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Papanikou, E., Karamanou, S. & Economou, A. Bacterial protein secretion through the translocase nanomachine. Nature Rev. Microbiol. 5, 839-851

December 2007
Nature Reviews Microbiology 5(11):839-51

December 2007
5(11):839-51

DOI:10.1038/nrmicro1771

Source
PubMed

Authors:

Effrosyni Papanikou

University of Chicago

All cells must traffic proteins across their membranes. This essential process is responsible for the biogenesis of membranes and cell walls, motility and nutrient scavenging and uptake, and is also involved in pathogenesis and symbiosis. The translocase is an impressively dynamic nanomachine that is the central component which catalyses transmembrane crossing. This complex, multi-stage reaction involves a cascade of inter- and intramolecular interactions that select, sort and target polypeptides to the membrane, and use energy to promote the movement of these polypeptides across--or their lateral escape and integration into--the phospholipid bilayer, with high fidelity and efficiency. Here, we review the most recent data on the structure and function of the translocase nanomachine.

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In cells from all three domains of life, more than one-

third of the proteome is secreted across, or inserted

into, biological membranes. Secretory proteins include

hydrolytic enzymes, periplasmic lipoproteins, toxins

and surface appendages such as pilli and flagella.

Integral membrane proteins mediate selective trans-

port, energy conversion, cell division, extracellular sig-

nal sensing, and membrane and cell-wall biogenesis.

In many cases, the plasma membrane is the main

— and in Gram-positive bacteria and archaea the

only — membrane that is involved. However, proteins

are also targeted to several other membranes, such as

those of eukaryotic sub-cellular organelles, the outer

membrane of Gram-negative bacteria and the inter-

nal-membrane enclaves of photosynthetic bacteria.

Moreover, in the case of bacterial pathogens that infect

eukaryotic hosts, bacterial exotoxins must negotiate

multiple self and host membranes before they reach

their target locations.

To successfully localize polypeptides extracytoplas-

mically, the cell must tackle five daunting tasks: discrimi-

nate the cytoplasmic-resident proteins from those that

are destined for export; deal with the inherent tendency

of polypeptides to fold rapidly; target exported proteins

to the membrane with specificity and fidelity; achieve

transmembrane crossing of these elongated, heteropoly-

meric substrates, which are several times as long as the

membrane is wide; and finally, manage a second sorting

event that releases membrane proteins into the lipid

bilayer and secretory proteins to the trans side of the

membrane.

Evolution has produced a remarkable array of

mechanisms to export proteins. Sixteen such systems,

which handle protein secretion, sorting and membrane

integration, are present in the Bacteria alone

1,2

(BOX 1).

Of these, only the Sec pathway is ubiquitous and essen-

tial for viability in all three domains of life. In addition,

the Sec pathway acts as the entry point for many of the

other protein export and sorting pathways (for Bacteria

see BOX 1). Over the past 30 years, all the genes that are

involved in the Sec pathway have been identified using

genetic and biochemical approaches. Export-specific

chaperones, pilot-like factors and signals in exported

proteins overcome the sorting and folding problem

and facilitate membrane targeting

3,4

. A dynamic mem-

brane-embedded nanomachine called the pre-protein

translocase or translocon (FIG. 1a) recognizes exported

proteins at the membrane and catalyses their export.

High-

5,6

and medium to low-

7–13

resolution structures

of the protein-conducting channel SecYEG, of both

eukaryotic and prokaryotic origin, and high-resolution

structures of the bacterial SecA motor

14–19

, are now avail-

able. Sequential recognition events and the expenditure

of energy allow the polymeric polypeptide substrate to

move through the translocase, effectively being threaded

through the membrane, and allow hydrophobic regions

to escape laterally into the lipid bilayer.

Here, we will review the latest advances in bacterial

protein secretion through the translocase, focusing

on the journey that secretory proteins take across the

bacterial plasma membrane. Membrane-protein inte-

gration through the translocase will not be discussed in

*Institute of Molecular

Biology and Biotechnology,

Foundation of Research

and Technology-Hellas,

PO Box 1385, Heraklion

GR-71110, Crete, Greece.

‡

Department of Biology,

University of Crete, Iraklio

GR-71110, Crete, Greece.

e-mail:

aeconomo@imbb.

forth.gr

doi:10.1038/nrmicro1771

Bacterial protein secretion through

the translocase nanomachine

Effrosyni Papanikou*, Spyridoula Karamanou* and Anastassios Economou*

‡

Abstract | All cells must traffic proteins across their membranes. This essential process is

responsible for the biogenesis of membranes and cell walls, motility and nutrient

scavenging and uptake, and is also involved in pathogenesis and symbiosis. The translocase

is an impressively dynamic nanomachine that is the central component which catalyses

transmembrane crossing. This complex, multi-stage reaction involves a cascade of

inter- and intramolecular interactions that select, sort and target polypeptides to the

membrane, and use energy to promote the movement of these polypeptides across — or

their lateral escape and integration into — the phospholipid bilayer, with high fidelity and

efficiency. Here, we review the most recent data on the structure and function of the

translocase nanomachine.

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REVIEWS

Box 1 | The bacterial protein-export and secretion-systems zoo

The figure shows a simplified

schematic summary of the

major protein-export, protein-

secretion and protein-

membrane-integration

systems in Bacteria. Peptide-

processing peptidases,

periplasmic chaperones and

folding factors are not shown.

For a comprehensive

presentation, the reader is

directed to REFS 1, 125. The

nomenclature of many

systems follows the type-‘n’-

secretion or type-‘n’-pilus

convention. The type IV

secretion system (T4SS) shares

ancestry with conjugation

systems and can also transport

DNA. The type VI secretion

system (T6SS) has only recently been identified and how exported proteins cross the cell envelope in this system is not

yet clear. The membrane protein YidC has homologues (known as Alb3 or Oxa1) that function in mitochondria and

chloroplasts but not in the archaeal or endoplasmic reticulum membrane

. The arrows indicate the path that is taken

by the exported protein. Arrows that initiate in the periplasm indicate that Sec (or rarely TAT)-dependent

translocation across the plasma membrane is a necessary first step for these systems.

Bam, beta-barrel assembly machine; CU, chaperone–usher pathway; Esx, specialized secretion system that is found

in Gram-positive bacteria (for example, mycobacteria); Fla, flagellum; HM, host cell membrane; LOL, lipoprotein

outer-membrane localization; OM, outer membrane; Omp85, also known as YaeT; Per, periplasm; PM, plasma

membrane; Sort, sortase; TPS, two-partner secretion; T2S, type II secretion; T3S, type III secretion; T4P, type IV pili;

T4S, type IV secretion; T5S, type V secretion (autotransportation), T6S, type VI secretion.

detail (BOX 2). These findings are mainly the result of the

focused dissection of the secretion pathway in a single

model bacterium, Escherichia coli. Nevertheless, genome

sequence data and some biochemical data suggest that

most of these Sec-pathway features are universal in the

Bacteria.

Overview of the Sec pathway

Secretory-protein export through the Sec pathway

is a multi-stage reaction that occurs mainly post-

translationally. This continuous process can be con-

ceptually and biochemically divided into three distinct

stages (FIG. 1b).

Stage one: protein sorting and targeting. Secretory proteins

are usually referred to as pre-proteins because they carry

removable amino (N)-terminal signal peptides. These

are 20–30-residue extensions, which contain 1 (or more)

positive charge at the N terminus and a short hydrophobic

core of 8–12 residues. One role of these ‘address tags’ is

to sort secretory proteins from cytoplasmic proteins.

Nascent pre-proteins are recognized directly by piloting

factors, such as the ribonucleoprotein signal-recognition

particle (SRP)

or the SecB chaperone

4,20

. Despite the fact

that in bacteria the SRP is predominantly involved in the

targeting of inner-membrane proteins (BOX 2), there are

examples of long, strongly hydrophobic signal peptides

of nascent secretory proteins, which are probably pref-

erentially bound by the SRP

21,22

. Other signal peptides

delay pre-protein folding and thus allow SecB to bind

to the mature region of the pre-protein

(FIG. 1b, part 1).

This process can occur while the polypeptide chain is still

being synthesized, but it is not mechanistically coupled to

elongation

. In both cases, the resulting SRP–pre-protein

and SecB–pre-protein complexes are targeted to the

translocase at the membrane (FIG. 1b, part 1). For SRP, this

is achieved by docking to its membrane receptor FtsY

and for SecB, by docking to the SecA subunit of the trans-

locase

. Although SecA does not contribute to the SRP-

targeting route

, when long, hydrophilic segments are

encountered, SecA is recruited to catalyse their export

Stage two: translocation. Irrespective of the targeting

route, all pre-proteins eventually reach the translocase at

the membrane (FIG. 1b, part 2). The translocase comprises

a membrane-embedded protein-conducting channel that

is built of the SecY, SecE and SecG polypeptides (corre-

sponding to the Sec61α, Sec61γ and Sec61β polypeptides,

respectively, in eukaryotes), through which pre-proteins

cross the membrane, and a flexible molecular motor, the

SecA ATPase, which drives translocation at the expense

of metabolic energy in the form of ATP and the proton-

motive force (PMF). Other cytoplasmic and membrane

subunits (FIG. 1a) optimize the translocation reaction.

Stage three: release and maturation. At the last stage,

pre-proteins are converted into mature proteins (FIG.1b,

part 3). After signal peptidase cleaves the signal peptides

correct folding of the polypeptide chain is initiated at the

trans side of the membrane

28–30

Nature Reviews | Microbiology

Per

T6S T4S Fla T3S CU T4P T2S LOL T5S TPS TAT Sec YidC Sort Esx Omp

+ + + + + + + + + + + + + – – +

– + – – – + – – – – + + + + + –

Gram-negative

Gram-positive

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The structure of the translocase and its parts

The translocase consists of the membrane protein

SecYEG, the SecA ATPase and a subset of partner pro-

teins (FIG. 1a). Each component will be discussed in turn

in the following section.

The SecYEG complex. The translocase core consists of

the integral membrane SecYEG heterotrimer. SecYEG

retains limited but detectable sequence homology from

bacteria to humans, and is remarkable in its ability to

translocate proteins not only through, but also laterally

into, the membrane (BOX 2), keeping the membrane bar-

rier intact throughout these processes. The purification

of E. coli SecYEG (ecSecYEG) and its functional reconsti-

tution into proteoliposomes

was a major breakthrough

that facilitated subsequent biochemical, biophysical and

structural analyses.

In a landmark study, the Rapoport group presented

a 3.2 Å structure of a monomer of SecYEG from the

archaeon Methanococcus jannaschii (mjSecYEG)

(FIG. 2a,b). This protein shares approximately 20%

sequence identity with, and contains 3 helices less than,

ecSecYEG. Nevertheless, the architecture of both chan-

nels is thought to be similar

and the missing helices

are not essential for ecSecYEG function

. M. jannaschii

SecY (mjSecY) is divided into 2 independent halves that

are connected with a loop — an N-terminal region

that contains transmembrane helices 1–5 (TM1–TM5)

and a carboxy (C)-terminal region that contains TM6–

TM10. The two subdomains share an inverted pseudo-

symmetry that resembles a clam shell (FIG. 2b, right), with a

narrow hourglass-shaped pore between the subdomains.

TM2 and TM7 are juxtaposed and are thought to form

the lateral gate of the channel (FIG. 2b, right).

The pore in a single SecYEG trimer is blocked from

its periplasmic side by a short loop–helix–loop substruc-

ture that is called the plug (shown in green in FIG. 2b). In

the crystal structure, the plug is in the ‘closed’ state and

SecYEG

YidC

SecDF

YajC

Nature Reviews | Microbiology

FtsY

SecA

Ribosome

ATP ADP

SecB

∆µH

−

Periplasm

Cytoplasm

SPase I

SecYEG

SecA

SRP

SRP-mediated targeting

(co-translational)

1 2 3

SecB-mediated targeting

(co- and post-translational)

Figure 1 | Bacterial protein secretion. a | A schematic representation of the bacterial pre-protein translocase subunits.

The translocase consists of the SecYEG pre-protein-conducting channel (yellow) and the ATPase motor SecA (red).

SecYEG can associate with the auxiliary proteins SecDFYajC and YidC (green)

95,98

. SecA, SecY and SecDFYajC are

known to form higher-order oligomers, but their actual oligomeric state in the functional holoenzyme has not been

conclusively determined. b | A general scheme for the secretion process. Secretory pre-proteins (thick orange line) are

synthesized with amino-terminal signal peptides (orange rectangle) and are targeted to the translocase either by the

ribosome-bound signal-recognition particle (SRP; blue) as soon as they emerge from the ribosome exit tunnel (this is

co-translational translocation, which occurs mainly with membrane proteins and proteins with extremely hydrophobic

signal peptides) or by the tetrameric SecB chaperone (pink) after translation has largely been completed (this is co- and

post-translational translocation, which occurs mainly with secretory proteins) (stage 1). Both targeting routes merge at

the membrane at SecYEG or SecYEG complexed with SecA. Some pre-proteins are targeted by SRP to SecYEG, but if

they have substantial hydrophilic stretches SecA is also required

. FtsY (pink) and SecA act as receptors for SRP and

SecB, respectively. For simplicity, SecYEG is presented without any auxiliary subunits. Pre-proteins are translocated

through SecYEG (stage 2) and they fold at the trans side of the membrane or integrate into the lipid bilayer after signal-

peptide cleavage by the signal peptidase

(SPase I; magenta) (stage 3). Signal peptides of lipoproteins require cleavage

by SPase II (not shown). Energy is provided by ATP binding and hydrolysis by the SecA ATPase and the proton-motive

force (∆µH

). The components are not drawn to scale.

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sits on top of a ring of hydrophobic residues (FIG. 2b) that

forms the narrowest constriction of the hourglass pore

(approximately 5–8 Å). Both the non-essential SecG

(shown in orange in FIG. 2a) and the essential SecE (vio-

let) bind peripherally to SecY, and SecE acts as an external

clamp. This form of the channel appears to be a ‘resting

state’, and there is no apparent ion conductance

The pore within SecY could be the export conduit

for polypeptides

. Strong evidence for this comes from

the observation that pre-proteins that stall during trans-

location can be crosslinked to residues in the pore

Moreover, several prl (protein localization) mutations,

which were isolated in genetic screens and allow the

secretion of polypeptides that contain defective, non-

functional signal sequences

36,37

, map on residues that

are located either on the helices that line the pore or are

on the plug

. This distribution led to the proposal that

prl mutations might ‘loosen’ the closed state or stabilize a

more open state of the export channel

. Signal sequences

could bind at a hydrophobic patch between TM2 and

TM7 (REF. 38), and this could trigger channel opening

It was proposed that the plug must leave the pore

during transmembrane crossing of the pre-protein

This was demonstrated directly by biochemical experi-

ments that showed that the plug can flip out towards

the periplasm

. Surprisingly, although the plug seems

to be important, and polypeptide translocation is

largely inhibited when the plug is crosslinked in the

closed state

, it is not essential for protein transloca-

tion or viability

40,41

. One possible explanation for this is

that in the mutated versions of SecY that lack the plug,

the residues that border the site of the deletion form a

functional pseudoplug

. Another concern is that the

~5–8 Å pore diameter is too small to accommodate a

pre-protein chain — having a minimal width of ~12 Å

in an extended conformation — or a disulphide-bonded

peptide loop (13 residues long) in a pre-protein

pre-proteins with covalent attachments

. Therefore,

for the single-SecY pore model, it is a prerequisite that

the pore dilates to become functional. This possibility

is supported by computer-based simulations

and elec-

trophysiological measurements

. The ‘diaphragm-like’

dilation of SecY could be facilitated by its several tilted

helices, the topological inversion of SecG

, the specific

binding of lipid molecules

, the partial detachment of

SecE, and by SecA, through ATP-driven conformational

effects (discussed below).

A single SecYEG trimer is necessary and, by reference

to Occam’s razor, might be sufficient for translocation.

Interestingly, however, fluorescence resonance energy

transfer (FRET)-based

and electron-microscopy (EM)

studies suggest the presence of higher-order oligomers.

By using cryo-electron microscopy

two-dimensional

8 Å crystals of

ecSecYEG in the lipid bilayer were viewed

and the contours of the 15 predicted TM helices (10 from

SecY, 3 from SecE and 2 from SecG) in the complex

were revealed. In this medium-resolution model,

ecSecYEG is arranged as a dimer of ‘back-to-back’

trimers (FIG. 2c, left). In another cryo-EM study, two

ecSecYEG trimers were shown to be bound to the exit

Box 2 | The sticky problem of membrane proteins

Integral membrane proteins (IMPs) account for approximately 20% of the total proteome of a bacterium such as

Escherichia coli. The key property that allows a protein to be selected for integration into the plasma membrane is the

presence of one or more transmembrane (TM) stretches of approximately 20 hydrophobic residues that form an α-helix

that is long enough (~3 nm) to span the hydrophobic core of the lipid bilayer

126

. Our current understanding of inner-

membrane biogenesis is based on the analysis of only a few model proteins. Membrane integration makes use of the

signal-recognition particle (SRP) and the protein-conducting channel SecYEG, with or without the membrane protein

YidC. In addition, YidC might act both on its own and/or together with the heterotrimeric complex SecDFYajC.

The ribosome-bound SRP identifies TM anchor sequences and long, highly hydrophobic signal peptides. It also targets

the ribosome–nascent chain complex to SecYEG at the membrane — mostly co-translationally

— where it binds to its

membrane-receptor protein (FtsY in bacteria; FIG. 1b). TM domains of IMPs are threaded through SecY during translation

and must eventually leave SecYEG and escape laterally into the lipid bilayer, possibly through the lateral gate of SecY, and

their orientation is partly regulated by phospholipids and the charged amino acids that lie at their termini and the

connecting loops

126–128

. Larger SecYEG assemblies could also facilitate the integration of TM bundles.

YidC can act as an auxiliary component of SecYEG

95–97,129

. Site-specific crosslinking places YidC and lipids in the vicinity

of the TM-anchor sequences of two model IMPs, whereas the hydrophilic regions of these proteins are located near SecA.

Interestingly, a bitopic membrane protein comes close to lipids and SecY before approaching YidC at a later stage of its

elongation

129

. Crosslinking data suggest that YidC is the core of assembly for multiple TMs of polytopic membrane

proteins such as MtlA and MalF, as it was found to be simultaneously attached to more than two consecutive TM segments

in these proteins

. YidC is thought to act as an independent insertase for small integral membrane substrates, such as the

Pf3 coat protein

and the c subunit of the F

ATP synthase

130

. The mechanism of this insertase function is unknown but it

is speculated that YidC provides a scaffold for the facilitation of the orientation and/or ‘flip flop’ movement of the helical

TM domains of newly inserted membrane proteins.

Proteins are also integrated into the outer membrane (OM) of Gram-negative bacteria (and the analogous membranes of

eukaryotic organelles). Proteins that are destined to enter this membrane still use the Sec translocase and, therefore, must

bypass lateral integration into the plasma membrane. To this end, the part of these proteins that crosses the OM in the

integrated state contains characteristic all-β-barrels (with only one known exception — the all-α-helical-barrel structured

Wza protein that exports capsular polysaccharides). A proteinaceous machinery is essential for this process. This

machinery consists of the Omp85 OM protein

131,132

, which is evolutionary conserved, as well as a subset of auxiliary

periplasmic chaperones

. Omp85 is required for the insertion of many, or all, of the β-barrel OM proteins that assemble

into the OM of E. coli (for a recent review see REF. 133).

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channel of a single ribosome complexed with a nascent

polypeptide chain

. Using a flexible fitting technique

and the high-resolution mjSecYEG structure

, a ‘front-

to-front’ arrangement of the two ecSecYEG trimers was

proposed (FIG. 2c, right). In this model, the two almost

juxtaposed lateral gates potentially form a transient

larger pore. This model would be compatible with

fluorescence-quenching analyses of eukaryotic Sec61p,

which are suggestive of the presence of a large, >40 Å,

pore opening during co-translational translocation

. In

both cases, allosteric communication between the two

SecYEG trimers could optimize translocation

From other EM studies of the homologous Sec61p

complex in eukaryotic cells

7–9

, and ecSecYEG

10,11

, it was

proposed that a pore is formed by 2–4 SecYEG trimers.

In eukaryotes, translocation is mainly co-translational

and the Sec61p structures were solved in the presence

of ribosomes

8,9

. Although it cannot be excluded that a

monomeric SecYEG is a peculiarity of M. jannaschii, the

fact that the EM studies were performed in the presence

of ribosomes

7,8

, or SecA and translocating pre-protein

10,11

implies that the recruitment of more than one SecYEG

might be more physiologically relevant. Obviously,

within these SecYEG oligomers, each of the SecYEG

trimers could represent an individual translocation

channel. Alternatively, larger pores might translocate

bulkier substrates or act as enclaves to trap auxiliary

factors. Another interesting possibility was proposed

after a recent crosslinking analysis of SecA-mediated

pre-protein delivery to SecYEG. These results support

the idea that in a SecYEG dimer there is division of

labour: one SecYEG complex provides the pore that is

used for pre-protein transport, whereas the other acts as

a docking station for SecA

In summary, a single SecYEG trimer can form an

export pore for exiting polypeptides. However, SecYEG

complexes also form dimeric or higher-order assemblies.

These might facilitate the docking of other translocase

subunits and the ribosome or have other roles that

remain poorly understood.

The SecA ATPase. SecA provides the chemo–mechanical

energy conversion that is required for translocation. The

domain organization of this molecular motor has been

revealed by biochemical analyses

50–54

and by the crystal-

lographic structures of SecA from Bacillus subtilis

14–16

Mycobacterium tuberculosis

, Thermus thermophilus

and E. coli

Each SecA protomer contains a central core — the

DEAD (DEAD-box) or helicase motor (FIG. 3a). This is

structurally homologous to the corresponding domains

in DEAD and DExD/H superfamily II proteins, which

include helicases (BOX 3). DEAD motors do not share

any significant primary-sequence similarity, other

than nine motifs, and are formed by two RecA-like fold

domains (BOX 3). In SecA, these domains are called NBD

(nucleotide-binding domain) and IRA2 (intramolecular

regulator of ATPase 2)

51,53

(FIG. 3b). A single nucleotide

binds at the NBD–IRA2 interface, which is lined by heli

case motifs that have been suggested to link ATP bind-

ing and hydrolysis to translocation

52,55

(BOX 3). Nuclear

magnetic resonance (NMR) spectra have revealed that

the DEAD motor is dynamic and that the C domain

partially suppresses this inherent flexibility

DEAD motors acquire specificity for different sub-

strates from non-homologous substrate-specificity

domains

. Such domains are usually fused at the N- or

C-terminal region of the DEAD motor or they protrude

from internal loops without disturbing the overall motor

structure (BOX 3). SecA has two specificity domains

(FIG. 3a–d): the C domain and the pre-protein-binding

domain (PBD). The α-helical C domain is fused at

the C terminus of IRA2 and contains 4 substructures

Nature Reviews | Microbiology

Periplasm

Cytoplasm

Periplasm

Cytoplasm

Bottom Front Side Top

Front Front Side

‘Back-to-back’ ‘Front-to-front’

Top

TM4

TM7

TM2

Plug

TM6–TM10

TM1–TM5

Figure 2 | Structure of the SecYEG protein-conducting channel. a | A space-filling

model of SecYEG from Methanococcus jannaschii (mjSecYEG; Protein Data Bank code:

1RH5)

. Four views are presented: bottom (from the cytoplasm); front; side; and top (from

the periplasm). SecY is in grey, SecE is in violet and SecG is in orange. In the bottom and

top representations the plug is removed to visualize the putative translocation pore.

b | A front-view space-filling model and ribbon diagrams of mjSecYEG

. The colours for

SecY, SecE and SecG are the same as in a, except that transmembrane helices 2 and 7

(TM2 and TM7), which are proposed to form the lateral gate of the SecY channel, are

coloured dark blue and red, respectively. TM4 is in cyan and the plug is green. In the

structure on the extreme right a dotted line reveals the pseudosymmetry of the two

subdomains of SecY (TM1–TM5 and TM6–TM10). c | A schematic representation of two of

the proposed modes of SecYEG dimerization: ‘back-to-back’(REF.12) and ‘front-to-

front’(REF.13). The two SecYEG trimers (dark and light grey space-filling models) were

arranged manually to allow visualization of the two proposed arrangements. A top view

is shown (as in b; right). The last TM helices of SecE (purple cylinders), SecG (orange;

shown as a filled circle in this view), and TM2, TM7 and TM4 of SecY (dark blue, red and

cyan cylinders, respectively) are presented. In the back-to-back arrangement, the second

TM helices of the two SecEs are juxtaposed and the lateral TM2–TM7 gates of SecY face

outwards (left), whereas in the front-to-front arrangement they are proximal to one

another (right). The presumed protein-conducting pore is in white.

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(FIG. 3b): the scaffold domain, which is a 46-residue

helix that acts as a molecular staple to control the open-

ing and closing of the DEAD motor

14,51,55,57

; the wing

domain (WD); the conserved helix–loop–helix IRA1

switch; and a short region of low conservation called the

C-terminal domain (CTD) or C tail, which is located at

the extreme C terminus and is not essential for cataly-

sis

. The structure of only the first half of the CTD in

one SecA structure has been determined by crystallog-

raphy

; its extreme 25 residues have been shown to bind

zinc ions by NMR

59,60

and biochemical studies

. The

PBD protrudes from the NBD between helicase motifs

II and III, binds pre-proteins

(BOX 3) and contains an

anti-parallel β-strand (stem) and a bilobate globular

domain (bulb) (FIG. 3c). Residues in (or from) the stem

and bulb appear to bind pre-protein segments

50,52,62

, with

the signal peptide binding mainly in a shallow groove

in the bulb

In the available SecA structures, the PBD bulb under-

goes a remarkable approximately 75° swivelling motion

between a closed (FIG. 3c) and an open (FIG. 3d) state. In

the open state, a large cleft is formed between the WD,

IRA1 and bulb structures. Part of this opening could

accommodate pre-proteins or regions of SecYEG

15,17

Order–disorder transitions of the DEAD motor are

allosterically transmitted to the PBD

. If pre-proteins

are bound, the PBD ‘talks back’ conformationally to the

DEAD motor

. Importantly, the two specificity domains

bind to one another, with the major contacts being pro-

vided by both IRA1 and the PBD bulb

. These physical

contacts ensure allosteric communication between these

domains, and between these domains and the DEAD

motor. This conformational flow lies at the core of

SecA-mediated catalysis

55,64,65

SecA oligomerization. SecA exists in solution in a

monomer to dimer equilibrium (the estimated equilib-

rium dissociation constant (K

) is approximately 1 µM

at 8°C in the presence of 300 mM K

acetate

) and, in

concentrated solutions, SecA is routinely isolated as a

dimer

51,66–69

. SecA is thought to partition equally between

the cytoplasm and the membrane. At the concentra-

tions that are believed to be found in the cell cytoplasm

(2–5 µM

66,69,70

), ecSecA is expected to be mainly dimeric.

Remarkably, the molecular determinants of SecA dimer

ization remain a conundrum. Despite the fact that all

the SecA protomers in the crystal structures are similar, the

dimers of the various SecAs present different dimeric

interfaces

14,16–19

(FIG. 3e). Moreover, B. subtilis SecA

crystallized as an apparent monomer in one study

, in

another it formed symmetrically related crystallographic

dimers

and in yet another it formed dimers in the

crystal unit cell

. Biochemical analysis of the different

domains of SecA favours possibilities for assembly

51,71,72

that are incompatible with most of the structures. For

example, an isolated C-domain polypeptide is stably

dimeric

51,72

. However, in none of the crystal structures is

the C domain used as an exclusive dimerization interface.

Finally, truncation of nine extreme N-terminal residues

that were thought to be important for dimerization only

in the symmetrically related dimeric bsSecA structure

14,15

C domain

Bulb

‘Closed’ ‘Open’

Stem

DEAD

motor

NBD

CTD

IRA1

IRA2

PBD

a b

c d

E. coli

B. subtilis I B. subtilis II

T. thermophilus M. tuberculosis

Figure 3 | The structure of SecA. Space-filling (a) and ribbon-representation (b)

models of the Escherichia coli SecA protomer (ecSecA) with its pre-protein-binding

domain (PBD) (modelled into Protein Data Bank (PDB) code 2FSF

as described in

REF. 17). The DEAD-motor domains are dark blue (nucleotide-binding domain

(NBD); residues 1–220 and 377–416) and mauve (intramolecular regulator of

ATPase 2 (IRA2); residues 417–621), the C domain (residues 622–901) is green and

the PBD (residues 221–377) is magenta. In b, the sub-structures of the C domain

are indicated: SD, scaffold domain (dark green); WD, wing domain (light green);

IRA1 (yellow); CTD, carboxy-terminal domain (orange). The swivelling of the bulb

sub-structure of the PBD in ecSecA from the ‘closed’ to the ‘open’ state is shown

against a space-filling model of the C domain (c and d). The closed state of ecSecA

was derived from structural modelling using Bacillus subtilis SecA (bsSecA)

as a

template (PDB code: 1M6N). The ‘open’ state was derived from the crystallographi-

cally determined structure

. In ecSecA, the stem comprises residues 221–227 and

370–377 and the bulb comprises residues 228–269. A comparison of SecA dimers is

also provided (e). Ribbon representation models are shown in which one protomer

is coloured as in a and the other is grey. The coloured protomer of each SecA

(shown in a top view looking down on the DEAD motor) was structurally aligned to

visualize the different orientations of the second protomer. The PDB codes that were

used are: B. subtilis SecA I (bsSecA I), 2BIM

; B. subtilis SecA II (bsSecA II), 1M6N

;

ecSecA, 2FSF

; Mycobacterium tuberculosis SecA (mtSecA), 1NL3 (REF. 18) and Thermus

thermophilus SecA (ttSecA), 2IPC

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Box 3 | Superfamily II helicases

SecA is a member of the superfamily II (SF2) DExH/D proteins. Most of the members of

this family are nucleic acid helicases

. Several other functions, such as the disassembly

of protein–RNA complexes and stabilization of RNA conformations, have been described.

Helicases use the energy from ATP hydrolysis to bind and unwind nucleic acid substrates,

whereas SecA uses this energy to bind and move along polypeptide substrates. As shown

in the figure all helicases share a structurally highly conserved domain that contains two

RecA-like subdomains that form the helicase or DEAD (Asp-Glu-Ala-Asp) motor. The

motor comprises nine conserved motifs, many of which line the ATP-binding cleft.

Several of these motifs are directly involved in nucleotide binding, whereas others

appear to be important in conveying conformational cross-talk with the substrates.

In the figure, panel a shows a ribbon diagram of the Escherichia coli SecA (ecSecA) DEAD

motor bound to ATP

. The helicase motifs are coloured according to panel b. Only the

beginning of the stem of the pre-protein-binding domain (PBD) is visible (pink). The side

chains of the residues discussed in the main text are shown in red. Gate 1 comprises D217

and R566, joined by a salt bridge

. Panel b shows a schematic representation of the nine

conserved motifs of SF2 DEAD helicases aligned with those of the ecSecA DEAD motor.

The site of insertion of the PBD is indicated, as well as the number of the first and the last

residue of each motif in SecA. Capital letters indicate more than 80% homology; lower-

case letters indicate 50–79% conservation. The ‘o’ represents threonine or serine. IRA2,

intramolecular regulator of ATPase2; NBD, nucleotide-binding domain; Q, glutamine.

did not affect ecSecA dimerization and function

73,74

(for

an alternative view, see REF. 75). However, truncation of

the nonapeptide could disfavour the formation of one

dimeric state but promote the formation of an alterna-

tive dimeric state. One possibility is that the revealed

dimerization interfaces correspond to different SecA

dimers that have specialized functions. Another possibil-

ity is that the extreme conditions used for crystallization,

such as elevated salt concentration, dissociate the SecA

dimer and favour different but physiologically irrelevant

arrangements of the protomers in the crystal lattice. In a

remarkable recent breakthrough, the Kalodimos labora-

tory has acquired a solution structure of the full-length

dimeric SecA, a protein of 204 kDa, using NMR spec

troscopy

. NMR studies will provide direct information

on the native state of dimeric SecA in solution.

The SecA–SecYEG holoenzyme. SecYEG and SecA form

the active holoenzyme

. SecA binds to the membrane with

low affinity at acidic phospholipids but with high affinity

(5–40 nM) at SecYEG

. Several cytoplasm-exposed loops

of SecY are available for a possible interaction with SecA

and/or the ribosome

5,12,13,32,76,77

(FIG. 2a,b). SecYEG makes

three connections with the ribosome — two between

ribosomal-RNA hairpins and the cytoplasmic SecY loops

and one between ribosomal proteins and the cytoplasmic

region of SecG

. Potential SecA-interacting residues in

the SecY cytoplasmic loops have also been identified

genetically, and biochemical data suggest that TM4 of

SecY, which is largely inaccessible from the cytoplasm,

might interact with SecA

(FIG. 2b,c). SecG, although not

essential for the initial binding of SecA to SecYEG, might

interact at a later stage of the translocation reaction

78,79

Several SecA domains might be important for binding to

SecYEG

65,80,81

. The DEAD motor was shown by biochemi-

cal methods to interact with SecYEG

49,65,80

. As the affinity

of wild-type SecA is four times higher than that of a SecA

that is missing its C domain

65,81

, the C domain might

also contribute to binding either directly or by allosteric

modulation of the DEAD motor.

The stoichiometry of the interaction between SecA

and SecYEG is largely unresolved. Several experi-

ments point to dimeric SecA being the active state

of the enzyme

67,82,83

. Nevertheless, the crystallization of

monomeric SecA

raised the possibility that SecA acts as

a monomer. The results of FRET and detergent-induced

monomerization experiments were interpreted as an

indication that SecA forms monomers during lipid bind-

ing

15,75,84–86

. In addition, SecA can bind to a single SecYEG

that is trapped in Nanodisc particles

. Nanodisc-

trapped SecA acquires an unusually elevated (several

fold) ATPase activity in the absence of a translocating

pre-protein. This is not detected when SecA is bound

to membrane-embedded SecYEG

64,87

. It has not been

reported whether Nanodisc-trapped SecA is still active

for the pre-protein interaction and translocation under

the same conditions. Monomeric SecA can accommo-

date two SecYEG trimers if it binds with its open PBD

conformation

15,49

(FIG. 4c). In other studies, all possible

combinations of SecA monomers and dimers that could

interact with SecYEG monomers or dimers have been

proposed

10,11,85,88

. Although a single SecA appears to be

competent in binding to a single SecY, the data that have

been obtained with low-resolution methods are not suf-

ficient to provide strong support for either combination

of SecA–SecY oligomeric assembly. The dimensions of

both SecYEG dimers

13,89

(FIG. 2c) are comparable to those

of both the monomeric and dimeric forms of SecA. As

an example, three possible arrangements of SecA bound

to dimeric SecY are shown in FIG. 4 (monomeric (part a)

and dimeric (parts b and c)). Addressing the assembly and

disassembly of a dynamic system at a membrane interface

is technically notoriously difficult.

Auxiliary components of the translocase. Another het-

erotrimeric protein complex called SecDFYajC has also

been found to be associated with the core SecYEG trimer

at the membrane

(FIG. 1a) under certain purification

Nature Reviews | Microbiology

NBD

ATP

R509

R566

D217

Gate 1

PBD

IRA2

R574

III

SF2 DEAD

SECA E. coli

Motifs

Q GG TPGR1 VlDEaDxm SAT liFxxo LvaTdvaaRGlD YxHRi GRogRxGAxoGoGKT PTRELAxQ

Q PG TNNEY LVDEVDSI TGT LVGTIS TIATNMAGRGTD ESRRIDNQLRGRSGRQGMKTGEGKT VNDYLAQR

Q I Ia GG Ib II III IV V VI

102

109

131

138

159

160

178

182

207

214

391

393

453

458

501

512

563

579

IRA2

C domain

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conditions. This complex is not required for the in vitro

reconstitution of the translocation reaction, but has a

stimulatory effect on translocation activity in the absence

of SecG

. However, viability and protein export are seri-

ously compromised in vivo when cells are depleted of

SecD or SecF

, although the same is not true for YajC.

SecDFYajC might be involved in the release of translo-

cated proteins from the membrane

or the regulation of

SecA membrane cycling

. Another membrane protein,

YidC (FIG. 1a), is involved in the biogenesis of both the Sec-

dependent and Sec-independent membrane proteins

94–96

and associates with SecDFYajC

(BOX 2).

The translocase binds multiple ligands

Translocase and phospholipids. Anionic phospholipids

are a significant component of the E. coli membrane

(20% phosphatidylglycerol (PG) and 5% cardiolipin

(CL)) and are necessary for protein secretion both in vivo

and in vitro

87,98

. Long-chain phospholipid analogues and

acidic phospholipids were shown to promote SecA mon-

omerization

84,86

. Acidic phospholipids bound at SecYEG

contribute to the binding affinity of SecA and the subse-

quent activation of its ATPase activity

46,87

, and can even

restore the activity of cold-sensitive SecA mutants

. In

contrast to PG, which gives rise to organized bilayers,

phosphatidylethanolamine (PE) — the major lipid com-

ponent (75%) of the E. coli membrane — cannot form

bilayer structures in vitro

. In the absence of PE, mem-

branes are functional for translocation only if they have

highly increased amounts of PG and CL, which, together

with divalent cations, promote non-bilayer structures

that are similar to those promoted by PE

100

The pre-protein–SecB complex. SecB is a general cellular

chaperone

that appears to be important for the export of

several secretory proteins, such as the outer-membrane

porin LamB

. It is not essential for viability and is not

present in all bacteria, but has a dual role: it maintains

secretory pre-proteins in a translocation-competent state

and interacts specifically with membrane-bound SecA

= 10–30 nM)

SecB crystal structures

101–103

revealed structural fea-

tures that could allow this chaperone to bind to extended

polypeptides and SecA. SecB is a stable tetramer (FIG. 5a)

that probably binds to pre-proteins by recognizing

exposed hydrophobic surfaces

104

. A groove has been

identified in the structures that could constitute the

pre-protein binding site. Each SecB

contains two such

grooves, which, as these sites are mainly solvent exposed,

could accommodate peptides without disturbing the

stable tetramer (FIG. 5c, pink cylinder). The grooves are

lined by several aromatic residues at one end and hydro-

phobic, but not aromatic, residues at the opposing end

(FIG. 5c, part 2). The aromatic-residue-rich sub-site of SecB

could accommodate the proposed SecB-binding motif

in pre-proteins, which, it is suggested, is a nonapeptide

that contains several aromatic and basic residues

105

. In

an acidic region on the top surface of the SecB tetramer,

several residues interact with the 25 C-terminal residues

of the SecA CTD

106,107

(FIG. 5b). The symmetry of SecB

(a dimer of dimers) favours the binding of 1 SecA dimer

per SecB

(FIG. 5b). Site-directed spin labelling in combi-

nation with electron paramagnetic resonance spectros-

copy was used to investigate how SecA and a pre-protein

docks on SecB

108

and the results partially confirmed the

sites that had already been proposed by crystallography

(FIG. 5b,c). Several routes for how the polypeptide wraps

its extended aminoacyl chain around SecB have been

proposed, as would be expected for a chaperone that

binds to numerous diverse substrates.

SecA–pre-protein complex. When SecA recognizes the

SecB-delivered pre-protein, it releases SecB in an ATP-

regulated reaction

107

. These events are thought to occur

at the SecYEG-bound form of SecA. However, some

evidence suggests that pre-protein–SecA complexes

might also form at the ribosome exit site

109,110

. SecA rec-

ognizes pre-proteins, at least in part, through its PBD

region

50,52,63

(FIG. 3a). The hydrophobic core of the signal

peptide acquires an α-helical conformation after binding

to SecA

111

. Signal peptides bind at an interfacial groove

between the PBD bulb and the IRA1 hairpin of the C

domain

. As part of the CTD occupies this groove

, this

could provide a mechanism to shield this region from

the premature binding of signal peptides to cytoplasmic

SecA. Mutational

112,113

and biochemical

52,114

studies with

a model mature domain suggest that SecA might bind

mature pre-protein segments or regions through its bulb

region.

SecA–nucleotide binding. The NBD of SecA contains

a high-affinity nucleotide-binding site (see motifs in

BOX 3). Most of the residues that contact the nucleotide

directly are located in the NBD. A few IRA2 residues

(BOX 3) directly contact ATP in the ecSecA crystal struc-

ture

. The putative arginine finger in IRA2 — R574 — is

indispensable for catalysis

53,55

, and faces away from the

pocket (BOX 3), implying that ecSecA crystallized in an

inactive state. However, it is clear from NMR studies of

ecSecA that the arginine fingers are important for nucle-

otide binding and for the nucleotide-dependent stabi-

lization of IRA2

(REF. 55). Other interactions between

Nature Reviews | Microbiology

~6.7 nm

~5 nm

~8.7 nm

Cytoplasm

Periplasm

a b c

SecYEG

Figure 4 | Hypothetical SecA–SecYEG interaction models. The approximate

dimensions for the SecYEG dimer (yellow) and the lipid bilayer are indicated. Hypothetical

interactions with a monomeric SecA (Bacillus subtilis SecA)

(a) and two different dimeric

SecAs (Mycobacterium tuberculosis SecA

(b) and Escherichia coli SecA (ecSecA)

(which

is approximately 15 nm x 8 nm) (c), are shown as examples. The ecSecA dimer

is the only

one in which both DEAD motors face the same side of the dimer and would thus both be

available for binding to a SecYEG dimer. In SecA, the DEAD-motor domains are dark blue

and mauve, the C domain is green and the pre-protein-binding domain is magenta.

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conserved residues that reside in opposing sites of the

motor ensure nucleotide-independent intra-domain

communication between NBD and IRA2

(REFS 14,17,65).

The crystal structures did not reveal major nucleotide-

driven conformational changes

14,15,17,18

. However,

nucleotides and other translocation ligands regulate the

equilibrium between the ordered and disordered states of

the DEAD motor

. In addition, a highly conserved salt

bridge (see gate 1 in BOX 3) controls the catalytic cycle

of SecA by regulating the opening and closing of the

nucleotide cleft

and is under the control of the PBD.

All of the above interactions between the trans-

locase and its peripherally associated ligands must be

ordered and sequential, and energy must be spent during

the ensuing step-wise mechanical work. The process of

energy generation is discussed in the next section.

Energizing the translocase machine

Stage two of the translocation reaction (FIG. 1b, part 2)

requires energy input in the form of both ATP and the

PMF. These energy sources function at different stages

of translocation

42,115

. ATP is essential for the initiation of

the translocation reaction

115

. Cytosolic SecA rapidly

hydrolyses ATP to ADP

. SecA–ADP is thermally stabi-

lized

53,116

and the release of ADP from the DEAD motor

is the rate-limiting step for the subsequent catalysis

53,116

SecA increases its ATP turnover in a pre-protein- and

SecYEG-dependent manner. This stimulated activity

is known as the translocation ATPase activity

64,87

. SecA

undergoes insertion and de-insertion cycles at SecYEG

during its catalytic cycle

93,117

. The inserted state is a con-

formation in which significant parts of SecA become

membrane integral and inaccessible to proteases. The

ATP- and ADP-bound states are thought to correspond

to inter-convertible extended and compact conforma-

tions that are able to insert and de-insert at the mem-

brane, respectively. The energy that is released during

these states is converted to mechanical work so that

pre-protein segments are inserted into the SecYEG pore

and eventually become translocated to the trans side of

the membrane after several SecA cycles of insertion and

de-insertion. When pre-protein translocation is substan

tially underway, the PMF can further drive the reaction

and even complete it in the apparent absence of ATP

115

A high concentration of SecA seems to render transloca-

tion largely PMF-independent

118

. Although the role of

the PMF during protein translocation is not completely

clear, it seems to promote SecA de-insertion either by

stimulating the release of ADP from the motor

119

by stimulating conformational changes in SecY

120,121

. In

the absence of a PMF and SecA, pre-proteins can slip

back towards the cis side of the membrane, which can

lead to uncoupled ATP consumption during translo-

cation

115

, and thus the PMF prevents back-slippage of

the translocating pre-protein

115,122

. Finally, some of the

energy that is released during export might be directed

towards active unfolding of pre-protein domains

122

Pre-protein translocation by the Sec translocase

Using the available information, we propose a mecha-

nistic model that describes SecA-dependent bacterial

pre-protein translocation in twelve stages (FIG. 6).

In the first, quiescent stage, SecA resides in the cyto-

plasm in its inactive and stable ADP-bound state

53,116

This is followed by a priming stage (stage 2), during

which SecA–ADP binds to SecYEG, mainly through

the DEAD motor

49,65,81

, and probably to the cytosolic

loops of SecY

5,76,77

. Priming leads to conformational

changes in the SecA C domain and, possibly, the partial

Nature Reviews | Microbiology

321

Figure 5 | The structure of SecB. a | A ribbon

representation of the Escherichia coli SecB (ecSecB)

tetramer, which is a dimer of dimers

103

(Protein Data Bank

code: 1QYN). The two dimers are green–yellow and

red–dark blue. b The SecB–SecA interaction. A ribbon and

space-filling model representation of the ecSecB tetramer

(grey)

103

in complex with the carboxy (C)-terminal peptide

of Haemophilus influenzae SecA (yellow)

101,102

is shown.

Two SecA peptides are in complex with one SecB tetramer.

The surface that binds the SecA C-terminal peptide is in

red. Other SecB residues that seem to interact with

SecA are in green

108

. c | The SecB–pre-protein complex.

A ribbon representation of the ecSecB

tetramer is shown

(protomers a–b, dark grey; c–d light grey) (1). The pink

cylinder marks the proposed peptide-binding channel that

is formed by two grooves on the same side of the tetramer

(a–d or b–c). A space-filling model of 1 is shown in 2, with

hydrophobic residues that line the 4 grooves (green) and

residues that seem to interact with a pre-protein indicated

(galactose-binding protein precursor; red)

134

. A space-

filling model of 1 is also shown in 3, but only protomers a

and d are shown so that the continuity of 1 peptide-

binding channel can be viewed. Each tetramer contains

two such channels.

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847

detachment of the scaffold domain

55,57,65

and conforma-

tional changes in the PBD

63,64

. These changes result in

moderate membrane ATPase activity and, importantly,

favour high-affinity binding of the SecB–pre-protein

complex

24,87

to the CTD

102,107

and/or the N-terminal

106,108

regions of primed SecA. In the third, triggering stage

(stage 3), SecA binds the signal peptide and the N-terminal

regions of the mature pre-protein through the PBD

50,52,62

and, possibly, the C domain. This binding alleviates

C-domain suppression and opens the gate 1 salt bridge at

the base of the DEAD motor

(BOX 3). As a result, IRA2

becomes disordered and detaches from the NBD, which

leads to loss of affinity of the DEAD motor for ADP

(stage 4)

51,53,55,64,65

. The loss of ADP drives SecA to the

pre-insertion stage (stage 5) in which the PBD, together

with the bound pre-protein, undergoes conformational

changes

52,55

and SecB is released from SecA

107

ATP binding to the empty DEAD motor (the ATP

stroke stage; stage 6) drives additional conformational

changes, and regions of SecA–ATP penetrate deeper

into the membrane plane at SecYEG

93,117

. Such regions

of SecA could include the two specificity domains — the

PBD and the C domain

117

— as well as the DEAD motor.

SecA membrane insertion could have two effects. On the

one hand it might prepare the SecYEG channel by initi-

ating the dilation of the SecY ‘diaphragm’ (stage 7). On

the other hand, however, it might deliver the pre-protein

to the primed channel. The release of the signal peptide to

the lateral gate of SecY, where it becomes stably bound

by its hydrophobic core

35,38,49

, causes the plug to flip out

away from the pore, thereby leading to a fully activated

channel (stage 8)

6,34,39

. Signal-peptide penetration in the

membrane drags along the N-terminal mature region

of the pre-protein

115

, and the hairpin that it forms

123

becomes positioned at SecY in such a way that the mature

region is threaded into the open SecY pore

35,49,124

. ATP

then becomes hydrolysed and the pre-protein is partially

released into the channel

49,115

(pre-protein-release stage;

stage 9). SecA returns to its previous ADP-bound state in

a reaction that is facilitated by the PMF

119

, the C domain

re-associates with the DEAD motor and the PBD is free

to move further down the pre-protein chain (de-inserted

state; stage 10)

93,117

. The attachment of the PBD onto the

succeeding pre-protein segment (stage 11), will therefore

trigger ADP release and a new round of catalysis (stages

4–11). The open and closed PBD states that were detected

in the crystal structures (FIG. 3c,d) and by NMR

might

correspond to pre-protein binding and release confor-

mations. SecA must perform multiple catalytic rounds,

alternating between the SecA–ADP and SecA–ATP

states and the conformational states of the holoenzyme

that they represent, in order for the whole length of the

polymeric pre-protein substrate to cross the channel in

segments of approximately 30 residues at a time

115,124

Several repeats of these events (stages 4–11) would lead

to the translocation of a complete pre-protein chain, with

SecA functioning as a typical processive motor. The cata-

lytic cycle is facilitated by the PMF, which can drive the

completion of translocation when the pre-protein is not

attached to SecA

115

. Finally, at the maturation stage (stage

12), the mature polypeptide chain, which is generated

after signal-peptide cleavage by signal peptidase, acquires

Cytoplasm

Periplasm

Nature Reviews | Microbiolog

SecB

SecA

1 2 3 4 5 6 7

9 10 11

D D

Plug

Figure 6 | SecA-dependent pre-protein translocation — a model for the sequence of events. See main text for

details. For simplicity, SecA and SecY are shown as monomers and the carboxyl terminus of SecA, where SecB is thought to

bind, is only shown in stages 1–4 and in stage 12. Only the initial stages of the process are shown, with a pre-protein

containing a signal peptide. However, similar multiple catalytic turnovers are expected to drive the complete secretion of

the mature region after signal-peptide cleavage. Irregular contours indicate conformational changes. The thick orange line

represents the secretory pre-protein and the orange rectangle represents the N-terminal signal peptide. D, ADP; T, ATP.

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its native three-dimensional structure at the trans side

of the membrane.

Conclusions

Despite recent progress in our understanding of the

mechanisms that are involved in bacterial protein secre-

tion, many fundamental questions remain and several

aspects of the catalytic cycle (FIG. 6) are unresolved. How

does the channel open to accommodate a passing pre-

protein? Which of the oligomeric states of SecYEG

and SecA defines an active channel? What are the

conformational changes that follow SecA–pre-protein-

complex binding to SecYEG and trigger translocation

through the channel? What is the role of the SecA-specificity

domains during the translocation reaction? What is the

role of the PMF? What is the molecular basis of SecA

processivity? To address these and other questions, high-

resolution structures of complexes must be determined

and biophysical real-time experiments, such as single-

molecule studies, should be performed. Bacterial protein

secretion remains an exciting research area and a unique

paradigm for macromolecular membrane transport.

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traffic in prokaryotes. Mol. Microbiol. 62, 308–319

(2006).

2. Holland, I. B. Translocation of bacterial proteins — an

overview. Biochim. Biophys. Acta 1694, 5–16 (2004).

Luirink, J. & Sinning, I. SRP-mediated protein

targeting: structure and function revisited. Biochim.

Biophys. Acta 1694, 17–35 (2004).

4. Randall, L. L. & Hardy, S. J. SecB, one small

chaperone in the complex milieu of the cell. Cell. Mol.

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Van den Berg, B. et al. X-ray structure of a protein-

conducting channel. Nature 427, 36–44 (2004).

The first crystal structure of the membrane-

embedded pre-protein-conducting channel. It

provided hypotheses for the channel-dilation

mechanism and its gating.

Li, W. et al. The plug domain of the SecY protein

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Acknowledgements
We are grateful to the Kalodimos laboratory for an exciting 
collaboration and to B. Kalodimos and T. Pugsley for stimu-
lating discussions. Research in our laboratory is supported 
by grants from the European Union (LSHG-CT-2005-037586), 
the Greek General Secretariat of Research and the European 
Regio nal   Deve lopm ent  Fun d  (01 AKMO N46  and 
PENED03ED623).
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus subtilis | Escherichia coli | Methanococcus jannaschii |
 
Mycobacterium tuberculosis
 
| Thermus thermophilus
Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=protein
ecSecYEG
Protein Data Bank: http://www.rcsb.org/pdb/home/home.do
bsSecA | bsSecA I | bsSecA II | ecSecA | ecSecB | mjSecYEG | 
mtSecA | ttSecA 
FURTHER INFORMATION
Anastassios Economou’s homepage:  
http://ecoserver.imbb.forth.gr
ALL LINKS ARE ACTIVE IN THE ONLINE PDF
RE V IE W S
NATURE REVIEWS 
|
 MICROBIOLOGY   VOLUME 5 
|
 NOVEMBER 2007 
|
 851

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine

Article

Full-text available

Sep 2023

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria–bacteria and bacteria–host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer’s patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms

Article

Full-text available

Jul 2023

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

The genome of Candidatus phytoplasma ziziphi provides insights into their biological characteristics

Article

Full-text available

May 2023
BMC PLANT BIOL

Phytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches’ broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete ‘Candidatus Phytoplasma ziziphi’ chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.

A REVIEW ON PROTEIN PRODUCTION AND SECRETION IN EUKARYOYTES

Article

Full-text available

Apr 2024

Protein secretion is an important process in any living organism, and this is mediated by numerous steps and several cellular components. This review provides an overview of protein production from nascent polypeptide synthesis stage to secretion of these proteins into extracellular space by vesicular sorting. This review also provides insights on the factors involved in ER and Golgi in this pathway and their role. It also covers responsibility of translocons and chaperones in stress response pathways such as UPR and ERAD. Beyond all these a series of quality control checks are performed by cellular machinery to ensure the quality of protein delivered. So essentially this review covers all the pathways involved from initiation of protein expression to secretion.

Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus

Article

Full-text available

Jun 2023

Bacterial predation impacts microbial community structures, which can have both positive and negative effects on plant and animal health and on environmental sustainability. Myxococcus xanthus is an epibiotic soil predator with a broad range of prey, including Sinorhizobium meliloti, which establishes nitrogen-fixing symbiosis with legumes. During the M. xanthus-S. meliloti interaction, the predator must adapt its transcriptome to kill and lyse the target (predatosome), and the prey must orchestrate a transcriptional response (defensome) to protect itself against the biotic stress caused by the predatory attack. Here, we describe the transcriptional changes taking place in S. meliloti in response to myxobacterial predation. The results indicate that the predator induces massive changes in the prey transcriptome with up-regulation of protein synthesis and secretion, energy generation, and fatty acid (FA) synthesis, while down-regulating genes required for FA degradation and carbohydrate transport and metabolism. The reconstruction of up-regulated pathways suggests that S. meliloti modifies the cell envelop by increasing the production of different surface polysaccharides (SPSs) and membrane lipids. Besides the barrier role of SPSs, additional mechanisms involving the activity of efflux pumps and the peptide uptake transporter BacA, together with the production of H2O2 and formaldehyde have been unveiled. Also, the induction of the iron-uptake machinery in both predator and prey reflects a strong competition for this metal. With this research we complete the characterization of the complex transcriptional changes that occur during the M. xanthus-S. meliloti interaction, which can impact the establishment of beneficial symbiosis with legumes.

Crafting negative training datasets for a high-performance classification of bacterial proteins secreted via non-classical pathways

Preprint

Full-text available

Jan 2023

Understanding protein secretion pathways are of paramount importance in studying diseases caused by bacteria and their respective treatments. Most such paths must signal ways to identify secretion. However, some proteins, known as non-classical secreted proteins, do not have signaling ways. This study aims to classify such proteins from predictive machine-learning techniques. Guided by the literature, we collected a set of physical-chemical characteristics of amino acids from the AA index site bolding know protein motifs, like hydrophobicity. In this work, we developed a six steps method (Alignment, Preliminary classification, mean outliers, two Clustering algorithms, and Random choice) to filter data from raw genomes and compose a negative dataset in contrast to a positive dataset of 141 proteins also gathered from the literature. Using a conventional Random Forest machine-learning algorithm, we obtained an accuracy of ~91% on classifying non-classical secreted proteins in a validation dataset with 14 positive and 92 negatives proteins - sensitivity and specificity of 91 and ~86%, respectively, performance compared to state of the art for non-classical secretion classification, but a less sophisticated algorithm allows us to classify bacterial proteins concerning secretion by non-classical pathways more rapidly. Therefore, this research has shown that selecting an appropriate descriptors' set and an expressive training dataset compensates for not using an advanced machine learning algorithm for the secretion by non-classical pathways purpose. The data and software from this work, available at https://github.com/santosardr/non-CSPs, can be downloaded for standalone use without needing third-party software.

Arabinan saccharification by biogas reactor metagenome-derived arabinosyl hydrolases

Article

Full-text available

Nov 2022
Biotechnol Biofuels

Background Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use as biotechnological feedstock is still limited. Results In order to identify efficient enzymes for polysaccharide breakdown, we have carried out functional screening of metagenomic fosmid libraries from biogas fermenter microbial communities grown on sugar beet pulp, an arabinan-rich agricultural residue, or other sources containing microbes that efficiently depolymerize polysaccharides, using CPH (chromogenic polysaccharide hydrogel) or ICB (insoluble chromogenic biomass) labeled polysaccharide substrates. Seventy-one depolymerase-encoding genes were identified from 55 active fosmid clones by using Illumina and Sanger sequencing and dbCAN CAZyme (carbohydrate-active enzyme) annotation. An around 56 kb assembled DNA fragment putatively originating from Xylanivirga thermophila strain or a close relative was analyzed in detail. It contained 48 ORFs (open reading frames), of which 31 were assigned to sugar metabolism. Interestingly, a large number of genes for enzymes putatively involved in degradation and utilization of arabinose-containing carbohydrates were found. Seven putative arabinosyl hydrolases from this DNA fragment belonging to glycoside hydrolase (GH) families GH51 and GH43 were biochemically characterized, revealing two with endo-arabinanase activity and four with exo-α- l -arabinofuranosidase activity but with complementary cleavage properties. These enzymes were found to act synergistically and can completely hydrolyze SBA (sugar beet arabinan) and DA (debranched arabinan). Conclusions We screened 32,776 fosmid clones from several metagenomic libraries with chromogenic lignocellulosic substrates for functional enzymes to advance the understanding about the saccharification of recalcitrant lignocellulose. Seven putative X. thermophila arabinosyl hydrolases were characterized for pectic substrate degradation . The arabinosyl hydrolases displayed maximum activity and significant long-term stability around 50 °C. The enzyme cocktails composed in this study fully degraded the arabinan substrates and thus could serve for arabinose production in food and biofuel industries.

SadB is a Pseudomonas aeruginosa global regulator

Article

Jul 2022

Maria Papangeli

Bacteria can adhere to almost any surface and form resistant biofilm communities. These pose a major clinical burden as they can form on medical devices such as catheters and are hard to prevent. Catheter- associated urinary tract infections (CAUTIs) are the most common nosocomial infection as they represent ~40% of all nosocomial infections. Hook et al., previously developed a series of polymers that prevent biofilm formation. Following a screen of 20,000 polymers and copolymers and subsequent scale-up, the hit acrylate polymer, EGdPEA has been clinically approved for the coating of urinary tract catheters and presents superior antibiofilm properties compared with silver-embedded silicone catheters. The bacterial biofilm resistance mechanism and surface sensing pathways involved are under investigation. In P. aeruginosa surface sensing includes the flagella, type IV pili and the wsp system. The present study sought to investigate SadB, an essential gene for biofilm formation and CheA, a histidine kinase of the chemotaxis system for their involvement in surface sensing. SadB is involved in the switch from reversible to irreversible surface attachment. Expression of sadB under a constitutive promoter overcomes the resistance of EGdPEA to biofilm formation. CheA is responsible for transmitting environmental signals to the cell and activating Pch phosphodiesterase, which is important in surface sensing. Biofilm and motility assays and monitoring on-surface gene expression were used as a test of potential involvement in surface sensing. In agreement with the literature, SadB was found to be essential for biofilm formation and to affect swarming motility. sadB was also found to be differentially expressed on the anti and pro-biofilm surfaces. In contrast, CheA affected swarming but did not affect biofilm formation on most polymer surfaces and glass. The promoter of cheA also controls the cheI operon with 13 more genes. The expression of the operon was found to be different on different surfaces but CheA is unlikely to be important in surface sensing. The literature and findings of the present study highlighted the importance of sadB in surface sensing, but the regulatory pathway was still unknown. Whole transcriptome analysis (RNAseq) was employed to investigate the downstream regulation of sadB in an untargeted manner. RT-PCR and phenotypic assays were used to validate the data. SadB was found to be a global regulator controlling up to 50% of the genome. sadB was shown to negatively affect the pqs system and subsequently the rhl system, rhamnolipid and pyocyanin production and the denitrification pathway. The levels of the PQS signal, pyocyanin and rhamnolipids were also shown to be affected by sadB. The denitrification pathway downregulation in the absence of sadB was shown to affect growth under microaerophilic conditions. Furthermore, sadB positively affected biofilm related targets such as Psl and c-di-GMP production. Advances were made in the present study in discovering the downstream targets of sadB that explain some of the observed phenotypes, however the function of SadB is still unknown. A study from Muriel et al., reported that a sadB homolog in P. fluorescens binds c-di- GMP. The sadB homolog of P. aeruginosa was successfully purified and tested using SPR and thermal shift assay. The results showed no binding of c-di-GMP. Additionally, attempts were made to resolve the crystal structure of SadB. SadB crystals were obtained after screening commercial crystal screens and optimisation of the conditions but the resolution of the diffraction was not sufficient. Finally, SadB was found to localise in the cytoplasm using an anti-SadB antibody and Western blot and a SadB fluorescent fusion and super-resolution microscopy.

PROTEIN-O-MANNOSYLATION BY NON-SEC/TAT SECRETION TRANSLOCONS IN ACTINOBACTERIA

Preprint

Aug 2023

Protein- O -mannosylation (POM) is a form of O -glycosylation that is ubiquitous throughout all domains of life and has been extensively characterized in eukaryotic systems. However, in prokaryotes this process has only been investigated in terms of pathogenicity (in Mycobacterium tuberculosis ) even though there are many non-pathogenic bacteria that are known to regularly carry out POM. To date, there is no consensus on what benefit POM imparts to the non-pathogenic bacteria that can perform it. Though the generation of a POM deficient mutant of Corynebacterium glutamicum – a widely utilized and known mannosylating actinobacteria – this work shows that even closely related actinobacterial GT-39s (the enzymes responsible for the initiation of POM) can potentially have different activities and substrate specificities for targets of POM. Moreover, presented here is evidence that POM does not only occur in a SEC-dependent manner; POM also occurs with TAT and non-SEC secreted substrates in a specific and likely tightly regulated manner. Together these results highlight the need for further biochemical characterization of POM in these and other bacterial species to help elucidate the true nature of its biological functions. Importance Both the mechanism and overall cellular function of protein- O -mannosylation, a ubiquitous subset of O -glycosylation, is poorly understood in bacterial systems. In Mycobacterium tuberculosis and other pathogenic actinobacteria, numerous secreted virulence factors were identified as mannoproteins, with protein- O -mannosylation deficient mutants displaying a less virulent phenotype due to these proteins lacking the modification. However, these findings do not offer any explanations as to why non-pathogenic strains of actinobacteria also perform this modification as in these organisms it is seemingly dispensable. Corynebacterium glutamicum is a widely utilized, industrially relevant actinobacteria that also performs protein- O -mannosylation. This manuscript describes the utilization of C. glutamicum as a Gram-positive recombinant host for the in vivo study of actinobacterial protein- O -mannosylation and demonstrates the distinct lack of first-hand biochemical data of the process in prokaryotes.

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Article

Full-text available

Apr 2023

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

Structure of the E. coli protein-conducting channel bound to a translating ribosome

Article

Full-text available

Oct 2006

Projection structure and oligomeric properties of a bacterial core protein translocase

Article

Full-text available

May 2001
EMBO J

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 Å projection structure was calculated using electron cryo-microscopy. The structure exhibits P121 symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

X-ray structure of a protein conducting channel

Article

Jun 2005

Bert van den Berg

Chem. Rev.

Article

Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm

Article

May 2004
BBA-MOL CELL RES

Hitoshi Nakamoto

Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source.

Oxal/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (Review)

Article

Jan 2005

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.

SecA, an essential component of the secretory machinery of , exists as homodimer

Article

Jan 1991

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical cross-linking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.

Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in

Article

Nov 1990

SecA is an essential component of the protein secretory machinery of . SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structure. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.

Tyr326 plays a critical role in controlling SecA-preprotein interaction

Article

Sep 2000

SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267–340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function. secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro. The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function

Article

Mar 2001
EMBO J

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its -helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2–NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.

Papanikou, E., Karamanou, S. & Economou, A. Bacterial protein secretion through the translocase nanomachine. Nature Rev. Microbiol. 5, 839-851

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