for SecYEG complex
stability (Gold et al., 2010; Ryabichko et al., 2020). A yet unsolved
question is how OMPs are transported to and inserted into the
OM without any apparent energy source due to the lack of ATP in
the periplasm and the absence of an ion gradient across the outer
membrane (Konovalova et al., 2017). The interaction between
the HTL and BAM could facilitate the energetic coupling of
inner membrane with outer membrane transport. Once OMP
precursors are translocated across the SecYEG complex and the
signal sequence is cleaved, the mature but yet unfolded protein is
bound by periplasmic chaperons, such as PpiD (Antonoaea et al.,
2008) and is then recognized by the BAM complex, forming a
trans-periplasmic supercomplex with SecDF as potential energy
supplier (Carlson et al., 2019; Alvira et al., 2020).
by site-directed in vivo cross-linking are basically identical to
the detected SecY-YidC contacts, which indicates that SecY can
either interact with YidC or PpiD. However, PpiD and YidC
show non-competitive binding to the SecYEG translocon in vivo
(Jauss et al., 2019), pointing to the possible presence of two
distinct SecYEG populations. This is also supported by BlueNative PAGE analyses, which found SecYEG either in contact
with YidC or PpiD/YfgM (Götzke et al., 2014) and by data
showing that the SecY-PpiD contact is lost when SecY is engaged
in inserting a membrane protein (Sachelaru et al., 2014). PpiD
contains an inactive prolyl-isomerase domain in its periplasmic
loop (Weininger et al., 2010) and does not seem to execute
any pulling force on SecY substrates (Jauss et al., 2019). Still
it improves translocation efficiency and the release of newly
translocated substrates into the periplasm, possibly by preventing
their backsliding into the periplasmic cavity of SecY (Furst et al.,
2018). PpiD was also found to cross-link to the periplasmic
chaperone SurA, providing further evidence for a role of PpiD
in connecting the translocation machinery to the periplasmic
folding machinery (Wang et al., 2016).
After their translocation across the inner membrane, β-barrel
OMPs have to be inserted into the outer membrane (Bos et al.,
2007; Konovalova et al., 2017). The β-barrel assembly machinery,
the BAM complex, is localized in the outer membrane (OM) and
facilitates the folding and insertion of OMPs into the OM (Ranava
et al., 2018; Ricci and Silhavy, 2019). The complex has a molecular
mass of around 203 kDa and comprises the core protein BamA
and the four additional lipoprotein subunits BamBCDE (Noinaj
et al., 2017; Figure 1). BamA contains a β-barrel domain and five
polypeptide-transport-associated (POTRA) domains protruding
into the periplasm. Even though only BamA and BamD are
essential in vivo, all five subunits are necessary for unrestrained
function of the complex (Iadanza et al., 2016).
The passage of OMPs from the SecYEG translocon to the
BAM complex has been analyzed in multiple studies (reviewed
in (Ricci and Silhavy, 2019). A direct interaction between the
SecYEG translocon and the BAM complex was first suggested
when a supercomplex consisting of BamA, BamB, SurA, PpiD,
SecY, SecE, and SecA was found by native gel electrophoresis
(Wang et al., 2016). Furthermore, cross-links between the
periplasmic chaperone SurA and BamA consolidated the idea
that translocation of OMPs across the inner membrane, passage
through the periplasm and the insertion into the OM could be
physically linked (Wang et al., 2016). BamA was furthermore
found to co-purify with the Sec translocon (Jauss et al., 2019)
and interactions between SecY and BamACD were identified in
a peptidisc approach combined with affinity purification/massspectrometry (Carlson et al., 2019). The existence of connecting
structures between the inner and outer membranes (so called
Bayer’s patches) that could aid the biogenesis of OMPs were first
postulated by Bayer (1968). However, they were controversially
discussed since their discovery, although some biochemical
evidence pointed to the existence of contact points between the
outer and inner membrane (Ishidate et al., 1986; Kellenberger,
1990; Malinverni and Silhavy, 2011). This was recently verified by
showing the interaction of the HTL with the BAM complex. This
transient contact was shown to be conferred by the periplasmic
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Further Contacts of the SecYEG
Complex
Functional and proteomic studies have identified several
additional proteins as potential contact partners of the SecYEG
complex (Kuhn et al., 2011; Carlson et al., 2019; Jauss et al., 2019),
although the functional relevance of some of these interactions
require further analyses (Table 1 and Figure 9).
The cytosolic protein Syd was shown to stabilize overexpressed
SecY in E. coli (Shimoike et al., 1995) and to prevent access
of SecA to an altered SecYEG translocon (Matsuo et al., 1998).
Syd is suggested to bind to the C4 and C5 loops of SecY (Dalal
et al., 2009), which are also part of the SecA binding site (Mori
and Ito, 2006; Kuhn et al., 2011) and it appears that binding of
SecA and Syd to SecY is mutually exclusive (Dalal et al., 2009).
The SecY-Syd interaction could provide a quality control system
for the correct assembly of the SecYEG complex, probably in
conjunction with the essential zinc-metalloprotease FtsH (Kihara
et al., 1995; Ito and Akiyama, 2005).
A cooperation between the SecYEG translocon and the Tat
transport system for folded proteins (Kudva et al., 2013) was
observed in Streptomyces coelicolor (Keller et al., 2012). Here,
the first two TMs of the Rieske iron-sulfur protein are inserted
via the SecYEG translocon, while TM3 is dependent on the
Tat machinery. The dual requirement for the Sec- and Tatmachinery appears to be common for membrane proteins that
contain globular, co-factor containing extracytoplasmic domains
(Tooke et al., 2017), which are abundant in both Gram-positive
and Gram-negative bacteria. TatA was also found co-purifying
with the SecYEG translocon in E. coli, supporting the concept
of a widespread cooperation between the Sec and Tat transport
systems (Jauss et al., 2019).
The F1 F0 -ATPase was also shown to interact with the SecYEG
complex (Chorev et al., 2018) and subunit b of F1 F0 -ATPase
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FIGURE 11 | Cellular response to impaired protein transport. Depletion of SRP/FtsY or SecYEG induce a multifaceted response. This includes membrane
stabilization via the induction of the phage-shock response (PspABC complex), the inhibition of translation via the induction of the ribosome-modulation factor (RMF)
upon FtsY depletion and the induction of the σ32 -response via the accumulation of misfolded proteins. Increased chaperone and protease production reduce the
cellular concentration of misfolded proteins and provide a negative feedback loop for declining the σ32 response. Chaperones inhibit σ32 directly and the membrane
bound protease FtsH degrades σ32 . Membrane targeting of σ32 for degradation by FtsH is dependent on SRP/FtsY and SecYEG. Thus, upon SRP/FtsY or SecYEG
depletion/saturation, elevated σ32 levels persist. FtsH also degrades misfolded/aggregated membrane proteins and SecY that is not in complex with its partner
protein SecE. Ffh, the protein subunit of SRP is also a substrate of the Lon protease; in particular when Ffh is in excess over the 4.5S RNA, the RNA subunit of the
bacterial SRP. “+” indicates increased production, “–” indicates reduced production, inhibition or degradation.
was enriched in a peptidisc approach (Young et al., 2020).
The interaction of the protein translocation machinery with
components of the respiratory chain has been extensively studied
in the mitochondrial inner membrane (Pfanner et al., 2019), but
the physiological importance of these interactions in the bacterial
membrane requires further analyses.
YibN and YicN are two single-spanning membrane proteins
of approx. 15 kDa that co-purify with SecYEG (Jauss et al., 2019;
Young et al., 2020), but their functions have not been elucidated.
A possible role of YibN in protein transport is supported by the
observation that YibN is up-regulated when YidC is depleted
(Wickstrom et al., 2011b) and in particular enriched when the
SecYEG translocon is purified from secDF-depleted E. coli strains
(Young et al., 2020). Nevertheless, the exact role of YibN/YicN in
the translocation machinery and how they interact with the Sec
translocon has still to be examined.
generated for some of the respective genes and were analyzed
for transcriptomic or proteomic responses. The depletion of SRP
induces the σ32 -response and leads to an up-regulation of several
chaperones and proteases, like DnaK, GroEL, GroES, ClpB,
IbpA, and FtsH (Bernstein and Hyndman, 2001; Wickstrom
et al., 2011a; Figure 11). It furthermore induces the phageshock protein A (PspA), which is generally associated with inner
membrane damage (Manganelli and Gennaro, 2017). However,
it does not lead to increased levels of stress-induced periplasmic
proteins, like DegP or Skp (Wickstrom et al., 2011a), suggesting
that the σE -dependent cell envelope stress response is not induced
(Hews et al., 2019). This is rather surprising, because the insertion
of SecY is dependent on the SRP/FtsY pathway (Koch and Muller,
2000) and SRP depletion should reduce the levels of SecY, which
subsequently should impair the translocation of OMPs (Kudva
et al., 2013). On the other hand, by promotor fusion experiments
it was shown that impaired SecY activity is not strictly linked
to the induction of the cell envelope stress response (Shimohata
et al., 2007). It appears likely that the σE -dependent cell envelope
stress response is only induced upon prolonged SRP-depletion
or when SecYEG-dependent transport largely ceased. The upregulation of chaperones and proteases is also observed in
a conditional FtsY-depletion strain. However, FtsY-depletion
CONNECTING PROTEIN TRANSPORT
TO THE PROTEOSTASIS NETWORK
The SecYEG translocon, SecA, SRP, and FtsY are essential
for cell viability, but conditional depletion strains have been
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recently shown that membrane targeting of σ32 is dependent
on SRP, FtsY, and SecY (Lim et al., 2013; Miyazaki et al., 2016;
Figure 11). Thus, depletion of SRP/FtsY increases the stability of
σ32 by reducing its degradation via FtsH. This allows for increased
chaperone and protease production when the SRP pathway or
the SecYEG translocon are saturated and links protein transport
directly to the proteostasis network.
The levels of SecY and SecE in E. coli are slightly higher on rich
media compared to minimal media and are reduced in stationary
phase (Yang et al., 2013; Crane and Randall, 2017). Thus, the
expression of secY and secE seem to mimic the expression of
house-keeping genes. A similar observation was made for secDF
expression in S. coelicolor (Zhou et al., 2014). This is different
for SecA; here an intriguing mechanism has been identified
that allows E. coli to tailor SecA-levels to reduced translocation
activity of the SecYEG translocon (Ito et al., 2010; Ito and
Chiba, 2013). This was first recognized by studies showing that
partial inactivation of SecYEG-dependent translocation by secY
mutations or by adding the SecA-inhibitor sodium azide, led
to an up-regulation of SecA (Oliver and Beckwith, 1982; Rollo
and Oliver, 1988). This regulation is achieved by the product
of the upstream secM gene, which is co-transcribed with secA.
Both genes are separated on the mRNA by a stem-loop- like
sequence that overlaps with the Shine-Dalgarno sequence of
secA. SecM (secretion monitor) is a signal-sequence containing
polypeptide that is translocated into the periplasm, where it is
rapidly degraded. A particular feature of SecM is the presence
of a stalling sequence at its C-terminus, which causes a transient
translation arrest that is released during translocation. However,
when translocation is compromised, translational arrest persists
and the formation of the stem-loop is blocked, allowing the
ribosome unhindered access to the Shine-Dalgarno sequence of
the secA gene and increases the production of SecA (Ito et al.,
2018). The use of monitoring substrates for adjusting the protein
transport capacity has also been shown in Vibrio alginolyticus,
where the substrate VemP controls the switch between a sodiumcoupled SecDF2 complex and a proton-coupled SecDF1 complex
in low Na+ environments (Ishii et al., 2015; Miyazaki et al., 2020).
Similar systems are also active in Gram-positive bacteria, like
B. subtilis. Here, the monitoring substrate MifM controls the
expression of the alternative YidC2 when YidC1 is compromised
(Chiba et al., 2011; Chiba and Ito, 2012, 2015).
Besides the minor growth-phase dependent regulation of SecY
and SecE as described above, entries in the E. coli gene expression
database do not reveal a strong transcriptional regulation of
the respective genes in response to different growth or stress
conditions (GenExpDB3 ). This is also validated by a proteomic
approach, which demonstrated comparable levels of SecY, SecE
and SecG over the entire growth phase of E. coli (Soufi et al.,
2015). This is rather surprising, because secY is encoded in the
spc operon together with genes for several ribosomal proteins
(Lindahl et al., 1990; Ikegami et al., 2005). These genes are
significantly down-regulated during stationary phase or when
cells encounter stress conditions (Coenye and Vandamme, 2005;
Ikegami et al., 2005; Starosta et al., 2014). The spc operon is under
additionally induced ribosome-inactivation via the ribosomemodulation factor (RMF) (Bürk et al., 2009). An up-regulation
of chaperones/proteases and down-regulation of translation is
also observed in eukaryotic cells upon SRP depletion (Mutka and
Walter, 2001). Importantly, the depletion of the SRP pathway in
either bacteria or eukaryotic cells does not cause a rapid decline
in the membrane proteome (Ulbrandt et al., 1997; Wickstrom
et al., 2011a; Costa et al., 2018). A possible explanation for this
conundrum is the intrinsic affinity of ribosomes for the SecYEG
complex (Prinz et al., 2000) and the presence of alternative
targeting systems in eukaryotes (Ast et al., 2013).
The cellular concentration of SRP is controlled by the Lon
protease, which is induced upon stress conditions. However,
Lon-dependent degradation of Ffh primarily occurs when the
Ffh levels exceed the concentration of the 4.5S RNA (Sauerbrei
et al., 2020) and it is unclear whether Lon also reduces the Ffh
levels upon stress conditions. FtsY is encoded in the ftsYEX
operon, upstream of the heat-shock sigma factor σ32 (Gill and
Salmond, 1987, 1990; Weinreich et al., 1994), however, they seem
not to be transcriptionally coupled (Gómez-Eichelmann and
Helmstetter, 1999). FtsE and FtsX are involved in the control of
peptidoglycan hydrolase activity and important for cell division
(Pichoff et al., 2019), explaining the filamentous phenotype of
ftsYEX mutations (Luirink et al., 1994). FtsY levels have been
shown to increase at low temperature (Liu et al., 2016; Zhong
and Zhao, 2019) and FtsY is subject to a proteolytic event,
which degrades its N-terminal membrane targeting sequence
(Weiche et al., 2008). However, the responsible protease and the
physiological significance of this degradation are still unknown.
Mutants lacking SecB or depleted for SecA also show an
up-regulation of the σ32 -response due to the accumulation of
secretory protein precursors in cytoplasm (Wild et al., 1992, 1993,
1996). SecB-deficient strains also show impaired growth on rich
medium (Kumamoto and Beckwith, 1985; Wild et al., 1993),
however, this is likely caused by a polar effect of the secB deletion
on the downstream gpsA gene, which is involved in phospholipid
biosynthesis (Shimizu et al., 1997).
The σ32 -response and the formation of cytosolic aggregates
containing many ribosomal proteins is also induced upon SecYE
depletion (Wild et al., 1992, 1993, 1996; Baars et al., 2008).
However, in comparison to SRP depletion, SecYE depletion
has a more drastic effect on the steady-state levels of inner
membrane proteins and secretory proteins (Baars et al., 2008).
SecYE-depletion primarily reduces the levels of multi-spanning
membrane proteins and the levels of membrane proteins
with large periplasmic domains. Intriguingly, these membrane
proteins cannot engage YidC as second integration site for
membrane proteins (Samuelson et al., 2000; Serek et al., 2004)
and are therefore strictly dependent on SecYEG. The levels of
single spanning and short membrane proteins are less impaired
by SecYE-depletion, because they can use YidC as alternative
integration site when SecYEG is depleted. This is also in line with
the observation that the SRP pathway can target both SecYEG
and YidC (Welte et al., 2012; Petriman et al., 2018).
The σ32 -response in E. coli is regulated by two feedback loops.
Free chaperones, like DnaK or GroEL bind and inactivate σ32 ,
while the inner membrane protease FtsH degrades σ32 . It was
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FIGURE 12 | (p)ppGpp-dependent regulation of translation and protein transport in bacteria. The alarmones ppGpp and pppGpp are synthesized upon amino acid
starvation by the ribosome-associated protein RelA or by the cytosolic protein SpoT upon carbon or fatty acid starvation. Allosteric regulation of RNA polymerase by
(p)ppGpp reduces ribosome biogenesis and increases the stability of the ribonuclease MazF, which degrades multiple mRNAs. This includes the mRNA encoding for
Ffh, the protein component of the bacterial SRP, or the ppiD mRNA, encoding for an accessory subunit of the SecYEG translocon. (p)ppGpp also increases the
activity of FtsH, which can degrade SecY and YfgM. YfgM forms a complex with PpiD that associates with the SecYEG translocon. Whether SecY is specifically
degraded by FtsH upon (p)ppGpp accumulation is not shown yet. (p)ppGpp also acts as competitive inhibitor of GTP-binding proteins like translation factors (IF2
and EF-G) or ribosome biogenesis proteins (ObgE). This leads to reduced ribosome biogenesis and reduced translation upon stress. Although not yet experimentally
shown, it appears likely that increasing (p)ppGpp concentrations also inhibit the two GTPases SRP and FtsY, which would fine-tune the protein targeting machinery
to the reduced translation rates.
control of the rplN promotor and binding of RNA-polymerase is
inhibited when cells enter stationary phase by the transcription
factor DksA and the alarmone ppGpp, a hyper-phosphorylated
guanosine derivative (Lemke et al., 2011; Haas et al., 2020). Thus,
secY expression is obviously disconnected from the regulation of
the other genes within the spc operon, probably by the presence
of an internal promotor.
In E. coli, the levels of the two alarmones ppGpp and pppGpp
are mainly controlled by the activity of two enzymes, RelA and
SpoT (Atkinson et al., 2011; Potrykus and Cashel, 2018; Pausch
et al., 2020). RelA primarily responds to stalled ribosomes upon
amino acid starvation (Starosta et al., 2014; Steinchen et al.,
2020), while SpoT activity increases upon fatty acid or carbon
starvation (Battesti and Bouveret, 2009; Figure 12). High levels
of (p)ppGpp induce a process called stringent response that is
associated with a significant re-programming of cellular activities
(Bennison et al., 2019; Irving et al., 2020). The (p)ppGpp levels
raise from approx. 40 µM during exponential phase up to
approx. 1 mM at the transition into stationary phase or upon
amino acid starvation (Varik et al., 2017; Haas et al., 2020;
Steinchen et al., 2020). Cellular re-programming is induced by
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two mechanisms: allosteric regulation of target proteins, like
RNA polymerase, which leads to reduced expression of the spcoperon (Liang et al., 1999; Steinchen et al., 2020), and competitive
inhibition of GTP-binding proteins, like the ribosome assembly
factor ObgE (Sato et al., 2005; Persky et al., 2009; Feng et al.,
2014), the initiation factor IF2 (Diez et al., 2020) or elongation
factor EF-G (Mitkevich et al., 2010; Steinchen et al., 2020). As
a result, ribosome biogenesis and translation are adjusted to
substrate limitation.
Increasing (p)ppGpp concentrations likely also interfere with
the activity of the GTPases FtsY and SRP and both proteins
were identified as potential targets of (p)ppGpp (Wang B. et al.,
2019). This would enable cells to adjust the protein targeting
machinery to the reduced protein synthesis rate upon entry into
stationary phase or during nutrient limitation. However, the
consequences of (p)ppGpp on SRP-dependent protein targeting
have not been studied so far. The accumulation of ppGpp
also activates the MazEF toxin-antitoxin system (Moll and
Engelberg-Kulka, 2012) and the mRNAs of both PpiD and
Ffh were identified as potential targets of the riboendonuclease
MazF (Sauert et al., 2016). This provides an additional link
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FIGURE 13 | Inhibitors of bacterial protein translocation. (A) Inhibitors of the ATPase SecA. (B) Inhibitors of the SecYEG-translocon. Ipomeassin F, decatransin,
eeyarastatin 1 and eeyarastatin 24 also act on the homologous Sec61 complex in eukaryotes. Chemical structures were retrieved from the Sigma Aldrich web
resource (https://www.sigmaaldrich.com/) or adapted from (Li et al., 2008; Van Puyenbroeck and Vermeire, 2018; Zong et al., 2019).
between stress conditions and the protein targeting and
transport machinery that requires further analyses. Bacteria
also produce hyper-phosphorylated adenosine derivatives, like
(p)ppApp, although less is known about the conditions of
synthesis and potential regulatory consequences (Travers, 1978;
Bruhn-Olszewska et al., 2018; Ahmad et al., 2019). Still, it is
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tempting to speculate that by accumulating (p)ppGpp or
(p)ppApp, bacteria can adjust protein transport by an allosteric
or competitive mechanism, rather than by transcriptional or
translational regulation. ppGpp also induces FtsH-dependent
degradation of the SecYEG-interacting protein YfgM when cells
enter stationary phase (Bittner et al., 2015). This is suggested
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SecYEG-Dependent Protein Transport
is active against E. coli and several clinically relevant pathogens
(Steenhuis et al., 2021). ES24 likely binds to the cytosolic part
of the lateral gate (Gamayun et al., 2019), but the antibacterial
activity depends on the presence of the nitroreductases NfsA
and NfsB, indicating that a specific reduction step is required
to activate ES24 (Steenhuis et al., 2021). Decatransin is a
naturally occurring fungal decadepsipeptide that was identified
in a cancer drug screen and later shown to inhibit SecYEG/Sec61.
Decatransin-resistant mutations mapped to the pore ring and to
the plug of the Sec channel, suggesting that decatransin interferes
with channel opening (Junne et al., 2015). However, whether
these SecA- and SecY-inhibiting compounds also have clinical
relevance requires further investigation.
to relieve the response regulator RcsB, thereby allowing cellular
protection by the Rcs phosphorelay system (Lasserre et al.,
2006; Wall et al., 2018). However, this would also reduce
the levels of the PpiD-YfgM complex and thus impact on
the SecYEG interactome under stress conditions. How stress
conditions influence the steady-state levels of the protein
transport machinery and the dynamic equilibrium between the
different SecYEG assemblies is largely a terra incognita, but a
promising area for future research.
INHIBITORS OF SECYEG-DEPENDENT
PROTEIN TRANSPORT
The rapid rise of antibiotic resistance is a major problem for
treating infections and novel antimicrobial strategies are of
crucial importance (Rodríguez-Rojas et al., 2013; Sulaiman and
Lam, 2021). Initial studies on exploring the protein transport
machinery as potential target were focused on SecA inhibitors,
because SecA homologues are absent in metazoans and SecA
inhibition would affect most periplasmic and OMPs as well as
some inner membrane proteins (Pohlschroder et al., 2005). Azide
was the first described inhibitor of SecA (Oliver et al., 1990),
but has no medical relevance due to its high toxicity (Chang
and Lamm, 2003). Additional small molecule SecA inhibitors
with broad-spectrum activity have been developed and include
compounds like SEW-05929 and CD 09529, which inhibit the
ATPase activity of SecA but are inactive on wild type E. coli strains
(Li et al., 2008; Figure 13). Further studies identified 4-oxo-5cyano thiouracils (Chaudhary et al., 2015), Fluorescein analogs
(Huang et al., 2012) and triazole-pyrimidine analogs (Cui et al.,
2016; Jin et al., 2016) as SecA inhibitors that are active against
E. coli and S. aureus (Rao et al., 2014; De Waelheyns et al., 2015;
Van Puyenbroeck and Vermeire, 2018).
The first characterized inhibitors of the Sec complex were
synthetic signal peptides that have been shown to inhibit the
eukaryotic Sec61 complex (Austen et al., 1984). The mammalian
Sec61 complex is also inhibited by lanthanum ions, which
stabilize the Sec61 channel in its open state (Erdmann et al.,
2009). Components that inhibit both the eukaryotic Sec61
complex and the bacterial SecYEG complex are the glycoresin
Ipomoeassin F (IpomF) (Zong et al., 2019; Steinberg et al.,
2020), eeyarestatin (Cross et al., 2009; Steenhuis et al., 2021) and
decatransin (Junne et al., 2015; Kalies and Römisch, 2015). IpomF
was isolated from the morning glory Ipomea squamosa and
shown to bind most likely near the lateral gate of Sec61α (Zong
et al., 2019). IpomF also inhibits SecYEG-dependent transport
in vitro, but this requires significantly higher concentrations than
required for inhibition of Sec61-dependent transport (Zong et al.,
2019; Steinberg et al., 2020). IpomF does not prevent the initial
contact of substrate proteins with the SecYEG translocon, but
rather blocks later stages of translocation (Steinberg et al., 2020).
Eeyarestatin I (ESI) was initially discovered as inhibitor of the
retrograde protein transport into the endoplasmic reticulum and
then shown to inhibit co-translational protein transport by the
Sec61 complex (Cross et al., 2009). ESI does not inhibit growth of
E. coli, but a smaller variant of ESI, ES24 (Gamayun et al., 2019),
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CONCLUSION AND OUTLOOK
The bacterial SecYEG translocon has been the focus of
intense research for decades and served as a paradigm for
genetic, biochemical and structural studies on protein transport
mechanisms. The progress that has been made from the early
genetic screens (Bassford et al., 1991; Beckwith, 2013) to
the currently available structures is incredible (Smets et al.,
2019; Tanaka and Tsukazaki, 2019). Snap-shots of the SecYEG
translocon in contact with its most prominent partner proteins
and of the SecYEG translocon in action during translocation
or insertion of protein substrates have been attained and
provide first insights into how these protein transport channels
work. Still, structural information of substrate-engaged larger
SecYEG assemblies, like the SecYEG-YidC complex, the SecYEGPpiD/YfgM complex or the HTL, are needed for understanding
how the SecYEG translocon handles the large variety of
potential substrates. Equally needed are structures of the SecYEG
translocon during the insertion of multi-spanning membrane
proteins. It is also evident that the current picture of the SecYEG
interactome is incomplete and includes only the most stable
and abundant partner proteins. Many transient interactions only
emerged upon improved mass spectrometry methods (Carlson
et al., 2019; Jauss et al., 2019) and the functional characterization
of these transient contacts will be a major challenge for the
future. This will be particularly demanding if these contacts are
only required for a specific subset of substrates, which are not
in the tool box of frequently used model substrates. Analysing
the transport of membrane proteins with large soluble domains
at the N-terminus (Facey and Kuhn, 2003; Maier et al., 2008;
Rawat et al., 2015; Wang S. et al., 2019) or very small membrane
proteins, which basically consist of just a single transmembrane
domain, has already revealed unexpected targeting and insertion
requirements (Steinberg et al., 2018, 2020). Despite the increasing
number of proteins interacting with the SecYEG translocon, the
number of identified contact sites on SecY is rather low and
mainly includes the cytosolic loop 5, the lateral gate and the
periplasmic vestibule. This suggests that some proteins either
compete for SecY binding, or interact with dedicated subpopulations of the SecYEG translocon and these subpopulations
need to be further characterized. Our current view on bacterial
20
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Oswald et al.
SecYEG-Dependent Protein Transport
protein transport pathways follows a rather strict dissection into
multiple separate transport pathways, but recent data suggest
that these pathways are intertwined. The best-studied example
is of course the SecYEG-YidC interaction (Scotti et al., 2000),
where YidC likely helps substrates to exit the SecY channel (Beck
et al., 2001; Houben et al., 2002), although YidC can also act
as SecYEG-independent insertase (Samuelson et al., 2000; Serek
et al., 2004). But there are more examples, like the SecYEG-Tat
interaction (Keller et al., 2012) or the SecYEG-Bam interaction
(Alvira et al., 2020), and the collaboration between different
transport systems needs to be further explored. Finally, it is
largely unknown how the protein transport machinery responds
to environmental changes or to stress conditions. Considering
the multifaceted responses that down-regulate protein synthesis
when cell encounter non-favorable conditions, it appears more
than likely that similar, but so far unexplored mechanisms, also
modulate the protein transport capacity of the cell. Thus, there is
still a lot to learn about the SecYEG translocon or, to cite famous
Isaac Newton: ”What we know is a drop. What we don’t know is
an ocean.”
AUTHOR CONTRIBUTIONS
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FUNDING
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG) to HGK (DFG grants KO2184/8,
KO2184/9 (SPP2002), SFB1381, Project-ID 403222702, and RTG
2202, Project-ID 278002225). Funding sources were not involved
in the design of the article, writing the article or decision to
submit the article for publication.
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