REVIEWS
Inserting proteins into the bacterial
cytoplasmic membrane using the Sec
and YidC translocases
Kun Xie and Ross E. Dalbey
Abstract | This Review describes the pathways that are used to insert newly synthesized
proteins into the cytoplasmic membranes of bacteria, and provides insight into the function
of two of the evolutionarily conserved translocases that catalyse this process. These highly
sophisticated translocases are responsible for decoding the topogenic sequences within
membrane proteins that direct membrane protein insertion and orientation. The role of the
Sec and YidC translocases in the folding of bacterial membrane proteins is also highlighted.
Department of Chemistry,
The Ohio State University,
100 West 18th Avenue,
Columbus, Ohio 43210, USA.
Correspondence to R.E.D.
e-mail: dalbey@chemistry.
ohio-state.edu
doi:10.1038/nrmicro1845
Published online 4 February 2008
Integral membrane proteins are responsible for a diverse
set of cellular functions and have key roles in energy
transduction, nutrient and ion transport, and protein
processing and quality control. These proteins vary in
complexity — for example, they span the membrane 1–18
times in the Gram-negative bacterium Escherichia coli1.
Approximately 20–30% of the proteins that are encoded
by the bacterial proteome are inner-membrane proteins1
and approximately 2% are outer-membrane proteins2.
Integral membrane proteins can contain many membrane
and extramembrane subunits, in addition to prosthetic
groups and metal clusters. These membrane protein
complexes must be assembled correctly to function in the
cell. If one polypeptide in the complex fails to assemble
accurately, the other polypeptides in the complex are often
degraded3. Proofreading mechanisms exist in the cell that
recognize when proteins misfold or are misassembled so
that they can be removed from the membrane4.
There are two types of integral membrane proteins in
cellular membranes: those that contain α‑helical transmembrane (TM) regions, which are widespread, and those
that possess multiple β‑strands, which are found predominately in the outer membranes of Gram-negative bacteria
and the mitochondrial outer membranes of eukaryotes. In
the past few years, there have been major developments in
our understanding of how β‑barrel proteins are assembled
into the outer membrane (BOX 1). In this article, we do not
discuss those outer-membrane proteins that contain covalently bound fatty acids, which anchor the proteins to the
membrane leaflet (reviewed in Ref. 5).
To understand how integral membrane proteins are
accurately inserted into a specific membrane it is essential to investigate the different membrane systems that
are found in cells. Gram-positive bacteria and archaea
have only one membrane system, the plasma membrane,
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which separates the inside of the cell from the outside
environment. Gram-negative bacteria contain two membranes — the inner (cytoplasmic) membrane and the
outer membrane. By contrast, in addition to a plasma
membrane, eukaryotic cells also contain numerous
internal membranes, such as the nuclear double membrane, the endoplasmic reticulum (ER) membrane, the
Golgi membranes, the lysosomal, endosomal and peroxisomal membranes, the mitochondrial inner and
outer membranes, and in plants, the chloroplast inner
and outer membrane, as well as the thylakoid membrane
(Box 2). Eukaryotes, therefore, face the most challenging task in targeting proteins to the correct organellar
membrane.
Cells solve the problem of protein targeting and membrane insertion by using molecular devices to carry out
these functions. These devices make membrane insertion
efficient, a process that otherwise would occur slowly, or
not at all, because of the barrier that the hydrophobic environment of the membrane interior poses to the passage of
the polar domains of membrane proteins. In E. coli, three
translocases have been discovered that insert proteins into
the cytoplasmic membrane such that they can obtain their
correct conformation. The Sec translocase is the general
translocase that transports proteins across the cytoplasmic
membrane in an unfolded state (reviewed in Refs 6,7)
(FIG. 1). Most proteins that are integrated into the cytoplasmic membrane are also inserted by the Sec apparatus. The
second translocase, the twin arginine translocation (Tat)
machinery, operates in a radically different way to the Sec
translocase and is responsible for translocating exported
proteins that are folded before translocation and typically
have bound metal cofactors (reviewed in Refs 8,9). The
Tat pathway is also involved in the biogenesis of a few
bacterial membrane proteins10. A third translocase, the
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YidC insertase, inserts Sec-independent proteins into the
cytoplasmic membrane (reviewed in Refs 11,12) (FIG. 1). In
addition to acting as an independent insertase, YidC also
assists in membrane protein insertion in the Sec pathway
(FIG. 1). Thus, there is a link between the YidC and Sec
translocases in the Sec pathway.
This Review will focus on the Sec and YidC systems
that are used to insert proteins into the cytoplasmic
membrane of E. coli. We will update readers on how
membrane insertion and the topology of membrane
proteins are achieved through topogenic sequences that
are encoded within membrane proteins, and discuss the
role of the translocase and insertase in this process. We
will also discuss the four biochemically distinct steps
that are used in membrane protein insertion: targeting
to the membrane; membrane translocation of the substrate polar domains; the lateral insertion of the apolar
regions into the lipid bilayer and protein folding; and the
oligomeric assembly of protein subunits.
Type I membrane protein
A protein which contains a
single membrane-spanning
domain that has its carboxyl
terminus orientated towards
the cytoplasm and its amino
terminus orientated towards
the lumen of membrane
compartments or in an
extracellular direction.
Type II membrane protein
A single-spanning membrane
protein that has the opposite
topology to a type I membrane
protein.
Signal-recognition particle
A complex that is responsible
for targeting nascent
polypeptides to the cell
membranes, and identifies an
amino-terminal signal
sequence that is carried by
proteins that are destined for
secretion or membrane
localization.
Two-partner secretion
system
A secretion system that is
composed of two distinct
proteins; one is secreted and
the other is its transporter.
Membrane topology and topogenic sequences
Whether in eukaryotes or prokaryotes, integral cytoplasmic membrane proteins all contain 1 or more TM
α‑helices, each of which is composed of 20–27 amino
acids. The α‑helices are typically perpendicular to the
plane of the membrane, although they can be tilted.
Bitopic membrane proteins contain one TM α‑helix
that is connected by two polar domains on opposite
sides of the membrane. Polytopic membrane proteins
contain two or more TM α‑helices that are connected
by extramembrane loops. Both types of membrane proteins typically have a unique topology with respect to
the membrane. Bitopic membrane proteins can span the
membrane once, with their amino (N) terminus facing
either the periplasmic space (type I membrane protein) or
the cytoplasm (type II membrane protein). Polytopic membrane proteins can span the membrane in many different
ways, depending on the number of TM spans and the
location of the N and carboxyl (C) termini. The topology
of these proteins is usually determined by the ‘positive
inside’ rule, in which the hydrophilic loops that border
the TM segments are enriched in positively charged
residues and are localized to the cytoplasm.
To be inserted into the membrane in the correct
orientation, integral membrane proteins require welldefined signals that are encoded within the polypeptide
chains; these are referred to as topogenic sequences.
These sequences are recognized and decoded by the
YidC or SecYEG protein translocation machinery during membrane biogenesis (TABLE 1). Some nascent membrane proteins are threaded into the Sec translocase in
an N‑terminal to C‑terminal direction. The topogenic
sequences specify the topology, whereas other membrane
proteins are inserted by a non-sequential mechanism.
Membrane targeting
In bacteria, both secreted and membrane proteins are initially synthesized in the cytoplasm and are then directed
to the inner membrane for translocation. Secretory proteins bind to the SecB chaperone and are then targeted
to the SecA ATPase, which is probably localized at the
surface of the cytoplasmic membrane13. Most membrane
proteins are targeted to the membrane by the ubiquitous
signal-recognition particle (SRP) pathway14,15. There is compelling evidence that membrane targeting by the SRP
is co-translational in bacteria16,17, although this has not
been demonstrated directly18.
Targeting to the membrane by the SRP pathway
requires the SRP and the SRP receptor FtsY (reviewed
in Ref. 19) (FIG. 1, left side). The SRP, which comprises
the SRP and a 4.5S RNA, binds to a hydrophobic region
of integral membrane proteins as they emerge from
the ribosome during protein synthesis20. The nascent
membrane protein–ribosome–mRNA complex is then
targeted to the SRP receptor, which, in most cases, is
located at the membrane surface21.
Box 1 | Assembly of b‑barrel proteins into the bacterial outer membrane
During the past 5 years, several exciting discoveries have been made regarding the machinery that facilitates the
assembly and folding of β‑barrel proteins into the outer membrane of Gram-negative bacteria. The first step in this
process is the transport of β‑barrel proteins across the cytoplasmic membrane into the periplasmic space by the Sec
machinery. β‑barrel proteins are synthesized with a cleavable signal peptide that is essential for export and is
proteolytically removed by signal peptidase.
The first important breakthrough in this field was the identification of Neisseria meningitidis Omp85, which is
essential for the assembly of β‑barrel outer-membrane proteins94 and cell viability94. Omp85, and its homologues
Tob55 and Toc75, exist in bacteria, mitochondria and chloroplasts, and are crucial for the assembly of β‑barrel
proteins in the mitochondrial outer membrane95–97, as well as translocation across the chloroplast outer
membrane from the cytoplasm98.
Another major step forward was the identification of Escherichia coli YfgL using a chemical–genetic approach99 in
which suppressors were identified by adding toxic molecules to bacterial strains that had a leaky outer membrane.
One such suppressor was a loss of function mutation in YfgL, which allowed the E. coli leaky mutants to grow slowly in
the presence of an otherwise toxic compound100. YfgL is an outer-membrane lipoprotein that forms a complex with the
Omp85 homologue YaeT and the outer-membrane lipoproteins YfiO, NlpB and SmpA100,101. How this complex
promotes folding and assembly into the outer membrane is not fully known.
Further progress was made with the recently solved X‑ray structures of the polypeptide-transported-associated
(POTRA) domain in YaeT102 and FhaC, another member of the Omp85/Tob55/Toc75 superfamily103. This structural work
on the periplasmically localized POTRA domains suggested how they might bind peptide sequences of the β‑barrel
proteins104,105. In addition, the 3.15 Å structure of FhaC provided a clue to how FhaC, the transporter in the two-partner
secretion system, translocates the filamentous haemagglutinin across the outer membrane in Bordetella pertussis103.
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In this SRP-mediated targeting pathway, the SRP binds
to the ribosomal exit site, where it can scan and bind to
hydrophobic signal anchor sequences. Membrane targeting
of the SRP–nascent membrane protein complex is achieved
by the interaction between the SRP and the receptor FtsY.
FtsY mediates membrane targeting through its ability to
bind membrane lipids and the SecYEG translocase (FIG. 1).
Targeting of the SRP–nascent chain–ribosome complex
requires GTP. After GTP hydrolysis by both the SRP
and FtsY component, the SRP is released from FtsY and
the nascent membrane protein is forwarded to the Sec
translocase to be integrated into the membrane bilayer.
This SecYEG transfer process is probably facilitated by
the ability of the ribosome to bind to the translocation
channel, although the details of this process are not
fully known.
Recently, the SRP has also been shown to target
Sec-independent proteins, for example, the mechanosensitive channel protein MscL, to the YidC insertase22.
However, several Sec-independent proteins are targeted
to the membrane independently of the SRP pathway;
this targeting is thought to occur by electrostatic interactions between the membrane protein substrate and
the head groups of the membrane phospholipids23. For
example, targeting of the Sec-independent M13 procoat
protein is unaffected when the targeting component Ffh
(discussed below) is inactivated, whereas the membrane
targeting of many Sec-dependent proteins is inhibited
under such conditions14,24,25.
YidC-only pathway for membrane insertion
In 2000, YidC was found to be a new translocase, or membrane insertase, that promotes the insertion of proteins
independently of the main Sec translocase in bacteria26–28.
When YidC was depleted from the cell membrane, insertion of the Sec-independent M13 procoat protein28,29 and
Pf3 coat protein30 was strongly inhibited. This was despite
earlier studies, which had suggested that the membrane
insertion of Sec-independent membrane proteins in bacteria was spontaneous31. Additional evidence that YidC
has a role in membrane protein insertion was provided by
the findings that YidC physically interacts with the TM
domain of the Sec-independent Pf3 coat protein during
insertion30 and that proteoliposomes that contain only
YidC are sufficient to insert the Pf3 coat protein into the
membrane32.
In the current model for the involvement of YidC in
membrane insertion of the Pf3 coat protein, YidC makes
Box 2 | The bacterial, archaeal and eukaryotic Sec systems
Signal anchor
A topogenic sequence that
signals the initiation of
translocation of the carboxyterminal region of a membrane
protein, and remains as a
membrane anchor with an
NinCout orientation. Also known
as a type II signal anchor.
Many of the components of the Sec translocase are conserved among Bacteria, Eukarya and Archaea106. In eukaryotes, the
internal endoplasmic reticulum (ER) membrane is analogous to the bacterial and archaeal plasma membranes. In plants,
homologues of SecY, SecE and SecA, but not SecG, are found in chloroplasts107–109. In the ER, Sec61α, Sec61β and Sec61γ
form the protein-conducting channel, in which Sec61α is homologous to SecY and Sec61γ is homologous to SecE110,111.
Although Sec61β is not homologous to SecG, it is its functional paralogue. Most archaeal genomes that have been
sequenced to date contain SecYEβ components and encode the SecD and SecF proteins of the bacterial Sec translocation
machinery (SecA is absent)112,113.
Variations exist in the components of the protein translocation machinery in yeast and mammals. In mammals, the
translocating-chain-associating membrane protein TRAM (which might be a chaperone that is involved in handling
transmembrane (TM) segments) is present in the ER, but YidC is missing. In yeast, the Sec62, Sec71, Sec72 and Sec63
components, and Kar2p (the yeast homologue of Bip), are required for post-translational translocation. SecA, SecD and
SecF are absent from the ER channel.
As for membrane protein insertion in bacteria, insertion of proteins into the ER is a dynamic process. Goder and
colleagues114 showed that an uncleaved TM signal is inserted into the translocase in the NoutCin orientation and then
reorientated within the channel to the NinCout orientation. The translocase is also an extremely dynamic structure. Laio
and co-workers115 found that at a certain stage of synthesis during membrane insertion the luminal gate of the ER
translocase opens. The gate remains open until the α‑helical TM region of the membrane protein has been synthesized
and located in the ribosomal tunnel; at a certain point the luminal gate of the translocase then closes. The folding of TM
regions can begin within the ribosomal exit channel116,117 and depends on the location within the tunnel, which suggests
the presence of folding zones inside the ribosome exit tunnel118.
Lateral exit of the TM domains from the translocase into the ER lipid bilayer can occur by a range of mechanisms, two of
which are similar to the mechanisms that are used in bacteria. The first mechanism is termination-coupled integration,
in which the TM domain is in contact with the Sec channel and TRAM until protein synthesis is completed119. TRAM has
been shown to stimulate the membrane insertion of certain proteins, and it also mediates membrane protein insertion, as
it can be crosslinked to these proteins during membrane integration. In bacteria, YidC takes the place of TRAM. The
second mechanism is the displacement model — which is similar to the sequential mechanism — in which the TM
segments enter the translocase sequentially and the preceding TM segment seems to be released simultaneously120.
Strikingly, some TM segments of membrane proteins, such as the aquaporin 4 water channel, exit Sec61α, but then seem
to interact with the translocase as protein synthesis and membrane insertion proceeds. This suggests that TM segments
are positioned at different locations of the Sec61 complex; this conflicts with the solved SecYEβ crystal structure, in which
the channel was predicted to tolerate no more than one TM helix at a time57.
From these ER studies, we now know that membrane protein biogenesis is a dynamic process and folding of a TM
domain of a membrane protein occurs within the ribosome, which can transmit information to the Sec translocase. It
will be interesting to investigate whether, in the bacterial system, movement of the TM domain within the ribosome
tunnel can cause dynamic changes within the SecYEG complex, thereby leading to changes in the gating of the
translocation channel.
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Sec–YidC
SecDF
YajC
Sec
YEG
YidC
FtsY
YidC
Periplasm
YidC
Cytoplasm
SRP
SRP
Figure 1 | The machinery that is involved in membrane protein insertion. The SecYEG–SecDFYajC complex is the
Nature
Reviews | Microbiology
general translocase that is used by Escherichia coli to insert newly synthesized polypeptides into
the cytoplasmic
membrane. The signal-recognition particle (SRP) and SRP receptor (FtsY) target most Sec-dependent proteins. The novel
insertase YidC can function with the Sec translocase or independently to promote membrane protein insertion. Some
Sec-independent proteins are also targeted by the SRP.
Reverse signal anchor
A topogenic sequence that
signals the initiation of
translocation of the aminoterminal region of a membrane
protein, and remains as a
membrane-spanning region
that has an NoutCin orientation.
Also known as a type I signal
anchor.
Signal peptidase II
A signal peptidase that
proteolytically removes
lipoprotein signal sequences.
contact with the reverse signal anchor domain of the Pf3
coat protein at the membrane and facilitates translocation of the short N‑terminal tail (TABLE 1). After the translocation step, the apolar domain of the Pf3 coat protein
is released laterally from YidC into the lipid bilayer. By
contrast, for the M13 procoat protein, YidC is thought
to catalyse the movement of the two closely spaced
hydrophobic domains into the membrane such that they
become perpendicular to the membrane (TABLE 1). The
hydrophobic domains form a helical hairpin that comprises a cleavable signal peptide and a membrane anchor
region in the mature region of the procoat protein. After
translocation of the short periplasmic domain, the signal
peptide and membrane anchor domain are released laterally into the membrane. The signal peptide is removed
from the procoat protein proteolytically, and the procoat
protein is then converted to a mature coat protein by
signal peptidase cleavage.
The YidC membrane insertase has a crucial role in the
assembly of energy-transducing membrane proteins. For
example, the depletion of YidC has a marked effect on
the ATPase activity of the F1F0 ATPase and the activity
of the cytochrome bo3 oxidase33. The role of YidC for
these membrane-bound enzymes might involve the
insertion of their membrane subunits. YidC depletion
inhibits the membrane insertion of subunit c of the F1F0
ATPase and subunit II of the cytochrome bo oxidase34–38.
Strikingly, YidC-only lipid vesicles were able to promote
the membrane insertion of subunit c, as well as the formation of the subunit-c oligomer39, which shows that the Sec
translocase is dispensable for subunit-c translocation.
A more complex mechanism is used for the membrane
insertion of subunit II of cytochrome bo3 oxidase (CyoA)
(Table 1). The N‑terminal region of the protein inserts
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into the cytoplasmic membrane by the YidC-only pathway, whereas the C‑terminal region uses the Sec pathway
(FIG. 2). Interestingly, a prerequisite for translocation of
the large periplasmic domain by the Sec pathway is prior
membrane insertion of the N‑terminal domain by the
YidC pathway36. CyoA is synthesized in a precursor form
called preCyoA by a cleavable signal peptide. Targeting
of pre-CyoA to the membrane requires the SRP pathway,
and the protein is processed by signal peptidase II and, subsequently, is modified by the addition of a lipid group to
form the mature CyoA lipoprotein.
YidC family members
In addition to bacteria, YidC family members are also
found in mitochondria and chloroplasts40,41, but are
probably not present in archaea. The chloroplast YidC
homologue Alb3 and mitochondrial YidC homologue
Oxa1 can substitute for YidC in E. coli42,43, and E. coli
YidC variants can replace Oxa1 and Oxa2 in yeast
mitochondria44. YidC, Oxa1 and Alb3 have a conserved
C‑terminal region that comprises five predicted TM
segments, although in E. coli, YidC has six N‑terminal
TM segments. In addition, YidC has a large periplasmic
domain that is not conserved. Interestingly, both Oxa1
and Alb3 typically have a long C‑terminal domain that
faces the mitochondrial matrix and chloroplast stroma,
respectively. In Oxa1, the matrix-exposed C‑terminal
domain that follows TM5 was shown to function as a
ribosome-binding domain45,46.
In the Gram-positive bacteria Bacillus subtilis and
Streptococcus mutans, there are two YidC homologues,
and either is sufficient for growth47,48. Strikingly, the
YidC homologue in S. mutans (YidC2) is crucial for
the growth of S. mutans under the stress conditions of
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Table 1 | The topogenic sequences that are used to generate the topologies of membrane proteins
Protein
Structure
Pf3 coat
N
Topogenic sequences
YidC
dependent?
Sec dependent?
Reverse signal anchor*
Yes
No
Uncleaved signal
anchor‡
Unknown
Yes
Helical hairpin§
Yes
No
Cleaved signal peptide||
and stop transfer¶
No
Yes
Reverse signal anchor*
and uncleaved signal
anchor‡
Yes
No
Reverse signal anchor*
and uncleaved signal
anchor‡
Yes
Yes
C
FtsQ
C
N
M13
procoat
N
C
M13 gpIII
preprotein
C
N
ATPase
subunit c
N
Lep
N
C
Tsr
Uncleaved signal anchor‡ Unknown
and stop transfer¶
Yes
Helical hairpin§
Yes
No
Helical hairpin§ and
uncleaved signal anchor‡
Yes
Yes
Uncleaved signal
anchors‡ and stop
transfers¶
Unknown
Yes
Helical hairpins§
Unknown
Yes
C
N
MscL
N C
PreCyoA
N
MalF
N
C
TetR
N
C
*Reverse signal anchor domains (also called type I signal anchors) initiate translocation of the amino (N)-terminal region of a
polypeptide chain and remain as a membrane-spanning region with an Nout Cin orientation. ‡Uncleaved signal anchors (also called
type II signal anchors) initiate the translocation of the carboxyl (C)‑terminal region of membrane proteins and remain as a
membrane anchor with an NinCout orientation. ||N-terminal cleavable signal peptides (NinCout orientation) initiate the translocation
of C-terminal regions of a polypeptide chain and are removed from the membrane protein by the action of signal peptidase.
¶
Stop-transfer sequences function to stop the translocation that was initiated by a preceding signal peptide; they remain as a
membrane anchor with an NoutCin orientation. §Helical hairpin domains comprise two closely spaced hydrophobic segments and
insert in the membrane in a folded manner. For a helical hairpin to be a topogenic sequence, both hydrophobic domains must be
present for insertion of the intervening polar region. After insertion, the two hydrophobic domains are oriented such that the
N and C termini are located in the cytoplasm. The arrows represent cleavage by signal peptidase.
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SPII
YidC
Sec translocon
Periplasm
SecA
Cytoplasm
N
N
SRP
N
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Figure 2 | Membrane insertion of the lipoprotein CyoA. CyoA uses distinct
mechanisms for membrane insertion of its amino (N)‑terminal and carboxy (C)‑terminal
domains. The N-terminal domain of preCyoA uses the YidC pathway, and the large
C‑terminal domain is translocated by the SecYEG–SecA translocase. During insertion,
preCyoA is cleaved by signal peptidase II (SPII) and a lipid is attached to the N‑terminal
end of the protein.
low pH or high salt. One hypothesis is that the sensitivity of growth of the YidC2-knockout mutant at low
pH is due to the fact that YidC2 is required for correct
membrane integration of the F0 components of the F1F0
ATPase. It has also been shown that the activity of the
H+ ATPase increases during acid stress to protect the cell
and sustain pH homeostasis49.
YidC structure and function
In E. coli, YidC contains six TM regions and a large
periplasmic domain between TM1 and TM2. So far,
the importance of these regions for the structure and
function of YidC has been investigated only to a limited
extent. As mentioned above, YidC functions both in
conjunction with the Sec translocase and on its own. A
region of YidC interacts with SecDFYajC, which links
it to the Sec translocase in vivo50. Recently, the nonconserved periplasmic domain of E. coli YidC was shown
to be required for the interaction with the SecF component of the SecDFYajC complex51. As determined by the
recent elucidation of the 2.5 Å X‑ray structure of the periplasmic domain of E. coli YidC52, the YidC region that
interacts with SecF maps to one side of a β‑sandwich.
Surprisingly, the interaction of YidC with SecF is not
required for cell viability or YidC functionality. The role
of the conserved C‑terminal TM domains was investigated using deletion and substitution mutants53, and it
was revealed that TM2, 3 and 6 are important for cell
viability and function. TM4 and 5 do not seem to be as
important for YidC function and viability, as they can be
replaced with unrelated hydrophobic domains.
Although these initial studies are important, they
do not reveal which regions of YidC bind to substrate
or the TM packing that occurs within YidC. There is
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also still a debate in the field as to whether YidC is
an oligomer. One study has shown that some purified
YidC appears as a dimer on a native polyacrylamide
gel50. However, the mitochondrial homologue Oxa1
appears as a tetramer if purified from Neurospora
crassa54. If YidC is an oligomer, it will be interesting
to determine which regions of YidC are important for
oligomerization.
The Sec pathway for membrane insertion
The Sec translocase is the main molecular machine
that is used to insert most bacterial proteins into the
membrane once they have been targeted there by
the SRP pathway. In Gram-negative bacteria, the Sec
translocase comprises the SecYEG core and the
accessory components SecDFYajC, SecA and YidC
(reviewed in Refs 55,56) (FIG. 1). SecYEG constitutes
the protein-conducting channel57, and SecDFYajC is a
trimeric complex that enhances the in vivo translocation of secretory proteins58 and insertion of membrane
proteins59. It has been proposed that SecDF prevents
pre-protein backsliding, thereby regulating SecA
cycling and releasing translocated proteins from the
translocation channel50. YajC is not essential for protein export or membrane protein insertion. SecA is a
molecular motor that uses ATP hydrolysis to promote
translocation of the polypeptide chain in steps of 20–25
residues (reviewed in Ref. 60). SecYEG, SecDF and SecA
are present in both Gram-negative and Gram-positive
bacteria.
An important advance in the protein translocation field has been the determination of the structure
of SecA. The structure revealed a nucleotide-binding
domain, substrate-specificity domain and C‑terminal
domain (reviewed in Ref. 60). In addition, the SecA
signal-peptide-binding region, which consists of an
elongated groove that is made up of apolar residues and
is surrounded by acidic residues, has been defined by
NMR spectroscopy61. SecA is required for translocation
of the large hydrophilic domains of membrane proteins62,63
and, in some cases, short loops64. For single-spanning
membrane proteins, SecA is required for translocation
of a short periplasmic loop (~13 amino acids), but is
not required if the protein contains a downstream TM
region65. However, exactly how SecA engages the SecYEG
channel and promotes translocation of a hydrophilic
domain through the channel interior is not known. In
addition, most of the data revealed that the SecA- and
ribosome-binding sites of SecY are almost certainly
overlapping in the C‑terminal region of SecY. Therefore,
SecA could promote translocation of the membrane
protein domain after the ribosome has been released
from the SecYEG channel. How release of the ribosome
and the subsequent binding of SecA to SecYEG are
regulated is not understood.
In addition to translocation, the Sec translocase also
functions in the integration of hydrophobic segments
into the lipid bilayer. It is not fully understood how,
after the translocation step, the hydrophobic region of
the inserting membrane protein is released from the
Sec channel to enter the bilayer. Recently, the features
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a
Sec translocon
N
YidC
YidC
Periplasm
YidC
Cytoplasm
b
Periplasm
YidC
N
C
Cytoplasm
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Figure 3 | The interaction of YidC with TM segments. YidC interacts with transmembrane
(TM) segments in two
different ways. In the sequential model, the first TM segment is released from YidC to enter the lipid phase before
binding the next TM helix (a). In the assembly-site model, YidC has an important role in packing the TM regions
into a bundle (b). The bundle of TM segments is released from YidC such that they enter the lipid bilayer at the
same time. In both cases, the TM segments are shown entering and leaving the SecYEG channel one at a time.
YidC is proposed to act as a chaperone, whereby it participates in the transfer of the TM domains from the
SecYEG channel into the lipid phase.
that determine whether or not a peptide segment is
integrated into the membrane were investigated in the
eukaryotic ER system66. The main conclusion was that
the recognition of a TM segment by the SecYEG protein
channel is determined solely by protein–lipid interactions; if a protein segment is sufficiently hydrophobic
it will integrate into the membrane and function as
a stop-transfer domain. Although the mechanism of
lateral transfer of hydrophobic segments from the Sec
channel into the lipid phase has been studied for only
a few proteins in bacteria, it seems that YidC acts as a
chaperone that mediates this transfer and might have
a role in stabilizing the TM segment after transfer
from the Sec channel67.
In the membrane-integration step, YidC has been
shown to interact with the apolar domains of membrane protein substrates in two different ways (FIG. 3).
For leader peptidase, which spans the membrane
twice, YidC interacts in a sequential manner with
the apolar domains before entering the lipid phase68
(FIG. 3a) . By contrast, for mannitol permease, YidC
forms an assembly site for hydrophobic domains
during the insertion process (FIG. 3b). Insertion of the
hydrophobic segments into the lipid phase occurs only
after they pack together to form a bundle69. In both of
these proteins, apolar domains enter and leave the Sec
channel individually.
240 | march 2008 | volume 6
SecYEG structure
In 2004, the structure of the Sec translocase from
Methanococcus jannaschii was elucidated at 3.2 Å resolution by X‑ray crystallography57. Strikingly, the translocation channel is contained within one SecYEβ (SecY
complex) and is not assembled from multiple copies
(FIG. 4). The translocation channel is formed mainly by
SecY. Half of the protein channel is formed by TM1–5
and half is formed by the region that contains TM6–10.
The opening in the centre of the SecY complex
constitutes the central pore through which hydrophilic
regions of exported or membrane proteins are moved
during the translocation process (FIG. 4a). Four isoleucines, one valine and one leucine line the pore ring.
Strategically introduced cysteines in the centre of SecY
can be crosslinked to peptide chains of the exported
protein that is trapped in the translocation channel70.
In the crystal structure, the translocation channel is
in the closed state, and a helix plugs the pore, which
presumably helps to maintain the permeability barrier. Recently, this helix was shown to contribute to the
gating mechanism, by keeping the channel in a closed
state71. As expected, locking the plug in the centre of
the channel through disulphide crosslinking results in
an inactive translocation channel. Moreover, deletion
of the plug results in a strain that has a protein location
A (PrlA) phenotype. Characterization of PrlA mutants
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REVIEWS
a
b
SecE
TM7
TM2
Signal
peptide
Secβ
Plug
L406
I260 I174
V79
I170
I75
Figure 4 | The SecYEβ structure and the lateral gate. a | The residues that line the
pore ring are highlighted. b | The lateral gate (TM2–7) interface region (view from
the cytosol) by which the transmembrane region would exit the channel. A signal
peptide is represented by a solid circle. Structures courtesy of M. Paetzel, Simon Fraser
University, British Columbia, Canada.
showed that they have higher translocation activity for
exported proteins that are with or without a functional
signal peptide, possibly because the SecYEG channel is
stabilized in the open state72,73.
In addition to the central pore, which is gated by the
plug, the structure also revealed a gate region that could
open laterally towards the lipid phase, thereby allowing a
hydrophobic domain of an inserted substrate to escape
into the lipid bilayer. This lateral gate region (FIG. 4b) comprises TM7 and TM2 of SecY, and is thought to be where
the signal peptide of an exported protein, or a hydrophobic
region of a membrane protein, intercalates before integrating into the membrane. Indeed, crosslinking studies have
provided evidence that the signal peptide of an exported
protein intercalates between TM7 and TM2 (Ref. 74).
Cryo-electron microscopy studies of the ER translocase revealed an oligomeric complex75 (for a discussion
of the concerted action of two Sec translocases in the
membrane biogenesis of proteins, see Refs 76,77). After
investigating the Sec61–ribosome complex, Beckmann
and colleagues78 discovered that the central pore of the
Sec61 complex aligns with the exit tunnel that is located
within a large ribosomal subunit. The central part of
the ER Sec61 translocase — through which the peptide
chain of an exported protein passes — is thought to be
an aqueous pore79. Interestingly, the crystal structure of
the archaeal SecY complex revealed an aqueous channel,
although the channel was constricted in its centre by the
hydrophobic pore ring57.
Folding of membrane proteins
During membrane biogenesis, membrane proteins
must fold into their three-dimensional conformations
(reviewed in Refs 80,81). For many multi-spanning
membrane proteins, this means that the α‑helical TM
segments must interact and pack together into a bundle.
In addition, the cytoplasmic and extracytoplasmic loops
must fold correctly. It has been proposed that folding
factors exist that can assist in the folding and assembly
of the TM domains of polytopic membrane proteins.
YidC has a crucial role in the folding of the sugar
transporter LacY, which spans the membrane 12 times82.
Although depletion of YidC does not seem to lead to a
defect in LacY membrane insertion through the Sec pathway, it does lead to a defect in the conformation of the
protein. Using two monoclonal antibodies that recognize
different conformational regions of the protein, Nagamori
and colleagues82 found that the binding of these antibodies was significantly perturbed when YidC was depleted.
Additionally, the proteolysis of this misfolded LacY under
YidC-depletion conditions was enhanced compared with
the proteolysis of LacY when YidC was present at wildtype levels. Taken together, these results show that LacY
cannot fold correctly if YidC is depleted. Given that YidC
makes contact with membrane proteins during insertion,
it is reasonable to propose that YidC forms an assembly
site for the TM regions.
It was suggested recently that SecY is involved in
membrane protein folding4. For example, in certain SecY
mutants, the LacY protein was found to be inserted into
the membrane, but was misfolded and susceptible to cellular proteolysis. These SecY mutants were not defective
in their translocation of the exported protein pro-OmpA,
but were defective in their membrane translocation of
a hydrophilic loop of MalF. Interestingly, these SecY
mutants show elevated membrane stress through the σE
stress-response pathway. Combined, these data suggest
that SecY and YidC play a part in the membrane integration and folding of membrane proteins. The exact folding
or insertion defect of LacY in these studies is not clear,
but could have been caused by improper insertion of a
TM segment that led to incorrect membrane topology.
Assembly of multi-subunit membrane proteins
Many proteins within the membrane are multi-subunit
membrane proteins that have essential cellular functions. After membrane insertion, the subunits of
membrane protein complexes must locate each other
and assemble into their correct oligomeric structure. To
illustrate the complexity of this problem, we consider
the formation of the F1F0 ATPase protein complex.
F0c, the c subunit of the F1F0 ATPase, inserts and assembles into an oligomer through the YidC-only pathway39.
F0a and F0b insert through the Sec pathway and require
YidC for efficient membrane insertion34. To assemble
the F1F0 ATPase complex it is likely that the subunit c
oligomer forms before the remainder of the F0 component, which comprises subunits a, b and c. Finally, the
entire complex is formed by the attachment of the F0
sector component to the F1 complex, which comprises
the α-, β-, γ-, δ- and ε-subunits.
Using 35S-labelling, native-gel electrophoresis and
SDS–PAGE, it was recently shown that the four subunits
(I–IV) of E. coli cytochrome bo3 oxidase are assembled
in a preferred order (III–IV, then I–III–IV and, finally,
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REVIEWS
I, II, III and IV)83. In the same study, Stenberg and colleagues83 also showed that insertion of the haem b cofactor facilitates the assembly of subunit I with the other
E. coli cytochrome bo3 oxidase subunits. The assembly of
cytochrome c oxidase has also been studied in other bacteria and in mitochondria84–87. The assembly pathway of
cytochrome c oxidase seems to differ between bacteria,
and in Rhodobacter capsulatus proceeds through subcomplexes which contain assembly proteins that mediate
the process84. Future studies will further elucidate the
functions of the chaperones and assembly proteins in
this process and determine which residues of the subunits
are necessary to form a stable protein complex.
Quality-control systems are used to check whether
membrane proteins are assembled and folded correctly,
and chaperones and proteases can be induced to assist
with misassembly and misfolding. YidC depletion,
which is thought to cause the misfolding of membrane
proteins, induces the σE stress-response pathway4. The
stress-induced FtsH protein is involved in degrading F0a
and SecY when they are expressed in the absence of their
partner proteins88,89. FtsH can use the energy from ATP
hydrolysis to dislocate a membrane protein substrate from
the lipid bilayer90. The YaeL, HtpX and GlpG membrane
proteases might also have quality-control functions.
These proteases can cleave within TM segments of
membrane proteins91–93. One open question is how great
a role FtsH, YaeL, HtpX and GlpG have in the degradation and quality control of the folding state of membrane
proteins.
Concluding remarks
In recent decades, the molecular devices that mediate
the insertion of proteins into the bacterial cytoplasmic
membrane have been discovered. The Sec translocase is
the general apparatus that is used for membrane protein
topogenesis in bacteria; it integrates the hydrophobic
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Acknowledgements
Work in the laboratory of R.E.D. was supported by National
Institutes of Health Grant GM63862-05. The authors thank
A. Kuhn for critical reading of the manuscript.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus subtilis | Escherichia coli | Methanococcus jannaschii |
Neisseria meningitidis | Neurospora crassa | Rhodobacter
capsulatus | Streptococcus mutans
Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=protein
Omp85 | SecA | SecB | SecE | SecF | SecG | SecY | YaeT | YajC |
YfgL | YidC
FURTHER INFORMATION
Ross E. Dalbey’s homepage: http://www.chemistry.ohiostate.edu/~dalbey/index.html
All links are active in the online pdf
www.nature.com/reviews/micro
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