the secy complex: conducting the orchestra of protein translocation

The SecY complex: conducting theorchestra of protein translocationKush Dalal and Franck Duong

Life Sciences Institute, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3,

Canada

Review

Like the conductor of an orchestra, the Sec proteintranslocation channel is the platform needed to bringtogether the many different players required for theconstitutive and obligatory process of protein transport.This conserved membrane channel, termed SecY in bac-teria and Sec61 in eukaryotes, creates a ubiquitousprotein-conducting pathway by which thousands ofnewly synthesized polypeptides make their way throughthe lipid bilayer. The channel is not a simple passivepore, however; it displays remarkable complexity byinteracting with numerous soluble partners, includingSecA, Syd, FtsY and the ribosome in bacteria. For severaldecades, scientists have been fascinated by the sophis-tication and versatility of this transport channel. In thisreview, we cover the current state of the field includingsome of the newest and most exciting findings on chan-nel structure and mechanism of action.

The Sec channel: a maestro conductorEach minute, hundreds of different polypeptides are tar-geted, transported across, or integrated into the bacterialplasma or the eukaryotic endoplasmic reticulum (ER)membrane with precision accuracy [1]. This accuracy iscritically dependent upon targeting signals such as N-terminal cleavable signal peptides and transmembranesegments (TMS) [2,3]. In fast growing cells such as bacteriaand yeast, translocation can be post-translational, whereasin mammals, transport is mostly co-translational [4]. Re-gardless of the mode of translocation, nascent proteins aretransferred to a membrane complex or channel, termedSecYEG in bacteria, SecYEb in archaea and Sec61abg ineukaryotes. The Sec assembly comprises a central subunit,SecY/Sec61a, made of 10 TMS associated with two smallersubunits: SecG/b, which consists of one or two TMSdepending on the organism, and SecE/Sec61g, which con-tains a single TMS, except in some Gram-negative bacteriawhere SecE possesses two additional N-terminal TMS[5,6]. Together, the three subunits associate into a 1:1:1stoichiometry to form a membrane channel and a bindingplatform for many partners, such as the SecA ATPase andthe ribosome that drives protein transport, or FtsY andSyd, which ensure the efficient delivery of substrates andthe proper assembly of the complex, respectively.

Like many other conserved cellular processes, proteintransport has mostly been analysed in bacteria and yeast,and considerable insight into the working mechanism of

Corresponding author: Duong, F. ([email protected]).

506 0962-8924/$ – see front matter � 2011 Elsevier Ltd. All rights reserv

translocation has been obtained by crystallographic andcryo-electron microscopy (cryo-EM) analysis. Genetic, bio-chemical and computational approaches have then linkedthis structural information to the dynamics of proteintranslocation, making the Sec channel one of the best-understood molecular machines in the cell. This reviewfocuses on the bacterial SecY complex with some of its bestcharacterized partners, and summarizes the major find-ings that have been obtained following the high resolutionstructural analysis of the channel [7].

An elegant composition: structure of the SecY channelThe first high resolution crystal structure of the SecYcomplexwas fromMethanococcus jannaschii and has beenfundamental for understanding the working mechanismof the channel [7]. Three main characteristics containedwithin the SecY subunit were identified (Figure 1): the‘pore ring’, made from six hydrophobic residues at themiddle point of the channel; the ‘plug’ domain, formedby a small a-helix seated on the pore on its extracytosolicside; and the ‘lateral gate’, creating an opening toward thebilayer at the interface between transmembrane seg-ments TMS2 and TMS7. Viewed from the top, the SecYsubunit resembles a clamshell, and when viewed from theside, it could be described as an hourglass topped by a plug(Figure 1). Sequence comparison and additional atomicstructures have confirmed that these structural charac-teristics are strictly conserved across evolution. The longTMSof the SecE/g subunit is also conserved, and embracesthe SecY subunit on the side opposed to the lateral gate,also termed the ‘back’ of the complex. The SecG/b subunit,which is the least conserved element of the channel withrespect to its sequence and possibly function, is located atthe periphery of the channel near SecE/g.

The discovery of the structural features of the SecYchannel made it possible to formulate specific hypothesesregarding the mechanism of channel gating. The firstprediction was that the insertion of a signal sequence ora transmembrane segment would occur at the lateral gate.This notion was supported by an earlier photocrosslinkinganalysis showing that a signal sequence can simultaneous-ly contact TMS2 and TMS7 during transport [8]. Site-directed cysteine crosslinking analyses have now con-firmed this prediction [9], and molecular dynamics (MD)simulations, which allow modeling of the subtle conforma-tional changes that might occur during transport, indicatethat the opening of the lateral gate is possible because of a

ed. doi:10.1016/j.tcb.2011.04.005 Trends in Cell Biology, September 2011, Vol. 21, No. 9

mailto:[email protected]

http://dx.doi.org/10.1016/j.tcb.2011.04.005

[(Figure_1)TD$FIG]

Pore ring

SecY

(a) (b) (c)

∗

∗∗

Cytoplasm

Periplasm

SecG

Plug

SecE

TM7

TM2

SecYSecβ

SecE

SecA

Finger

TRENDS in Cell Biology

Figure 1. Crystal structures of the SecY complex. The SecYEb complex from Methanococcus jannaschii (PDB: 1RHZ) viewed (a) from the cytosol or (b) from the plane of the

membrane. The complex is colored as follows: SecY TMS 1–5 (grey); SecY TMS6–10 (cyan); SecE (yellow); Secb (orange); SecY pore ring (red); SecY plug domain (blue). Asterisks

(* and **) denote the locations of the hinge region and cytosolic loops of SecY, respectively. The position of the membrane surface is represented by the dark grey lines. (c) The

co-crystal structure of the SecYEG–SecA complex from Thermotoga maritima (PDB number: 3DIN) viewed from the plane of the membrane. A single SecYEG complex (grey,

yellow, and orange) is bound to a single SecA molecule (blue). Note that the SecA two-helix finger (red) is inserted into the cytoplasmic funnel made by the SecY subunit.

Review Trends in Cell Biology September 2011, Vol. 21, No. 9

flexible hinge sequence located between TMS5 and TMS6at the back of the complex [10].

The structural information also led to the predictionthat the pore ring, which is only 5 to 8 A in diameter in theresting state, would need to expand in diameter to allowinsertion of the polypeptide chain. This widening wouldfollow the opening of the lateral gate, because TMS2 andTMS7 contain three of the six amino acids that create thepore structure (Figure 1). Although not yet experimentallytested, the interior of the channel during polypeptidetransport would be expected to approach an inner diameterof 20 A � 15 A following amotion of 15 degrees at the hinge[7]. This seems to be the case at least in silico, because MDsimulations show that the pore ring can expand to allowthe passage of beads up to 16 A in diameter [11].

The plug domain, which is linked to the channel withunstructured loops, was another element predicted to beinvolved in the gating of the channel. During transport,this domain (TMS2a) was found located near the carboxylterminus of the SecE subunit, more than 20 A away fromits normal position [12,13]. The use of crosslinking agentsof different lengths has now confirmed that a plug displace-ment of at least 13 A is necessary to allow unrestrictedpolypeptide transport [14] and, as expected, a plug domainlocked in its resting position adjacent to the pore ringinactivates the channel [14,15]. The opening of the channelis largely influenced by the association between the plugand the pore, because some mutations in these structurescan destabilize the closed state of the channel. MD simula-tions indicate that perturbations in the hydrogen bondingnetwork between the SecY TMS can eventually be trans-mitted to the pore and plug to destabilize the closed state ofthe channel [16]. This destabilization might in turn allowexport of substrates with defective signal sequences (prlmutations) [17]. Under normal conditions, disruption ofthe hydrogen bonding network might be facilitated by thedocking of SecA or the ribosome onto the channel.

The SecY SecA duetThe cytosolic SecA ATPase is essential for recognising,targeting and pushing polypeptide substrates across the

SecY channel. These polypeptide substrates generally car-ry a signal sequence that is moderately hydrophobic [18].The recognition by SecA might begin as early as the pointwhen the polypeptide emerges from the ribosome, becauseco-sedimentation experiments show that SecA has affinityfor the protein L23 at the ribosome exit tunnel [19]. Certainsubstrates are also transferred to SecA via the exportchaperone SecB, which prevents polypeptides from acquir-ing their tertiary structure, and keeps them in a translo-cation-competent and unfolded state [20,21]. SecA wouldthen accept the unfolded substrate-SecB complex by recog-nising the signal sequence. NMR and Forster resonanceenergy transfer (FRET) analysis confirm the existence of asignal sequence binding site at the surface of SecA [22,23].A recent crystal structure of SecA further shows that ahydrophobic clamp, formed by the adjacent pre-proteinbinding domain (PPXD) and a-helical scaffold domain(HSD), is capable of interactions with the mature segmentof the substrate [24,25].

A recentmilestonewas reachedwith the co-crystal struc-tureofSecAbound toSecYEG,providingnewcluesas tohowthese two proteins interact together to catalyze transport(Figure 1c) [26]. In the structure, a two-helix hairpin or‘finger’, contained within the HSD domain of SecA, wasfound inserted at the entrance of the SecY cytoplasmicfunnel. The insertion of the finger allowed partial openingof the lateral-gate interface and a slight shift of the plugdomain toward the periplasmic side of the channel. It isreasonable to assume that the conformational change pro-voked by the finger domain is crucial for the gating of SecYand therefore, that the finger might act as an allostericactivator capable of ‘priming’ the channel during the initialsteps of translocation [27]. This priming step might berelated to the SecY-dependent SecA ATPase activity thatis observed in the absence of protein substrate, both indetergent solution and Nanodisc particles (soluble nano-scale lipid bilayers) [28–30]. During transport, the polypep-tide chain captured inside the SecA clamp would betransferred to the two-helix finger, then to the center ofthe channel. An elegant and systematic cysteine crosslink-ing analysis designed tomap the protein translocation path

507


revealed that the polypeptide chain is most probablythreaded in an extended conformation between the SecAclamp and the pore ring [31]. A similar crosslink strategywas used to show that the SecA finger moves toward theSecY pore upon binding of ATP, and probably drags thepolypeptide chain with it [32]. Analysis of the thermody-namic energy released by SecA upon docking of the poly-peptide onto SecYEG indicates that the signal sequencecontributes to lowering of the activation energy barrierfor translocation initiation [3]. With the energy barrierovercome, SecA would then be able to initiate cycles ofATP hydrolysis to push consecutive segments of the proteinsubstrate across the membrane (Figure 2a).

Acidic lipids might also contribute to reducing the acti-vation energy barrier of translocation [33]. These lipids,whose deficiency severely impairs protein transport both in[(Figure_2)TD$FIG]

(b)

(a)

5' 5'

3'

SecY

SecA

FtsY

SRP

Ribosome

Figure 2. Post-translational and co-translational modes of translocation in bacteria. (a) Sec

two-helix finger is not shown in this drawing. Left: the SecA nucleotide binding domain

channel (blue), as described in ‘The SecY channel in stereo’ section. Middle: upon bindin

active copy of the SecY channel. Insertion of the signal sequence might induce the movem

ATP, with each cycle pushing a segment of polypeptide chain across the SecY channel.(b) R

emerges from the ribosome (green) is recognized by SRP (orange) and targeted to the mem

mRNA with 50 and 30 ends is shown bound to the small 40S ribosomal subunit. Middle: t

followed by the insertion of the signal sequence. Right: elongation of the polypeptide ch

508

vivo and in vitro, cause dissociation of the SecA dimer intomonomers [28,34] and stimulate the ATPase activity ofSecA when bound to the channel [35,36]. Recently, it wasshown that the binding of SecA to the SecYEG complex isenhanced by cardiolipin (CL) in detergent solution [35].This acidic lipid is tightly bound to the channel, as itremains associated even during purification of the Seccomplex in detergent solution. Additional work is neces-sary to better understand the contribution of this particu-lar lipid to the quaternary structure and activity of theSecYEG–SecA complex.

The quartet: SecY–ribosome–nascent chain and signal-recognition particleSecA is only found in bacteria but the signal-recognitionparticle (SRP) is found inall organisms.TheSRP-dependent

5'

3'

3'

ATPADP


A-driven post-translational translocation through the dimeric SecY channel. The SecA

(dark green) docks onto the cytosolic loops of a non-translocating copy of the SecY

g of ATP, the SecA clamp (light green) transfers the signal sequence (yellow) to the

ent of the plug (red) away from its central position. Right: SecA binds and hydrolyses

ibosome driven co-translational translocation. Left: the signal sequence (yellow) that

brane-associated FtsY (pink). The ribosome exit tunnel is shown as a dashed line. The

he ribosome nascent chain (RNC) is transferred to the SecY channel (blue), which is

ain resumes after the SRP dissociates from the signal sequence.

Box 1. The pathway for membrane protein integration

During membrane protein synthesis, hydrophobic TMS are inserted

into the channel and moved through the lateral gate into the lipid

bilayer. For this to occur, the lateral gate must be partly or

permanently open to expose the TMS to the hydrophobic environ-

ment of the membrane. Photocrosslinking experiments show that

hydrophobic amino acid sequences that occupy the channel simulta-

neously contact TMS2, TMS7 and phospholipids [86,87]. The primary

factor for membrane integration is the hydrophobicity of the TMS

sequence and its capacity to dissolve into the lipid phase [88]. During

transport, factors other than TMS hydrophobicity influence mem-

brane protein integration: the pore ring residues, which were shown

by mutagenesis to influence the hydrophobic threshold for integra-

tion [89]; the length of the TMS [90] and its flanking sequences [91];

and the kinetics of transport itself [92]. MD simulations confirm that a

TMS in transit through the channel can stabilize the open conforma-

tion of the lateral gate, whereas hydrophilic peptides preferentially

stabilize the closed state [93]. Interestingly, the position of the plug

domain within the channel depends on whether the transiting

sequence is hydrophobic or hydrophilic, suggesting that the plug

domain contributes to the partitioning of the polypeptide segment

across or into the membrane.

Most membrane proteins have positively charged residues located

in their cytosolic loops [94]. This ‘positive inside’ rule is the primary

factor that determines membrane protein topology. For multispan-

ning proteins, it has been shown that Nlum–Ccyt helices do not

completely exit the lateral gate until the following Ncyt–Clum segment

is synthesized [95]. This has suggested that multiple helices might

simultaneously occupy the translocation channel. Using arrested

membrane integration intermediates, it was shown that the Sec61

channel can accommodate one hydrophilic sequence and two

hydrophobic segments at the same time [96]. This result highlights

the astonishing flexibility of the Sec channel and perhaps, the

possibility that multiple copies of Sec61 are necessary for the

biogenesis of multispanning membrane proteins.


pathway delivers protein substrates in a co-translationalmanner to the membrane (Figure 2b) [37–39], and in bacte-ria, this mechanism serves for the integration of mostmembrane proteins (Box 1). Targeting begins when SRP(also termed Ffh) binds and arrests the translation of thefirst TMS that emerges from the ribosome. The ribosome–

nascent chain (RNC) complex, when boundwith the SRP, isthen targeted to the SRP receptor at themembrane (termedFtsY in bacteria). Following GTP hydrolysis, the SRP dis-sociates and the RNC is transferred to the SecY channel,where translation of the polypeptide chain resumes [40].FtsY has a crucial role in this sequence of events, becausethis receptor provides the bridge between the soluble SRP–

RNC complex, the membrane and the Sec channel. FtsYinteractswith the phospholipid bilayer via two lipid-bindinghelices locatedat theNandAdomains of theprotein [41,42],and FtsY displays low but significant affinity for the cyto-solic loops of the channel [43]. It is noteworthy that the[(Figure_3)TD$FIG]

H

(a) (b)

tRNA

40S

Sec61

60S

Figure 3. Cryo-EM structure of a translating ribosome bound to the Sec61 channel. (a) Th

the 60S and 40S ribosomal subunits and the tRNA are shown. The model was taken from

ribosome junction (PDB: 2WWB). The Sec61 cytosolic loops C4 and C5 (green) are show

rRNA helices H50 and H7 (blue). The arrow shows the location of the ribosome exit tu

association of the lipid-binding helices with liposomes isstrongly enhanced by phosphatidyl-glycerol (PG), as shownby photocrosslinking and surface plasmon resonance (SPR)experiments [42,44]. The presence of acidic lipids in thevicinity of SecYmight be functionally important becausePGlipids were found to increase the GTPase activity of theSRP–FtsY complex [44], and therefore might contribute tothe unloading of the nascent chain to the channel.

The understanding of themechanism of co-translationaltranslocation has been greatly aided by progress in thefield of cryo-EM. The first snapshots of the RNC bound tothe yeast Sec61 channel were obtained at low resolution[45–47], leading to the conclusion that 2 to 4 Sec complexeswere attached beneath the ribosome. With better instru-mentation and resolving power, cryo-EM analysis nowshows that the RNC–Sec61 complex contains of only oneSecY/Sec61 heterotrimer [48], in agreement with the X-raystructure, which indicates that a single complex is suffi-

C5 C4

L23

59

Sec61

H7

H50

L19

L39

L35

Exit tunnel


e RNC complex is shown bound to the mammalian Sec61 channel. The positions of

[38] and modified to fit the purpose of this review. (b) The structure of the channel–

n to be in proximity to the ribosomal proteins L23 (red) and L35 (cyan), and to the

nnel. The color code used for the Sec channel is the same as that in Figure 1.

509

[(Figure_4)TD$FIG]

K348 R255R255

Y299S300

S350D351

E55

A163

K202

C5

C5

C4C4

A48

SecYEG SecYEG

SecA

NBD1

PPXD

Active PassiveTRENDS in Cell Biology

Figure 4. Representation of contact points between SecA and the back-to-back

SecY dimer. The structures of SecY and SecA are derived from the SecYEG–SecA

crystal structure (PDB:3DIN). Proteins and domains are colored as follows: SecY

(grey); SecE (yellow); SecG (orange); SecA (blue); SecA PPXD domain (purple);

SecA NBD1 domain (brown); space-filling representation of amino acids (green).

All residues are numbered according to E. coli proteins. Active SecY copy: the

cytosolic loops C4 and C5 of the active SecY copy interact with the SecA PPXD

domain [26], but for simplicity, only a few interactions are represented here.

Residues in the PPXD domain of SecA that are in proximity to the SecY cytosolic

loops were selected if they are within a distance of 4 A from the indicated SecY

residue. For example, residue R255 on the C4 loop of SecY is close to residues

S350–D351 of SecA, whereas residue K348 on the C5 loop is close to the SecA

residues Y299–S300. The position R357 is not shown because this residue does not

interact with SecA in the SecYEG–SecA crystal. Passive SecY copy: the location of

the cysteine crosslinks between the passive SecY copy and SecA [9] are shown.

The residue R255 in the C4 loop of the passive SecY copy establishes crosslinks

with the positions A48, E55, K163 and G202 in the nucleotide binding domain 1

(NBD1) of SecA.


cient to create a protein-conducting channel. In these highresolution pictures, the Sec61 loops were found in proximi-ty to the ribosomal proteins L23 and L35, and to specificrRNA helices (Figure 3). L23 is now considered a generaldocking platform for factors that act on nascent chains,such as SecY/Sec61, SRP and SecA [19,49]. Image recon-structions also show that the channel pore is aligneddirectly below the ribosome exit tunnel, thus creating acontinuous pathway for the transfer of the polypeptidesubstrate [48,50]. In the latest cryo-EM analysis, the chan-nel was found bound to a ribosome with the nascent chaininserted into the translocation pore, causing the middlepart of the lateral gate to open toward the lipid bilayer(�5 A) [48]. In a crystal structure of SecYEb from P.furiosus, the lateral gate was also found to be open, butthroughout its entire length [51]. In this latter case, acrystal packing artefact, in which the C-terminal tail ofone SecY is inserted into the lateral gate of a secondjuxtaposed SecY, might havemimicked the conformationalchange that normally occurs when a nascent chain transitsthrough the channel. It is noteworthy that these channels,seemingly trapped in their active state, were still cappedwith their plug domain in closed position. This last obser-vation has suggested that the lateral gate and the plugdomain might move independently from each other at theearly stage of membrane protein insertion.

The SecY channel in stereoThe Sec complex can form various oligomers in the mem-brane or in detergent solution, making the analysis of itsfunctional quaternary structure difficult. Dimers andhigher-order SecY oligomers have been detected by nativePAGE, analytical ultracentrifugation [52,53] and EM [54–

56]. In two-dimensional crystalline membranes, SecY com-plexes were related to each other by a two-fold symmetry,with their interface formed by the long TMS of SecE [57].This dimeric arrangement was termed ‘back-to-back’ ori-entation. Several studies have since focused on under-standing the role of SecY oligomerization for proteintransport. Earlier EM analysis concluded that SecA andprotein substrates play an important role in modulatingthe oligomeric status of the complex in the membrane [54–

56]. Later, it was shown using a genetically fused SecYEGdimer that a cysteine crosslink between a polypeptidesubstrate and the pore of a mutant channel, otherwisedefective for binding of SecA, can occur only if a wild-typeSecY copy is present in trans [9]. This structural comple-mentation is explained if SecA binds asymmetrically to theSecY dimer, with one copy actively engaged with the two-helix finger and the other (the passive copy) simply servingas a docking site for the nucleotide binding domains ofSecA [9]. The second and passive SecY copy could thereforeact as an anchor to prevent the complete dissociation ofSecA when the two-helix finger disengages from the activecopy upon ATP hydrolysis. Additional experimental sup-port is needed, but the model is consistent with theSecYEG–SecA atomic-resolution structure showing thatthe two-helix finger is inserted into the translocation pore[26], while the remaining unbound portion of SecA, includ-ing the first nucleotide binding domain, is available to bindanother SecY copy. This other copymight not solely act as a

510

docking site for SecA, however, because residue R357,located on the SecY cytosolic loop C5, seems to be impor-tant for triggering a conformational change that leads tothe activation of SecA [58].

The question regarding the oligomeric state of SecYremains unsettled and the orientation of SecY protomerswithin the oligomers further complicates the problem. Aback-to-back dimer can spontaneously form in the mem-brane, as judged by the high efficiency of cysteine cross-linking between two SecE subunits when the complex isoverproduced [59,60]. Recent single-molecule analysis inreconstituted liposomes showed that a crosslinked back-to-back dimer is functional, whereas its monomeric counter-part displays only 40% of the activity of the dimer [60].Recent in silico models of the back-to-back mode of associ-ation have been constructed, based on the atomic-resolu-tion structure of the SecYEG–SecA complex [35,60]. These


models are consistent with various cysteine crosslinkinganalyses that have identified the contact points betweenSecY and SecA in the translocation active complex[9,58,61]. In the back-to-back model (Figure 4), the passiveSecY unit would provide an additional surface for thebinding of SecA [26]. It is interesting that the acidicphospholipid cardiolipin facilitates the dimerization ofSecYEG in detergent solution, but also strengthens thebinding of SecA to the channel and enhances SecA ATPaseactivity in the membrane [35]. It is possible that the SecYdimer bound to this particular lipid creates the optimalsurface for the binding and activation of SecA [35]. Theback-to-back orientation might not be an exclusive mode ofinteraction, and other conformations might exist, or be-come stabilized, upon binding of the ribosome and poly-peptide substrates. For example, a ‘front-to-front’orientation can be fitted into the low-resolution cryo-EMstructure of the SecY–ribosome complex [62], and thismode of association seems to be supported by recent invivo crosslinking analysis, which used a photoreactiveprobe incorporated into SecA [63].

Variations on a theme: the ion gate-keeping activity ofSec channelsRecent studies have addressed the problem of membranepermeability to small molecules during protein transport,a notion particularly important in bacteria because themembrane needs to maintain a stable electrochemicalgradient. Pioneering studies have shown that syntheticsignal sequence peptides can open large ionic conductingchannels, whereas channels jammed with a translocationintermediate were leaky for chloride ions [64,65]. Thelatter observation is still puzzling, because the SecY atomicstructure would rather suggest that the pore ring, made ofsix hydrophobic side-chains that project toward the chan-nel interior, creates a tight seal around the polypeptidechain in transit, which would prevent permeation of smallmolecules. These lateral side-chains are indeed within thedistance of a disulphide bridge to the polypeptide chainduring translocation [66].

The role of the pore-plug assembly in the gating of theresting channel has been better characterized. Electro-physiology experiments in planar lipid bilayers haveshown that a partial deletion of the plug domain, or lockingthe plug in its open conformation, is sufficient to render thechannel conductive to ions [67]. In vivo, the stabilization ofthe plug domain in its open state is lethal, suggesting thatthe pore ring on its own is insufficient to seal the channel[12,15]. Certain amino acid substitutions in the pore ringproduce ion-conductive channels, probably because thesemutations destabilize the interactions that keep the plugin its closed position [67,68]. These leaky channels arespecific for chloride ions only [68,69], which would explainwhy they can be produced in the membrane [70]; althoughchloride ions are neither necessary nor stimulatory forprotein translocation, the mutations that increase chlorideconductance decrease the activation energy required toopen the channel.

Unlike bacteria, yeast can tolerate Sec61 channels withdefective and even missing plug domains [71,72]. Thepresence of permanently open channels might be less

crucial for yeast growth because the solute compositionacross the ER membrane might be similar. In fact, electro-physiology experiments indicate that the resting Sec61complex conducts a diversity of small molecules, includingCa2+ [73–76]. A recent study has identified calmodulin as abinding partner of Sec61a [77]. Molecular modeling sug-gests that calmodulin might fit into an open cavity thatnormally exists at the junction between the ribosome andthe Sec61 channel. Binding of calmodulin at this interfacewould regulate Ca2+ conductance across the channel. Onthe opposite side of the membrane, the ER chaperone,binding immunoglobulin protein (BiP), might serve to sealthe luminal face of the Sec61 channel both at rest andduring the early stages of protein translocation [78].

Quality control of SecY, with Syd in minor keyChannels that are constitutively open or jammed with apolypeptide substrate are lethal in Escherichia coli. TheFtsH protease degrades the SecY channels that are non-assembled with SecE [79], or blocked with a translocationintermediate [80]. In addition to this proteolytic mecha-nism, which eliminates defective and potentially toxicchannels, some Gram-negative bacteria contain a smallprotein termed Syd, which verifies the proper assembly ofthe SecY complex in the membrane. Syd was originallyisolated as a suppressor of a dominant-negative mutationinto SecY, capable of sequestering SecE into an inactivecomplex (hence the name suppressor of SecY dominance)[81]. Accordingly, Syd interferes with protein translocationonly when the channel displays abnormal SecY–SecE asso-ciations [82]. In detergent micelles, Syd was found toinduce the dissociation of the SecYEG heterotrimer intosingle subunits [83]. These observations have suggestedthat Syd belongs to a quality-control system that proof-reads the SecY complex, leading to its disassembly andsubsequent degradation by the FtsH protease when thecomplex is compromized by abnormal heterotrimeric asso-ciations. The crystal structure of Syd reveals a globularprotein with an electronegative concave surface that formscontacts with the electropositive loops of SecY [83]. TheSecY electropositive loops thus seem to be a general dock-ing platform for all cytosolic binding partners of the chan-nel, including SecA and the ribosome (Box 2).

Allegro: the increasing tempo of translocation researchFrom the ability to decipher structures of the channel athigh resolution to the finest cysteine crosslinking maps,the roadway to understanding translocation has been sol-idly paved. There are now several outstanding questionsthat remain regarding the channel’s interactome, the dy-namics of its assembly and the mechanism of gating. Inbacteria, the SecY membrane partners SecDFyajC andYidC (which are involved in the stimulation of proteintranslocation and integration of certain membrane pro-teins, respectively) await further biochemical and struc-tural characterization. Clarifying the role of the SecG/bsubunit, which is essential in stimulating protein translo-cation, at least in vitro, is also necessary for a completepicture of SecY channel activity. In eukaryotes, moredetails are needed on how the Sec61 complex interactswith the ER luminal ATPase BiP to ‘pull’ the substrate

511

Box 2. The general SecY docking platform

The structures of Syd and the SecYEG–SecA complex serve to

highlight the electrostatic interactions between the channel and its

binding partners. In particular, the C4 and C5 cytosolic loops on SecY

and Sec61a contain several conserved arginine and lysine residues

(position K250, K268, R340, K347, K348 and R357; Figure I). The SecY

mutation R357E, which introduces an acidic residue into the C5

electropositive loop, disrupts the interactions with the negatively

charged ribosomal RNA [50], and causes defects in the activation of

SecA by SecY [97]. Furthermore, deletion of R357 reduces the binding

affinity of Syd for the channel [83].

The SecYEG–SecA crystal structure from Thermotoga maritima

reveals how the cytosolic loops of SecY establish contacts with the

PPXD domain of SecA [26]. Analysis of the electrostatic potential on

the surface of SecA reveals discrete areas of negative charge density

in the PPXD domain (Figure I, middle). In the SecYEG–SecA crystal

structure, the positively charged residues on the C4 and C5 loops of

SecY are within salt bridge distance (�4 A) to the negatively charged

regions of SecA. For example, the conserved basic residues at

positions 250 and 255 in the C4 loop of SecY interact with SecA near

the conserved serine and aspartate residues at positions 350–351. The

conserved arginine at position 357 of SecY does not bind directly to

SecA; however other basic residues exist in the C5 loop, such as R348,

which are located near the negatively charged region of the PPXD

domain containing a tyrosine and serine at positions 299–300. These

observations suggest that electrostatic interactions play an important

role during the binding of SecA to SecYEG.

Syd possesses a concave electronegative groove that could form

electrostatic interactions with the cytosolic loops of SecY (Figure I,

right). Binding analysis shows that the interaction between Syd and

the SecYEG complex is diminished in the presence of high salt

concentrations [83]. The interaction between the SecY C4 loop and the

concave surface of Syd was shown by disulphide crosslinking

analysis to occur between position 255 of SecY and position 115 of

Syd [83].[(Box_2)TD$FIG]

N115

SecY SecA Syd

Finger

90° 90°45°

Finger

S350 &D351

Y299 &S300

Periplasm

Cytosol

C4

C4

C5

C5

R357

R357

R255

R2t55

K250

K250

K348

K348

PPXD

PPXD

Concave groove


Figure I. Electrostatic interactions between SecY and SecA, and SecY and Syd. The SecY complex, SecA and Syd were analysed using the Pymol software (version 0.99) and

the electrostatic representation was generated with the APBS plug-in. Blue and red represent electropositive and electronegative potential, respectively. The surface

potential was set between�4.0 and +4.0 kT/e for the SecYEG complex, and between�2.5 and +2.5 kT/e for SecA and Syd. The solvent-accessible area option of the software

was used in the calculation. Surface representations were constructed from the Thermotoga maritima SecYEG–SecA crystal (PDB: 3DIN) and E. coli Syd (PDB: 3FFV). All

residues are numbered according to E. coli proteins. The residues colored in yellow where chosen to highlight the possible electrostatic interactions taking place between

SecY and SecA, or SecY and Syd (see text). Left: on the SecY protein, conserved basic residues were highlighted to show the positive charge density on the C4 and C5

cytosolic loops. Middle: the indicated residues in the SecA PPXD domain lie in regions of negative charge density that are in proximity to the SecY cytosolic loops. Right: the

amino acid at the position 115 on the electronegative and concave surface of Syd interacts with residues located in the cytosolic loop C4 of SecY.


across the channel, and on how the membrane-boundSec62p complex regulates BiP function [1]. Given theinherent difficulty of analysing protein interactions inthe lipidic environment, questions such as the oligomericstate of the channel are likely to remain an area of impor-tant investigation, largely depending on parallel technicaladvances in membrane biology. Now that almost all theatomic structures of the cytosolic participants are avail-able, drug discovery should represent another fruitfulavenue of research, while providing additional tools to

512

dissect the translocation mechanism. For example, theSecA ATPase is an attractive target for discovery of smallmolecules with potential antibacterial activity [84]. TheSec62p complex represents another drug target because itsactivity is linked to cancers and neurodegenerative dis-orders [85]. With improved membrane-related methodsand the possibility of high-throughput screening, the paceof research down the roadway of protein transport is likelyto quicken, with additional breakthroughs expected incoming years.


AcknowledgmentsWe thank Ms Kailun Jiang for art work. K.D. was supported by theAlexander Bell Canada graduate scholarship. F.D. is a Canada ResearchChair Tiers II. This work was supported by the Canadian Institutes ofHealth Research.

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the secy complex: conducting the orchestra of protein translocation

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