Inserting proteins into the bacterial cytoplasmic membrane using the ...

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 www.nature.com/reviews/micro

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REVIEWS 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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REVIEWS 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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REVIEWS 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


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 volume 6 | march 2008 | 237


REVIEWS 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.



REVIEWS 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 nature reviews | microbiology

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 volume 6 | march 2008 | 239


REVIEWS a Sec translocon

N YidC

YidC

Periplasm

YidC Cytoplasm

b Periplasm YidC

N

C

Cytoplasm

Nature Reviews | Microbiology 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 www.nature.com/reviews/micro


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,

Nature Reviews | Microbiology nature reviews | microbiology


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

1. 2.

3. 4.

5. 6. 7. 8. 9.

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segments laterally into the lipid bilayer and transports the hydrophilic chains across the membrane. The topogenic sequences of the membrane proteins that enable the correct membrane topology to be achieved during insertion are as important as the translocases. The process of assuming correct topology during insertion can be extremely complex and dynamic, especially for membrane proteins. Interestingly, the YidC component mediates membrane protein insertion and integration directly, and recent studies have shown that both YidC and the Sec translocase play crucial parts in the folding of Lac permease into its correct conformation. To move the field of membrane protein topogenesis forward, it is essential to characterize the translocases that actively insert membrane proteins. Does the oligomeric state of SecYEG and YidC vary depending on the membrane protein substrate? To understand how YidC works at a molecular level, it will be essential to solve the structure of YidC with and without bound substrate. The structure would reveal how this important protein facilitates membrane insertion and shed light on the organization of the six TM segments, as well as the substrate-binding region. It will be interesting to find if YidC, like SecYEG, contains a lateral gate that allows TM domains to leave the YidC insertase and integrate into the membrane. Finally, the field is moving towards an understanding of the assembly of membrane protein complexes. This is a large frontier, as there are many multi-subunit membrane proteins. Some of the key questions are: do the subunits assemble in a preferred order; are there chaperones that facilitate the assembly process; what happens if the complexes fail to be fully assembled; and are there cellular processes that respond to and eliminate these incomplete complexes? Solving these questions will require a combination of structural, biochemical and genetic tools.

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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
