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Protein TranslocationAcross the BacterialCytoplasmic MembraneArnold J.M. Driessen and Nico NouwenDepartment of Molecular Microbiology, Groningen Biomolecular Sciences andBiotechnology Institute and the Zernike Institute for Advanced Materials, Universityof Groningen, 9751 NN, Haren, The Netherlands; email: a.j.m.driessen@rug.nl,nico.nouwen@mpl.ird.fr

Annu. Rev. Biochem. 2008. 77:64367

First published online as a Review in Advance onDecember 13, 2007

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This articles doi:10.1146/annurev.biochem.77.061606.160747

Copyright c 2008 by Annual Reviews.All rights reserved

0066-4154/08/0707-0643$20.00

Key Wordschaperone, membrane protein, proton motive force, SecA, SecY,translocase

AbstractAbout 25% to 30% of the bacterial proteins function in the cell en-velope or outside of the cell. These proteins are synthesized in thecytosol, and the vast majority is recognized as a ribosome-boundnascent chain by the signal recognition particle (SRP) or by thesecretion-dedicated chaperone SecB. Subsequently, they are tar-geted to the Sec translocase in the cytoplasmic membrane, a multi-meric membrane protein complex composed of a highly conservedprotein-conducting channel, SecYEG, and a peripherally bound ri-bosome or ATP-dependent motor protein SecA. The Sec translocasemediates the translocation of proteins across the membrane and theinsertion of membrane proteins into the cytoplasmic membrane.Translocation requires the energy sources of ATP and the protonmotive force (PMF) while the membrane protein insertion is cou-pled to polypeptide chain elongation at the ribosome. This reviewsummarizes the present knowledge of the mechanism and structureof the Sec translocase, with a special emphasis on unresolved ques-tions and topics of current research.

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Translocase: amembrane-embedded proteincomplex thatmediates thetranslocation ofpolypeptides fromone side of themembrane to theother side

ContentsINTRODUCTION. . . . . . . . . . . . . . . . . 644TARGETING AND

RECOGNITION. . . . . . . . . . . . . . . . 645Signal Sequences . . . . . . . . . . . . . . . . . 645Targeting Routes . . . . . . . . . . . . . . . . . 646SecB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646Signal Recognition Particle

and Its Receptor . . . . . . . . . . . . . . . 648SecA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649

TRANSLOCATION. . . . . . . . . . . . . . . . 651SecYEG . . . . . . . . . . . . . . . . . . . . . . . . . 651SecDF( yajC) . . . . . . . . . . . . . . . . . . . . . 655Mechanisms and Energetics . . . . . . . 656

MEMBRANE PROTEININSERTION . . . . . . . . . . . . . . . . . . . . 659Mechanisms and Lateral Pore

Opening . . . . . . . . . . . . . . . . . . . . . . 659YidC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660

SUBCELLULAR LOCALIZATIONOF THE SEC SYSTEM . . . . . . . . . 661

INTRODUCTIONBacteria are generally simple structures thatlack a membrane-bound nucleus. Becauseof this, they are described as prokaryotes,which also include the Archaea that rep-resent a separate kingdom of life. Bacteriamay be conveniently divided into two furthergroups, depending upon their ability to retaina crystal violet-iodine dye complex, termedthe Grams stain. Gram-negative and gram-positive bacteria have fundamentally differ-ent structures, related to the composition ofthe cell envelope. Gram-positive bacteria havemany layers of peptidoglycan that surroundthe cytoplasmic membrane, whereas the cellenvelope of gram-negative bacteria is morecomplex. Above the cytoplasmic membraneis a periplasm with some layers of peptido-glycan, and beyond this layer lies an outermembrane, which contains phospholipids andlipopolysaccharides. Some bacteria have anatypical cell envelope, such as the mycobac-teria that contain a thick hydrophobic my-

colic acid layer surrounding the cytoplasmicmembrane. Each of these envelope struc-tures represents a subcompartment in the cellwith its own set of resident proteins. Proteinsare synthesized at ribosomes localized in thecytosol, and all proteins that function out-side of the cytosol either need to insert orneed to pass the cytoplasmic membrane toreach their final destination. The major routefor protein transport across and into the cy-toplasmic membrane is the Sec translocase(Figure 1). In its minimal form, the Sectranslocase consists of a protein-conductingchannel, the SecYEG complex, and a periph-erally associated component that delivers en-ergy for the transport process. This compo-nent is either a translating ribosome or SecA,an ATP-driven motor protein. Other Sec pro-teins may associate with the Sec translocase toprovide it with additional functionalities. Theprotein-conducting channel is a highly con-served complex with homologs present be-yond the prokaryotic kingdom, such as in thechloroplast thylakoid membrane and the en-doplasmic reticulum of eukaryotes.

An excellent historical overview of the firstdiscovery of the Sec translocase in bacteria un-til the establishment of a reconstituted in vitrotranslocation system, based on purified com-ponents, was written by Wickner and cowork-ers (1) more than 15 years ago for the AnnualReview of Biochemistry. Extensive genetic anal-ysis, using Escherichia coli as a model organism,has resulted in the identification of all majorgenes (and proteins) (for a review, see Refer-ence 2). Another major milestone has been thecomplete functional in vitro reconstitution ofthe protein translocation reaction using puri-fied components (3). This opened a new eraof research allowing an understanding of theprotein translocation process at the molecularlevel. Since the early 1990s, major progresshas been made, and recent high-resolutionstructures of subunits of the Sec translocaserepresent new milestones (4, 5). Supportedby extensive biochemical analysis, the mostcomplete view of the molecular basis of aprotein translocation and membrane protein

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PMF

GTP

5'

3'

FtsY

SRP

SecB

SecYEG

SecDF(yajC)

YidCYidC SPase

ATP

N

N

NC

C

SecA Cytoplasm

CM

Periplasm

a

b

c

Figure 1Scheme of protein targeting to the Sec translocase. The bacterial Sec translocase is a protein complex inthe cytoplasmic membrane (CM), which comprises a peripheral motor domain SecA ( green), theprotein-conducting channel, SecYEG (orange), and the accessory proteins SecDF(yajC) ( pink) and YidC(red ). Signal peptidase (SPase) is a membrane-bound peptidase that cleaves the signal sequence frompreproteins at the periplasmic face of the membrane. (a) Secretory proteins ( yellow) areposttranslationally targeted to the Sec translocase by virtue of their signal sequence, which is recognizeddirectly by SecA, the motor domain of the Sec translocase, or by the aid of the molecular chaperone SecB(blue). (b) Membrane proteins and some preproteins are cotranslationally targeted to the Sec translocaseas ribosome-bound nascent chains by the SRP and the SRP-receptor FtsY ( purple). (c) Some membraneproteins insert into the cytoplasmic membrane via YidC. Abbreviation: PMF, proton motive force.

insertion pathway is now possible. This re-view summarizes the recent progress on theSec translocase, with an emphasis on thestructural and mechanistic aspects, and doesnot discuss other translocases of the cyto-plasmic membrane involved in the transloca-tion of folded proteins or subsets of envelopeproteins.

TARGETING ANDRECOGNITIONSecretory proteins (preproteins) and mem-brane proteins are synthesized at ribosomesin the cytosol. To reach their destinationoutside of the cytosol or in the cytoplasmic

Preprotein: theprecursor form of asecretory protein,which is usually anunfolded state of theprotein with anN-terminal signalsequence

Signal sequence:an N-terminalextension ofsecretory proteinsthat functions as asorting andrecognition signalfor the translocase

membrane, these proteins need to be recog-nized and targeted to the Sec translocase thattranslocates them across or inserts them intothe cytoplasmic membrane. Below, we dis-cuss the features that distinguish preproteinsand membrane proteins from cytosolic pro-teins and how they are targeted to the Sectranslocase.

Signal SequencesSecretory proteins (preproteins) are synthe-sized with an N-terminal extension, the sig-nal sequence, which is removed after translo-cation (for a review, see Reference 6). Signalsequences have a tripartite structure with an

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Transmembranesegment (TMS):stretch ofhydrophobic aminoacid residues thatanchors membraneproteins in thephospholipid bilayer

Ribosome-nascentchain (RNC): acomplex of aribosome, anmRNA, and apartially synthesizedpolypeptide chain

Signal recognitionparticle (SRP):complex of a 4.5SRNA molecule andthe GTPase P48,which recognizeshydrophobictransmembranesegments inribosome-nascentmembrane proteins

N-terminal region encompassing one to threepositively charged amino acid residues (the Ndomain), a hydrophobic core region consist-ing of 1015 residues (the H domain), anda more polar C terminus, which constitutesthe signal peptidase cleavage site (C domain).These domains show little sequence conserva-tion, but the presence of an N-terminal signalsequence can conveniently be predicted withcomputer algorithms such as SignalP (7). Sig-nal peptidase is a membrane-bound enzymethat utilizes a Lys-Ser catalytic dyad for sig-nal peptide cleavage (8). As shown by struc-tural studies, the signal peptide cleavage siteat the 3 and 1 positions contains aminoacid residues with small neutral side chainsthat allow the signal peptide to dock intothe catalytic site of signal peptidase. An -helix-destabilizing amino acid residue is of-ten found at position 6 and/or in the mid-dle of the H domain (6). Nuclear magneticresonance studies on the conformation of sig-nal peptides in membrane mimetic environ-ments suggest the presence of two structuraldomains: a stable -helix N-terminal domainand a more flexible C domain (9, 10). Al-though mutagenesis studies have clearly es-tablished the importance of various domainsof the signal sequence for translocation, de-ficiencies in one region can often be com-pensated by improving the quality of an-other region (11). This suggests a multifacetedmechanism of signal-sequence recognition bythe Sec translocase. Integral membrane pro-teins generally do not contain a signal se-quence, but instead, their hydrophobic trans-membrane segments (TMSs) function as aninternal signal for targeting and insertion.

Targeting RoutesE. coli contains two major targeting routesthat direct proteins to the Sec translocase(Figure 1). Most preproteins are targetedvia the molecular chaperone SecB (Figure 1,step a) whereas cytoplasmic membrane pro-teins are targeted as ribosome-bound nascentchains (RNCs) by the signal recognition par-

ticle (SRP) (Figure 1, step b). These twopathways diverge at an early stage when thenascent chain emerges from the ribosome (12)because of a competition between the SRPand the peptidyl-prolyl cis-trans isomerasetrigger factor for binding to the nascent chain(13). The strength of interaction of the SRPwith signal sequences increases with the hy-drophobicity of the H region (14). Owing tothis high selectivity, the SRP binds directlyto hydrophobic TMSs of nascent cytoplas-mic membrane proteins, directing them intothe cotranslational targeting pathway. Trig-ger factor shunts nascent preproteins into theSecB pathway by blocking the interaction be-tween the SRP and the signal peptide (13).While the nascent chain grows, SecB or otherchaperones may associate with the nascentpreproteins, which are then targeted post-translationally to the Sec translocase.

SecBIt has been known for a long time that syn-thesis and translocation of preproteins in E.coli are uncoupled events (15). Because pro-teins need to be translocated in an unfoldedconformation and because unfolded proteinsare unstable in the cytosol, the unfoldedstate of preproteins is stabilized by molecu-lar chaperones prior to translocation. SecB isa secretion-dedicated chaperone (for a review,see Reference 16) found mostly in the -, -,and -proteobacteria (17). SecB is a homote-trameric protein that is organized as a dimer ofdimers (18) (Figure 2). The structure of theHaemophilus influenzae SecB shows the pres-ence of 70-A-long channels located on ei-ther side of the SecB tetramer that may be in-volved in the binding of unfolded polypeptidesegments. Consistent with biochemical data,each binding groove seems to contain twosubsites that may recognize distinct featuresof preprotein substrates. One subsite is a deepcleft, lined with mostly conserved aromaticresidues and suited for binding of hydropho-bic and aromatic regions of polypeptides, andthe other subsite forms a shallow open groove

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SecAC-terminal

peptide

Polypeptide-bindinggrooves

a b

Figure 2Structure of SecB, a secretion-dedicated molecular chaperone. A ribbon drawing of the structure of theH. influenza SecB tetramer is shown in complex with peptides that correspond to the C-terminalSecB-binding domain of SecA (Protein Data Bank number 1OZB) (29) in two orthogonal views.(a) Front view showing the four-stranded -sheet of each monomer and the packing of the dimer. (b) Sideview showing the dimer-dimer interface formed by -helices, and as indicated by the shaded ellipse, thelong putative polypeptide-binding grooves on the sides of the SecB tetramer. The two SecA C-terminalpeptides are shown as ribbon drawings in gray with the central zinc ion indicated as a sphere. Eachsubunit in the SecB tetramer is shown in a different color.

with a hydrophobic surface that might beinvolved in the binding of -pleated sheets(18). The position of the binding sites sug-gests that polypeptides are wrapped aroundthe tetrameric SecB protein and supported byelectron paramagnetic resonance experiments(19). The mechanism by which SecB differ-entiates between secretory and nonsecretoryproteins remains poorly understood. Variousmethods have been used to identify SecB-binding sites in preproteins (for an overview,see Reference 16). SecB only interacts withunfolded polypeptides and appears to asso-ciate with mature regions that are normallyburied in the folded structure (20). On the ba-sis of peptide-binding studies, a general SecB-binding motif has been defined that consistsof approximately nine amino acid residues en-

riched in aromatic and basic residues, whereasacidic residues are strongly disfavored (21).Such peptide sequences are found with sim-ilar frequency in both cytoplasmic and se-cretory proteins, and this situation does notexplain the high selectivity of SecB in vivowhere it is associated with only a subset oflong nascent secretory proteins (22). Selectiv-ity in binding has been attributed to a kineticpartitioning between polypeptide folding andassociation with SecB (23). Herein, the signalsequence could indirectly affect the selectivityof SecB by retarding the folding of the pre-protein. Because the rate of SecB associationwith polypeptide substrates is limited only bythe rate of collision, which is much fasterthan folding (24), the specific and selectiveinteraction with preproteins is likely a

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complex and delicate mechanism with molec-ular features that remain unknown.

Possibly more general chaperones substi-tute for the SecB function in bacteria that lackSecB (25). A general chaperone activity hasalso been associated with SecB in chaperone-compromised cells (26), but a key featurethat distinguishes SecB from other chaper-ones is its ability to interact with high affin-ity [Kd 1030 nanomolars (nM)] with theSecYEG channel-bound SecA protein (27).The highly conserved 22 C-terminal aminoacid residues of SecA constitute a genuineSecB-binding domain (28). Tetrameric SecBbinds the dimeric form of SecA (28), consis-tent with the ability of SecB to bind two C-terminal SecA peptides (29) (Figure 2). TheSecA C terminus is a highly flexible, posi-tively charged region with three cysteines anda histidine residue that together coordinatea zinc atom that is needed for SecB bind-ing (30). The latter stabilizes the fold of theC terminus of SecA (29). SecA-SecB bindingis mediated through an electrostatic interac-tion of the positively charged SecA C terminiwith the negatively charged patch presenton both sides of the SecB tetramer (29, 31)(Figure 2). This SecA-SecB complex can beenvisaged as a symmetric structure whereinthe C termini of the two SecA protomers em-brace the tetrameric SecB with the postulatedpeptide-binding grooves on SecB aligned withthe protein-binding sites on SecA (29).

SecA and SecB interact in solution withlow affinity (Kd 1.6 M) (16). This in-teraction involves the C terminus of SecB(32), which may insert at the dimer inter-face of SecA and bind the N terminus ofSecA (29, 33). SecB does not interact withATP. The transfer of the secretory proteinfrom SecB to the SecYEG-bound form ofSecA therefore occurs independently of en-ergy. However, for membrane release, SecBdepends on the binding of ATP to SecA, andin this respect, it seems to act as a cochap-erone of SecA (28). Also, high-affinity SecA-SecB binding involves the dimeric SecA (31),whereas low-affinity binding is observed only

with the monomeric form of SecA (34). Thishas led to the suggestion that a SecA dimer-to-monomer transition underlies the mechanismof SecB release from the membrane (34).

Signal Recognition Particleand Its ReceptorApproximately 20% the proteome of E.coli concerns cytoplasmic membrane proteins(35). Most of these proteins enter the mem-brane via the Sec translocase. Unlike prepro-teins, membrane proteins are targeted to themembrane in a cotranslational manner. Thisinvolves a bacterial homolog of the eukary-otic SRP (for a review, see Reference 36). Thebacterial SRP is of a lesser complexity com-pared to its eukaryotic counterparts. The E.coli SRP is composed of a complex of a 4.5SRNA and a 48-kDa GTPase P48 or Ffh (forfifty-four homolog) (37) that interacts specifi-cally with the signal sequence or hydrophobicTMSs of nascent proteins (14, 38). In eukary-otes, this interaction results in a translationalarrest, but this phenomenon has not been ob-served in bacteria. RNC-bound SRP interactswith FtsY (39), a prokaryotic homolog of the-subunit of the SRP receptor. Bacteria lack ahomolog of the -subunit of the SRP recep-tor, and it is generally believed that FtsY ful-fills the function of both the SR- and SR-subunits of the SRP receptor (40). FtsY bindsto membranes via the anionic phospholipids(41) but also interacts directly with the Se-cYEG channel (42). The interaction betweenFtsY and Ffh changes the nucleotide-bindingaffinity of both proteins and allows them tobind GTP (43, 44). Upon GTP hydrolysis byboth the SRP and FtsY, the RNC-SRP-FtsYcomplex dissociates, and the released RNCcomplex is transferred to SecYEG (45), whichbinds the ribosome directly (46).

Ffh is composed of two domains, the NGdomain (N, amino terminal; G, GTPase),which contains a GTP-binding site, anda methionine-rich C-terminal M domain,which is involved in the binding of the sig-nal sequence and SRP RNA (47). The central

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G domain is related to the p21Ras GTPasefamily. FtsY contains a similar NG domainand a very acidic N-terminal domain that isinvolved in membrane targeting (48). In bothFtsY and Ffh, the N domain is closely asso-ciated with the G domain, and the connect-ing region between the two domains is highlyconserved (49, 50). The interaction betweenboth proteins appears to occur mainly via theirstructurally related NG domains (49, 51). The4.5S RNA and the M domain of Ffh are notrequired for the stimulation of GTPase activ-ity in vitro (52, 53). However, the 4.5S RNAcoordinates the interaction of the SRP andFtsY (52) with recruitment of the ribosomeand the subsequent transfer of the RNC tothe SecYEG complex (54).

The signal-sequence-binding site of Ffhlocalizes to the M domain. The structureof the M domain of Thermus aquaticus Ffhshowed that it contains a deep groove that islined almost exclusively with the side chainsof conserved hydrophobic residues and thatincludes all conserved methionines (55). Thedimensions and hydrophobic character ofthe groove suggest that it forms the signal-sequence-binding pocket of the SRP (56).Close to this groove, part of the conservedRNA IV domain binds to the M domain. Thissuggests that the functional signal-sequence-binding site consists of both protein and RNA.Recent cryo-electron microscopy structuresof RNC complexes with the SRP of the E.coli (57) and the mammalian system (58) in-dicate that the signal sequence is presentedat the ribosomal tunnel exit in a manner thatallows it to slide directly into the proposedsignal-sequence-binding site on the SRP. Fur-ther analysis awaits high-resolution structuresof the SRP with a bound signal sequence.

SecASecA is a central component of the Sectranslocase, which functions as an ATP-dependent motor protein (Figure 1). It inter-acts with nearly all other components involvedin protein translocation, and its ATPase activ-

ity is allosterically regulated by unfolded pre-proteins, the SecYEG complex, acidic phos-pholipids, and by SecB (59, 60). In recentyears, high-resolution structures of SecA pro-teins of various species have been solved (4,6164), providing detailed insight into theiroverall domain organization. In most of thesecrystal forms, SecA is dimeric with its pro-tomers arranged in an antiparallel fashion,except for the Thermus thermophilus SecA,which has been crystallized as a parallel dimer(64). The SecA protomer (Figure 3) can besubdivided into several structural subdomains[nucleotide-binding folds 1 and 2 (NBF1and NBF2); the preprotein cross-linking do-main (PPXD); the -helical scaffold domain(HSD); the -helical wing domain (HWD);and the C-terminal linker (CTL)]. The actualmotor function of SecA, i.e., the conversion ofchemical energy into movement is performedby the DEAD motor core that is also foundin DNA/RNA helicases (65). The DEAD mo-tor consists of two RecA-like NBFs, NBF1and NBF2. At the interface of these two do-mains, ATP can be bound and hydrolyzed toinduce the conformational changes necessaryfor preprotein translocation. SecA interactswith preproteins via its PPXD (66, 67), a re-gion that is inserted in NBF1 but that forms aseparate domain in SecA (4) (Figure 3). Inter-estingly, an extended -helix of the HSD con-tacts all other domains of SecA and thereforelikely plays an important role in the catalyticcycle of SecA.

SecA is a soluble protein that localizesboth to the cytosol and the cytoplasmic mem-brane (68). Its association with the membraneoccurs via low- and high-affinity interac-tions with anionic phospholipids (59) and theSecYEG complex (27), respectively. TheCTL domain of SecA has a dual function, in-cluding its involvement in lipid binding (69)and in SecB binding, as described in the previ-ous section. SecA exists in a dynamic equilib-rium between a monomeric and dimeric form(Kd 0.1 M) (70). In the cytosol andwhen purified, SecA is mainly dimeric (71).Although the exact features of the dimer

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PPXD NBF2 HWD CTLNBF1

DEAD domain C domain

HSD

1 223 378 416 621 684 756 832 922

PPXD

NBF2HWD

IRA1

NBF1

HSD

N

C

Centralopening

Figure 3Structure of SecA, the motor domain of the Sec translocase. The structure of the Mycobacteriumtuberculosis SecA (Protein Data Bank number 1NKT) (61) shows the different subdomains (NBF1 andNBF2, nucleotide-binding folds 1 and 2; PPXD, preprotein cross-linking domain; HSD, -helicalscaffold domain; HWD, -helical wing domain) in color and the corresponding linear display of thedomains. The C-terminal linker (CTL) domain was not resolved in the structure. The second protomerof the dimeric SecA is represented as a ( gray) ribbon. The intramolecular region of ATP hydrolysis 1(IRA1), which controls hydrolysis of ATP at NBF1, localizes to the HSD (as indicated) and to a centralopening at the SecA dimer interface (shaded region).

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interface differ between the various crystalstructures of SecA, the C-terminal part of theextended HSD participates in dimer forma-tion (4, 61, 62).

ATP binding and hydrolysis involves boththe NBF1 and NBF2 of SecA. Both NBFscontain the highly conserved Walker A andthe less-conserved Walker B motifs orga-nized in a RecA structural fold (72). BothNBFs are essential for the ATPase activity ofSecA and translocation (73). NBF1 is the cat-alytically important nucleotide-binding site(Kd, ADP 0.13 M) (74) that is regulated byNBF2 (73, 75). Both NBFs operate in concertto bind a single nucleotide comparable to theDEAD-box helicases (4, 62).

Several functionally important regions inSecA have been defined. The intramolecularregulator of ATP hydrolysis 1 (IRA1) regionis a global regulator of the ATP turnover ofSecA. This region localizes in the HSD. Itforms a helix-loop-helix structure that con-tacts on one side NBF2 and on the other sidethe PPXD (Figure 3). For soluble SecA, dele-tion of IRA1, or disturbance of the IRA1-NBF2 or IRA1-PPXD interaction, resultsin an elevated preprotein-uncoupled ATPaseactivity (76). Therefore, the IRA1 domainlikely prevents uncontrolled ATP hydroly-sis of SecA in the cytosol. The central fea-ture of the mechanism used by preproteinsto control the ATPase cycle of SecA is, how-ever, a highly conserved salt bridge, termedGate 1, that controls the opening/closure ofthe nucleotide-binding cleft (77) in conjunc-tion with the binding signal generated at thePPXD. This relay mechanism is only ac-tive after binding of SecA to SecYEG andallows the functional coupling between thepreprotein binding and release mechanismand the ATPase cycle. Structural and func-tional data indicate an allosteric communica-tion between the DEAD motor domain andthe PPXD of individual monomers withinthe SecA dimer, wherein conserved regionslining the nucleotide cleft undergo cycles ofdisorder-order transitions (78). In a crystalform of a high [Mg2+]-enforced monomeric

and nucleotide-free Bacillus subtilis SecA (79),the PPXD, HSD, and HWD have undergonea drastic conformational change as comparedto the protomer in the crystallized dimer,whereas the NBFs have remained at the sameposition. This form has been proposed to rep-resent the open conformation where theHSD/HWD and the PPXD constitute a largegroove that in its dimensions is similar topeptide-binding grooves observed in chap-erones and peptide-binding proteins. SecAcontains separate binding sites for the signalsequence and for the mature domain of pre-proteins (59), and an additional potentialpeptide-binding site locates to the interfaceof the NBF1, the HSD, and the PPXD (4).

Although it is generally accepted that SecAis dimeric in the cytosol, its translocation-active quaternary structure is currently underdebate. In vitro, dimeric SecA dissociates intomonomers upon an interaction with anionicphospholipids (80, 81), whereas signal pep-tides have been reported to either dissociatethe dimer (81, 82) or promote its oligomeriza-tion (80). Also, it has been argued that SecAfunctions as a monomer because of the simi-larity of the DEAD motor domain with that ofhelicases (79). However, various lines of evi-dence indicate that in in vitro translocation as-says SecA functions and remains dimeric (71,83, 84). In addition, attempts to create a stablemonomeric SecA form by site-directed muta-genesis and truncations result in a severe lossof activity (34, 81, 85).

TRANSLOCATIONAfter targeting to the cytoplasmic membrane,preproteins are translocated across this mem-brane via a complex that is composed ofseveral integral membrane proteins. Below,we describe the structure of this integral mem-brane protein complex and how it may facili-tate protein translocation.

SecYEGThe E. coli protein-conducting channel con-sists of three integral membrane proteins,

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Front to front

Back to back

SecY

Centralpore

Lateralgate

b c

SecE

a

Pore ring

Plug

Plug

Hinge

Cytosol

Periplasm SecG(Sec)

SecE

SecY(TMSs 15)

SecY(TMSs 610)

SecA/ribosome-associating groups

Late

ral

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Figure 4Structure of SecYEG, the protein-conducting channel of the Sec translocase (5). (a) Membrane crosssection and (b) a cytosolic view of the structure of the M. jannaschii SecYE (Protein Data Bank number1RHZ) (5) and (c) a schematic presentation of possible SecYEG dimer configurations. Theprotein-conducting channel consists of three subunits: the SecY (Sec61) that is embraced by the SecE(Sec61) subunit and the peripheral bound SecG (Sec61) protein. The channel forms an hourglass-likestructure with a pore ring of hydrophobic amino acid residues at its constriction. The pore is closed atthe periplasmic side by a plug formed by a short -helix of a periplasmic loop that folds back into thefunnel. The two halves of the clamshell-like structure of SecY are indicated as TMS15 and TMS610and are connected by a hinge region in the back. The clamshell opening in the front may form a lateralgate to the lipid bilayer. Signal-sequence insertion into lateral gate is thought to widen the central poreopening and to destabilize the plug, resulting in the opening of a vectorial water-filled channel (5).

termed SecY, SecE, and SecG, that togetherform a stable complex (3). The heterotrimericorganization of this complex, which corre-sponds to Sec61 in eukaryotes, is con-served in all three kingdoms of life (86).The X-ray crystallography structure of anidle SecYE heterotrimer of the archaeonMethanococcus jannaschii (5) (Figure 4) andthe cryo-EM structure of a active E. coliSecYEG complex, bound cotranslationally toan RNC (87), have revealed detailed aspectsof the structure of the heterotrimeric com-plex and the highly dynamic nature of thetranslocation pore. In agreement with its uni-versal conservation, the overall structure ofthe M. janaschii SecYE superimposes withthe low-resolution two-dimensional cryo-EMelectron density map of the E. coli SecYEGcomplex (88). The two complexes differ onlyslightly in conformation; however, compared

to that of M. janaschii, the E. coli complexcontains three additional TMSs: two fromSecE and one from SecG. The structure con-firms the presence of 10 -helical segmentsin SecY (Sec61) with the N- and C-terminalends at the cytosolic side of the membrane,and the single TMSs of SecE (Sec61) andof Sec61 (SecG) expose only their N ter-minus to the cytosol. In E. coli, SecE con-sists of three TMSs, but a truncate, whichresembles Sec61 with only the C-terminalTMS, suffices for functionality (89). SecY isorganized into N- and C-terminal domains,compromising TMS15 and TMS610, re-spectively (Figure 4). Both domains are con-nected by a periplasmic loop between TMS5and TMS6, yielding an overall structure thatresembles a clamshell with a central funnel-like pore. Both domains of SecY are heldtogether by SecE, which acts as a molecular

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clamp. One part of the clamp is formed bythe conserved amphipathic cytoplasmic loopthat connects TMS2 with TMS3 of SecE, andthe other part is formed by TMS3 of SecE atthe back of SecY (5, 88). SecG makes onlylimited contact with SecY and localizes at theperiphery of the complex (88). The M. jan-naschii SecYE structure likely represents theclosed state of the translocation channel (5),but its overall organization suggests a possi-ble mechanism of channel opening. At the cy-tosolic face of the membrane, the proposedchannel is shaped like an inverted funnel witha diameter of 2025 A at its widest point and4 A at it narrowest point, which is formedby a ring of isoleucine residues that constrictthe channel (pore ring). At the periplasmicface of the membrane, the structure shows acavity, which is closed by the first periplas-mic loop of SecY that folds back as a distorted-helix (TMS2a) into the funnel-like cavity.This structure is termed the plug and is be-lieved to be displaced from the periplasmiccavity upon the initiation of protein translo-cation (90), which would result in the open-ing of the pore to the periplasmic side of themembrane thus forming a continuous vecto-rial aqueous path across the membrane. Poreopening is probably accompanied by an over-all expansion of the SecY clam-like structure,allowing insertion of the signal sequence plusthe early mature region of a preprotein as ahairpin-like structure. Opening of the channelmight be achieved by a hinge movement of theloop region that connects TMS5 and TMS6of SecY (5). A small conformational changein this region may move the two domains ofSecY away from each other, thereby creatingan opening between TMS2 and TMS7 towardthe lipid bilayer, which is at the front of theSecYEG complex. Intercalation of the signalsequence between TMS2 and TMS7 at thelipid bilayer interface (91) and displacement ofthe plug region would result in a channel thatis sufficiently large to permit the translocationof unfolded polypeptide segments through itscenter. This open state of the channel has anhourglass shape with hydrophilic funnels on

both sides of the central constriction. Thehydrophobic pore ring may fit like a gasketaround the translocating polypeptide chain,thereby providing a seal that restricts the pas-sage of ions and other small molecules duringprotein translocation. Mutations in the porering indeed result in transient openings of theSecYEG channel (92). However, the diame-ter of the pore ring is likely too small to allowpassage of an extended polypeptide chain, andtherefore, additional widening of the pore isnecessary. The latter may occur by the move-ment of the -helices to which the pore re-gion residues are attached. The high plasticityof the SecY channel also follows from obser-vations that, in the absence of SecE, SecY isintrinsically unstable and readily degraded bythe membrane-bound protease FtsH (93).

Cysteine cross-linking studies with the E.coli SecY, based on the M. jannaschii SecYEstructure, and fluorescence studies confirmthat a translocating nascent polypeptide chainpasses through the membrane by the postu-lated water-filled channel and suggest that thepore ring residues form a tight hydrophobicring that surrounds the translocating polypep-tide chain (94, 94a). Double cysteine SecYmutants showed that the plug domain movesaway from the channel opening upon initia-tion of translocation (95) and that, in the dis-placed state, there is a constant channel open-ing (92). Moreover, deletion studies of theplug domain suggest that it mainly serves tostabilize the closed state of the SecYEG chan-nel (96). Based on the X-ray structure, themaximum dimensions of the pore are 1520 A (5), which accommodates polypeptidesegments with an -helical secondary struc-ture. However, the Sec translocase can alsotranslocate larger disulfide bond-stabilizedtertiary loops in secretory proteins (97) as wellas proteins, conjugated with bulky fluorescentdyes (98), and organic molecules (99). A mul-titude of biochemical (100102) and struc-tural (87, 103105) data demonstrate that theSecYEG (and Sec61 complex) complexassembles into highly ordered oligomers, withtetramers and dimers as the most dominant

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forms. SecA and the ribosome recruitSecYEG monomers to form such oligomers(105), but SecYEG also has a strong ten-dency to dimerize in the absence of theseligands. A larger pore might be generatedwhen two (or more) SecYEG complexes as-sociate into a front-to-front organization inwhich the lateral openings fuse to form a con-solidated pore (106). In the two-dimensionalcrystals of the E. coli SecYEG, the moleculesare arranged as a back-to-back dimer (88)(Figure 4c), consistent with cross-linkingstudies on the idle state of the complex (107).However, a cryo-EM study of the E. coliSecYEG complex bound cotranslationally toa ribosome-nascent polypeptide complex sug-gests a dimeric arrangement of SecYEG chan-nels in a front-to-front arrangement in itsactive state (87) (Figure 4c). But only onechannel is used to translocate the polypep-tide chain, which is consistent with cross-linking data (108). In addition to dimericSecYEG complexes, higher ordered, possi-bly tetrameric complexes have been observed(100, 102, 104). In eukaryotes, such complexeshave been observed when the Sec61 com-plex is in association with ribosomes, but theirfunctional role is not known. Obviously, inorder to settle controversies on subunit sto-ichiometry, the X-ray structure of an activeSecYEG channel is the ultimate goal forfuture research.

Because SecYEG does not utilize nu-cleotides to generate energy, it must asso-ciate with cellular components that providethe driving force necessary for polypeptidetranslocation or insertion. For cotranslationalmembrane protein insertion, SecYEG asso-ciates with the ribosome (46), whereas in theposttranslational mode, it interacts with SecA(27). The ribosome and SecA interact withsimilar regions of SecYEG, which may sug-gest a similar type of channel-opening mecha-nism. Structural and biochemical data indicatethat ribosomes associate with the SecYEGcomplex at three distinct sites, two of whichare formed by the pairs of long cytoplasmicloops of SecY (C4 and C5, respectively) (87,

109, 110) (Figure 4). The third connectionis mediated by the cytoplasmic loop of SecGand the two N-terminal TMSs of SecE. SecAbinds the same C4 and C5 loop regions ofSecY (111, 112) with SecG (113) and with a re-gion at the interface between TMS4 and C3 ofSecY that is in direct contact with SecG (111).Importantly, interacting regions are located inboth halves of the SecY clamshell (111), whichprovides a means of communication betweenSecA and SecY to sense or induce the transi-tion from a closed to an open state of the pore.In a dimeric arrangement of SecYEG, sepa-ration of the two SecY domains takes placeat the dimer interface, and thus the openingof one subunit is directly transmitted to theneighboring subunit. It should be noted thatthe features that mediate the third ribosome-SecYEG connection (SecG and the SecE ex-tension) are not essential for cell viability orprotein translocation (114, 115). Ribosome-induced opening of the SecYEG channel maybe mediated primarily by the C4/C5 connec-tions, and the third connection plays an aux-iliary role. The stimulatory role of SecG inSecA-dependent translocation may be basedon similar interactions.

The classical view is that channel open-ing is mediated by the signal sequence andthat the ribosome and SecA merely functionto release this domain to the SecYEG chan-nel. In another view, the ribosome and SecAhave been proposed to be actively engagedin the channel-opening mechanism (106) (seealso the section Mechanisms and Energet-ics). The SecA-dependent opening mecha-nism might, however, differ in several as-pects from that of the ribosome as SecA hasbeen proposed to penetrate deeply into theSecYEG channel (116, 117). Moreover, SecYmutants of the C4 loop are severely defec-tive in SecA-mediated posttranslational pro-tein translocation (118) but normally allowthe SecA-dependent translocation of periplas-mic domains of membrane proteins that aretargeted to the Sec translocase via the SRPpathway (119). The latter involves the ribo-some instead of SecA to initiate translocation,

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suggesting different requirements for SecAand the ribosome in channel opening. SecGis in close proximity to C3 and TMS4 ofSecY, which is the region where SecA is postu-lated to (partially) insert or interact with theSecYEG complex (120, 121). This may ex-plain why SecG promotes the SecA confor-mational cycle. SecG has been proposed to in-vert the membrane topology of its two TMSsduring a catalytic cycle of the Sec translo-case (122). Structural studies, however, pro-vide no clues on how this process may oc-cur (5). Moreover, a topologically fixed SecGvariant has been shown to be fully functional(123). Therefore, the presumed topology in-version most likely represents a conforma-tional change within a highly dynamic regionof the SecYEG complex.

Genetic studies have identified a series ofmutations in sec genes that can suppress thetranslocation defect of preproteins with a de-fective signal sequence (124). Interestingly,many of these mutations are in secY (prlA) andlocalize to the pore-facing side of TMS7 andthe plug domain (TMS2a) (for a review, seeReference 2). In these mutants, the SecY-SecEinteraction is destabilized (125). Prl muta-tions do not merely compensate for the signal-sequence defect by restoring the recognition,but also are thought to facilitate the openingof the channel and either stabilize the openor destabilize the closed state of the chan-nel. With the native complex, the open stateis supposed to be stabilized by the signal se-quence, SecA, and/or the ribosome (5). In-deed, several of the prlA mutations have beenshown to stabilize the interaction betweenSecY and SecA, and in vitro these mutants arehyperactive (126) and less dependent on theproton motive force (PMF) (127) for translo-cation. It thus appears that prlA mutations al-ter the channel conformation, and as a resultof this conformational change, SecA is boundtightly to SecY, which permits a more effi-cient initiation of translocation. This resultsin a more efficient translocation of wild-typepreproteins and of the translocation of signal-sequence defective preproteins above back-

Polytopicmembrane protein:a membrane proteinthat contains morethan onetransmembranesegment

ground levels. This concept has been termedproofreading (124) and might be directly re-lated to the mechanism by which SecY acti-vates the ATPase catalytic cycle of SecA. SecAbinds with high affinity (Kd 10 nM) to theSecYEG complex. This binding reaction con-verts SecA to an activated state (128) that isprimed to interact with preproteins and SecB(27, 28). Priming is manifested by an accel-eration of the rate of nucleotide exchange onSecA (128), which removes the rate-limitingstep in ATP hydrolysis, namely ADP release(129). The tight link with SecYEG bindingprevents the uncontrolled hydrolysis of ATPby the cytosolic SecA. Possibly, prlA mutantsresemble a constitutively activated state of theSecYEG-bound SecA.

SecDF( yajC)The SecYEG channel can associate with an-other heterotrimeric membrane complex con-sisting of the SecD, SecF, and YajC proteins(130). In E. coli, SecD and SecF are polytopicmembrane proteins with a large periplasmicdomain (131). This structural organizationis reminiscent of the Resistance-Nodulation-Cell Division family of membrane proteinsthat includes multidrug-resistance pumps(132). YajC is a membrane protein with asingle TMS and a large cytosolic domain.Although YajC associates with SecDF, it isnot needed for functionality, but cells lack-ing SecD and SecF are cold sensitive, whichprevents growth, and are severely defectivein protein translocation (133). Membranesdepleted of SecDF(yajC) (134) or contain-ing SecDF mutants are severely defectivein in vitro protein translocation (135), butSecDF(yajC) is not needed for translocationper se, as shown by reconstitution studies(3). Possibly SecDF(yajC) functions in a stepdownstream of the primary translocation re-action, after the release of proteins from theSecYEG channel exit site (136), or in theclearance of the pore for processed signal se-quences or phospholipids. SecDF(yajC) havebeen proposed to regulate the catalytic cycle

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of SecA, thereby controlling the movementof the polypeptide chain through the SecYEGchannel (137). However, this effect is presum-ably indirect as Archaea lack a SecA homologbut contain the SecD and SecF proteins thatare needed for protein translocation (86, 138).Another possibility is that SecDF(yajC) is cat-alytically involved in the formation or regu-lation of an oligomeric SecYEG pore com-plex, which would be consistent with theirlower abundance as compared to the num-ber of SecYEG complexes in the cytoplasmicmembrane.

Mechanisms and EnergeticsThe driving force for preprotein transloca-tion is provided by ATP hydrolysis at SecAand by the PMF (139). These energy sourcesare employed at different stages of the pro-cess. ATP is essential for the initiation of thetranslocation and is used throughout the pro-cess. Several of the intermediate steps duringATP-dependent translocation have been re-solved (140, 141). Protein translocation startswith the binding of ATP to SecA, which al-lows the insertion of a hairpin-like loop struc-ture of the signal sequence with the earlymature protein region into the transloca-tion pore. This step can be stimulated bythe PMF, which likely affects the orienta-tion of the signal sequence in the transloca-tion pore (142, 143). ATP hydrolysis resultsin a release of the bound preprotein fromSecA (140), after which SecA can either dis-sociate from SecYEG or rebind to the par-tially translocated preprotein trapped in theSecYEG pore. This rebinding results in anATP-independent translocation of a polypep-tide segment of about 22.5 kDa, andsubsequent ATP binding causes the translo-cation of another 22.5 kDa (140, 141). Mul-tiple rounds of nucleotide binding and hydrol-ysis by SecA drives the stepwise translocationof the preprotein, whereby each turnover ofSecA results in the translocation of 5 kDain two consecutive steps (141). The exact stepsize, however, has not yet been precisely de-

fined, but the time frame for translocation in-creases linearly with the length of the pre-protein as predicted for a stepping mecha-nism (144). Physiochemical properties of thetranslocating polypeptide segment as, for in-stance, the presence of relatively hydrophobicsegments likely influence also step size andtranslocation kinetics (145). Once ATP hy-drolysis has dissociated the preprotein fromSecA, the PMF can drive translocation fur-ther and, in late stages, even complete translo-cation in the absence of ATP (97, 140, 141,146). Although PMF-driven translocation oc-curs independently of SecA and ATP, thesetwo modes of translocation are tightly interre-lated. For instance, the PMF has been shownto stimulate the release of ADP from SecA(129), and it promotes a translocation-relatedconformational change of SecA (147). ThePMF also seems to modulate the opening orformation of the translocation channel (97,127). The intermediate stages of translocationare reversible, and temperature-dependentretrograde movements of the polypeptidechain can occur in the absence of SecA, ATP,and the PMF (140, 146).

Importantly, the Sec translocase functionsin the translocation of unfolded polypep-tides, whereas stably folded structures gener-ally cause a translocation arrest (148). How-ever, the Sec system is capable of unfoldingand translocating the tightly folded humancardiac Ig-like domain I27 when fused to theC terminus of a preprotein (149). Becauseof the requirement for unfolding, this pro-cess is accompanied by an increased utiliza-tion of ATP, suggesting the participation ofSecA in active unfolding, which is similar tothat shown for some cytosolic chaperones. In-deed, a chaperone activity has been suggestedfor SecA; its proposed function is in a qual-ity control mechanism that assists the fold-ing of signal-sequence-less proteins, therebyexcluding them from the secretion process(150).

The exact molecular mechanism bywhich SecA promotes translocation is largelyunresolved. On the basis of protease resistance

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and labeling with chemical reagents addedfrom outside of the cell, it has been pro-posed that SecA inserts, with almost its en-tire mass, deeply into the SecYEG channel(116, 151). Considering the molecular di-mensions of SecA, it seems unlikely that aSecYEG pore can accommodate such a largestructure. Probably, SecA adopts a protease-resistant conformation upon SecYEG, pre-protein, and nucleotide binding (152) and isaccessible from the periplasmic face of themembrane to small labeling reagents that per-meate the SecYEG pore (153). Indeed, thechemical modification sites are spread outover the entire SecA molecule (4, 154).

The structural information on SecA andthe SecYEG complex, either alone or in as-sociation with the ribosome, now providessome clues concerning the possible mode ofaction of SecA. Basically there are two majormechanistic models that describe the role ofSecA as a motor protein, i.e., the power-strokemodel and the Brownian ratchet (155). In thepower-stroke mechanism, ATP binding andhydrolysis induce conformational changes inSecA that are translated into a mechanicalforce, which is imposed on the bound pro-tein substrate. This pushing force then drivesthe movement of the preprotein through theSecYEG channel. With the Brownian ratchet,SecA biases the random Brownian motion ofa translocating unfolded polypeptide chain(156). In this model, spontaneous reversiblemovement (hysteresis) of the polypeptidechain in the SecYEG channel is coupled toenergy-dependent trapping by SecA. Trap-ping prevents retrograde movement, therebyproviding directionality to the translocationprocess. The observed step-wise translocationmechanism linked to nucleotide-dependentconformational changes in SecA suggest apower-stroke mechanism (140, 141, 144). Be-cause of the homology of the NBFs of SecAto the corresponding RecA domains of theDEAD helicase family, it has been postu-lated that the translocation-active SecA ismonomeric (79) and functions according tothe inchworm mechanism proposed for the

helicase PcrA (72, 157). However, DEADhelicases contain two substrate-binding siteswith different affinities, and so far for themonomeric SecA, only one peptide-bindingsite has been detected. For this reason, theSecYEG channel has been implicated in theformation of the second peptide-binding site(81, 108).

Other models implicate the dimer as thetranslocation-active state of SecA (71). Thestructures of the antiparallel SecA dimer showa central opening (4, 61). Another view of apower-stroke mechanism is the piston model,which proposes that the central SecA openingaligns with the SecYEG pore and that it trapsthe preprotein in the initial SecA-bound state.By means of a nucleotide-dependent powerstroke, the SecA-bound preprotein is pushedthrough the pore (61). This model was fur-ther refined in the molecular peristalsis model(106) (Figure 5). According to this model,the dimeric SecA docks onto a (front-to-front)SecYEG dimer creating a large vestibule be-tween the two protein complexes (Figure 5a).An unfolded preprotein gains access to thePPXD regions of the SecA dimer that areexposed to the inner surface of the vestibulevia the central opening. Subsequently, bind-ing of ATP induces a conformational changein SecA, resulting in a different dimer inter-face (Figure 5b). This closes the central open-ing in the SecA dimer, thereby trapping thebound preprotein, with a concomitant reduc-tion in the volume of the vestibule and anopening of the SecYEG channel. The latterwould result from a reduction of the distancebetween the interaction sites of the individualSecA monomers and the cytosolic loops of twoSecY proteins. The strict coupling betweena reduction in cavity volume and openingof the SecYEG channel directs the polypep-tide segment present in the central cavity intothe translocation pore, and backsliding is pre-vented by the closure of the central openingin the SecA dimer. Next, ATP is hydrolyzed(Figure 5c), which results in a reversal ofthe SecA dimer conformational change, andcoupled with the opening and closing of the

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SecYEG1

SecB

SecYEG2

P ADP

SecAdimer

ADPADP ADP ADP ADDPADP ADP

C

C

C

C

Cytoplasm

Periplasm

a b c d

N

Figure 5Model for Sec-mediated protein translocation. The peristalsis model, proposed in Reference 106, isbased on SecA dimer structures from M. tuberculosis (open pore) (61) and B. subtilis (closed pore) (4) andproposes that the nucleotide-dependent changes in the SecA dimer conformation coordinate theSecYEG channel opening and closing. (a) The SecA dimer in an open-pore conformation is bound to thedimer of SecYEG, creating a large central cavity in between SecYEG and SecA. In this state, it acceptsthe preprotein from Sec B. Because of Brownian motion, the polypeptide passes through the centralopening in the SecA dimer into the cavity where the signal sequence of the protein binds to the PPXD ofone of the SecA protomers while the cavity fills up with protein. The PPXD of the second SecAprotomer may also participate in the binding of the protein in the central cavity. (b) Conformationalchanges (arrow) owing to ATP binding (ADP/ATP exchange) to SecA result in the closure of the SecAcentral opening concomitantly with an opening of the SecYEG channel. The conformational change ofthe SecA dimer results in a reduction of the cavity volume and the release of secretory proteins from thePPXD(s) of SecA. The signal sequence and early mature region of the secretory protein are forced tomove into the open SecYEG pore as a looped structure. (c) ATP hydrolysis reverses the SecAconformational change, which results in the reopening of the central SecA channel and closure of theSecYEG pore, allowing a new stretch of secretory protein to enter the cavity. (d ) This cycle ofnucleotide-dependent alternating opening and closing of the central opening in the SecA dimer and thepore in SecYEG is repeated until translocation of the polypeptide is completed. The proton motive force(PMF) may drive the translocation of larger polypeptide segments when SecA has released the secretoryprotein after ATP hydrolysis. How this translocation energy is coupled is unclear, but this activity may beeffected by a PMF-dependent opening or widening of the SecYEG pore. The figure was adapted withpermission from Reference 106.

channels in SecA and SecYEG, respectively.Further diffusion of nontranslocated polypep-tide substrate into the cavity and subsequentbinding of ATP (Figure 5d ) result in thetranslocation of another segment. Alterna-tively, ATP hydrolysis releases SecA from thepolypeptide chain and the SecYEG channelwhereafter SecA is replaced by another SecAprotein or possibly, in the case of a nascentmembrane protein, the (re-)association of the

ribosome with SecYEG. Notably, in the peri-stalsis model, the actual movement of thepolypeptide is driven by Brownian motion,whereas a power stroke is employed to de-crease the cavity size and to open the Sec-YEG channel. In the inchworm and pistonmodels, the step size of translocation dependson the size of the lever arm. To account fora step size of 25 amino acids (140, 141), avery large conformational change is needed

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that reaches a distance of 85 A (one aminoacid is 3.3 A). Interestingly, the large step sizemight be explained by the peristalsis mech-anism, as it can be determined only by thevolume of the vestibule formed between SecAand SecYEG. Because these models are builton static structures of SecA and SecYEG, ex-periments should be designed to provide di-rect experimental evidence for the postulatedmechanistic models.

MEMBRANE PROTEININSERTIONA remarkable feature of the SecYEG complexis that it is capable of two seemingly oppo-site functions, i.e., the translocation of polarpolypeptide segments across the cytoplasmicmembrane and the insertion of hydrophobicTMSs of membrane proteins into the cyto-plasmic membrane. Membrane proteins ex-ist in many different topologies with one ormultiple TMSs. In contrast to the N termi-nus of a signal sequence that always remainsin the cytosol, the first TMS of a nascentmembrane protein will either face the cytosolor will be translocated to the periplasm. Al-though there are some exceptions, the firstTMS of a polytopic membrane protein of-ten determines the orientation of subsequentTMSs, which need to alternate in orientation.The topology of membrane proteins mostlyfollows the positive-inside rule (158). Statis-tical analysis of bacterial membrane proteinsshowed a very strong distribution of posi-tively charged residues in cytoplasmic loopsrather than in periplasmic loops (158). Thishas been attributed to the orientation of thetransmembrane electrical potential (negativein the cytoplasm) (159) and to the presenceof negatively charged phospholipids in cyto-plasmic membrane, which may interact withthe positively charged amino acid residues inthe cytosolic loops (160). The alteration of thecharge distribution in the regions flanking aTMS often results in a reversal of the topol-ogy. However, other features of a TMS such

as its length and mean hydrophobicity are alsoimportant for membrane insertion (161).

Mechanisms and Lateral PoreOpeningThe requirement for specific components ofthe Sec translocase for membrane integrationvaries for different classes of membrane pro-teins. Many membrane proteins integrate intothe membrane independently of SecA (162,163), but SecA is required for the translo-cation of large periplasmic loops (164). Themechanism by which hydrophobic TMSs par-tition from an aqueous interior of the translo-cation channel into the hydrophobic interiorof the phospholipid membrane is largely un-resolved. Hydrophobic regions in preproteinscan serve as stop-transfer sequences, caus-ing a translocation arrest and a lateral re-lease from the translocation channel (161,165, 166). The stop-transfer function of thesesequences correlates to their mean hydropho-bicity and is reinforced by the presence ofpositive charges in the flanking region of thehydrophobic domain (167). In E. coli, thepresence of a synthetic stop-transfer regionin a preprotein induced the release of SecAfrom SecYEG, whereupon the hydrophobicdomain partitioned into the membrane (165,166). Membrane integration is likely a ki-netically controlled phenomenon in whichslow translocation of hydrophobic sequencesis critical for stable integration into the lipidbilayer. Indeed, moderate hydrophobic re-gions can escape membrane insertion as a re-sult of rapid translocation (165).

The structure of the SecYEG channelsuggests that the channel has a lateral gatethrough which TMSs can partition into thelipid bilayer (5). This lateral gate is formedby the relative short helices of TMS2b,-3, -7, and -8, which localize to both SecYhalves (Figure 4). The gate may undergoopening and closing during the translocationof a polypeptide chain (92, 96), and possi-bly even fluctuates thereby temporarily expos-ing translocating polypeptide segments to the

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hydrophobic interior of the lipid bilayer. Thismay enable the equilibration between the twophases. Alternatively, TMSs may already besensed at an early stage by the ribosome (orSecA), which may alter or open the lateralgate to the lipid bilayer in a coordinated man-ner. TMSs likely integrate into the mem-brane by a simple partitioning between theSecYEG pore and the lipid bilayer. Thispassive-partitioning model is supported by theobservation that a hydrophobicity scale, de-rived from peptide interactions with an or-ganic solvent, can be used to predict thetendency for a TMS to integrate into themembrane (168).

The insertion of membrane proteins withmultiple TMSs is probably much more com-plicated because a translocation pore consist-ing of a single SecYEG channel is too smallto store several TMSs. Cross-linking stud-ies suggest that the TMSs of multispanningmembrane proteins leave the channel one ata time or perhaps in pairs (169). After mov-ing through the lateral gate, some TMSs aredirectly exposed to lipids, and others remainassociated with SecYEG (or bind to the YidCproteinsee the YidC section, below.) un-til termination of translation (169). Anotherpossibility is that the TMSs of multispan-ning membrane proteins are assembled by anoligomeric complex of SecYEG and are re-leased into the lipid bilayer after dissociationof the ribosome and the SecYEG oligomer.While the newly synthesized membrane pro-tein inserts into the membrane, it folds intoits final conformation. This not only involvesextensive helix-helix packing but also specificinteractions with phospholipids that are criti-cal for the proper topological folding of mem-brane proteins (169a).

YidCAlthough most membrane proteins insert intothe membrane via the Sec system, in recentyears, YidC has been identified as a novel andessential membrane protein that facilitates theinsertion of a subset of membrane proteins

on its own and in cooperation with the Secsystem (Figure 1, step c) (for reviews seeReferences 170 and 171). YidC is function-ally and structurally homologous to Oxa1 inmitochondria and Alb3 in chloroplasts. Thelatter two proteins function as membrane pro-tein insertases in these organelles and playan important role in the membrane assem-bly of energy-transducing complexes. YidCcan also associate with SecYEG (172) andwith SecDF( yajC) (173), where it contactsthe TMSs of newly synthesized membraneproteins and, in some cases, facilitates in-sertion. In E. coli, YidC is involved in thefunctional assembly of the F1F0 ATPase andcytochrome bo3 quinol oxidase (170). Remark-ably, YidC catalyzes the membrane insertionof small phage coat proteins, such as M13 pro-coat and Pf3, that previously were thoughtto insert spontaneously into the membrane(174, 175). The membrane-insertase functionof YidC was demonstrated by reconstitutionstudies in which purified YidC was shown tocatalyze the membrane insertion of the F0subunit c of the F1F0 ATPase (176) and thePf3 coat (177). Finally, YidC has also beenimplicated in the proper folding of LacY,a lactose/H+ symporter (178). In its Sec-independent activity, YidC seems to functionprimarily in the insertion of small (mono- andbitopic) membrane proteins that need to as-semble into larger membrane protein com-plexes. Possibly, it also actively participatesin the assembly of membrane protein com-plexes as a chaperone. Within the context ofSecYEG, YidC may stabilize moderately hy-drophobic TMSs after they are inserted intothe membrane. This could facilitate their as-sociation with subsequently inserting TMSs,whereafter they can be released as a properlyfolded membrane protein into the lipid bi-layer. The mitochondrial Oxa1 was shown tobind the ribosome via its C-terminal tail (179),a region lacking in bacterial YidC proteins.Some substrates of the YidC only pathway re-quire the SRP for membrane targeting, andone of the remaining questions is how theseproteins are targeted correctly to YidC instead

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of to SecYEG, which by default accepts RNCcomplexes. Other YidC substrates do not de-pend on the SRP for targeting (176, 177) andmust be recognized directly by YidC.

SUBCELLULAR LOCALIZATIONOF THE SEC SYSTEMAsymmetric localization of proteins is essen-tial for many basic biological processes inbacteria, such as motility and chemotaxis.Also, some bacterial infection-related pro-teins seem to locate specifically to one of thecellular poles. This led to the suggestion thatcomponents of the Sec system may be spatiallyorganized in the cytoplasmic membrane. Bymeans of immunofluorescent microscopy andprotein fusions with green fluorescent pro-tein, several of the Sec components were lo-calized into helical arrays in the rod-shaped

bacteria E. coli (180) and B. subtilis (181). ThisSec coil appears distinctly different fromthe MreB coil, an actin-like cytoskeletal pro-tein. However, other studies suggest that theSec system is evenly distributed within thecytoplasmic membrane of E. coli (182). Inthe coccoid Streptococcus pyogenes, the Sec sys-tem localized to a unique single microdomaintermed the ExPortal site close to the divisionseptum (183). Possible explanations for thisphenomenon include Sec system interactionswith murein, with actin cytoskeletons, or withspecific lipid domains. Clustering of the Secsystem in the cytosolic membrane with spe-cific associated factors might allow for the se-lection of particular secretory proteins thatcan be targeted to unique subcellular sites,such as the pole(s) or the mid cell. However,the mechanisms responsible for this spatial lo-calization remain mysterious.

SUMMARY POINTS

1. Bacterial secretory proteins are synthesized in the cytosol with an N-terminal signalsequence and are targeted to the Sec translocase by the molecular chaperone SecB oras RNCs by an SRP.

2. The Sec translocase is embedded in the cytoplasmic membrane and consists of aprotein-conducting channel (SecYEG) and an ATP-dependent motor domain, whichalso functions as a preprotein receptor (SecA).

3. SecB transfers the preprotein to the SecYEG-bound form of SecA and dissociatesfrom the membrane upon the binding of ATP to SecA.

4. Protein translocation is a step-wise process whereby cycles of ATP binding and hy-drolysis by SecA permit the translocation of polypeptide segments with a discretelength.

5. Proteins translocate in an unfolded conformation and pass through the membrane viaan aqueous pore formed by the SecYEG complex.

6. Bacterial cytoplasmic membrane proteins are targeted to the Sec translocase as RNCsby an SRP and cotranslationally integrate into the cytoplasmic membrane via theSecYEG protein-conducting channel.

7. Some cytoplasmic membrane proteins, most notably subunits of the main energy-transducing complexes, integrate into the membrane with the aid of the YidCprotein.

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FUTURE ISSUES

1. How does SecA drive translocation of polypeptide chains through the SecYEG chan-nel? How are the energy sources, ATP and the PMF, employed in this process?

2. How are TMSs of membrane proteins inserted into the cytoplasmic membrane? Howis the lateral pore in SecYEG gated? How are the actions of SecA and the ribosomecoordinated during membrane protein insertion and translocation of extracellularloops?

3. How do SecYEG complexes assemble into functional units? What is the stoichiometryof the active SecA-SecYEG complex? How do the interacting partners SecDF(yajC)and YidC cooperate with the SecYEG pore?

4. How does YidC recognize its substrates, and how does it catalyze membrane proteininsertion?

5. How are certain secretory and membrane proteins targeted to specific subcellularsites?

DISCLOSURE STATEMENTThe authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTSWe apologize to authors whose work we were unable to cite because of space limitations.

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