Binding,activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase

The bacterial preprotein translocase is comprised of a membrane-embedded oligomeric SecYEG structure and a cytosolic dimeric SecA ATPase. The associations within SecYEG oligomers and SecA dimers, as well as between these two domains are dynamic and reversible. Here, it is shown that a covalently linked SecYEG dimer forms a functional translocase and a high affinity binding site for monomeric and dimeric SecA in solution. The interaction between these two domains stimulates the SecA ATPase, and nucleotides modulate the affinity and ratio of SecA monomers and dimers bound to the linked SecYEG complex. During the translocation reaction, the SecA monomer remains in stable association with a SecYEG protomer and the translocating preprotein. The nucleotides and translocation-dependent changes of SecA-SecYEG associations and the SecA dimeric state may reflect important facets of the preprotein translocation reaction.     

Determination of the Oligomeric State of SecYEG Protein Secretion Channel Complex Using in Vivo Photo- and Disulfide Cross-linking

SecYEG protein of bacteria or Sec61αβγ of eukaryotes is a universally conserved heterotrimeric protein channel complex that accommodates the partitioning of membrane proteins into the lipid bilayer as well as the secretion of proteins to the trans side of the plasma or endoplasmic reticular membrane, respectively. SecYEG function is facilitated by cytosolic partners, mainly a nascent chain-ribosome complex or the SecA ATPase motor protein. Extensive efforts utilizing both biochemical and biophysical approaches have been made to determine whether SecYEG functions as a monomer or a dimer, but such approaches have often generated conflicting results. Here we have employed site-specific in vivo photo-cross-linking or cysteine cross-linking, along with co-immunoprecipitation or SecA footprinting techniques to readdress this issue. Our findings show that the SecY dimer to monomer ratio is relatively constant regardless of whether translocons are actively engaged with protein substrate or not. Under the former conditions the SecY dimer can be captured associated with a translocon-jammed substrate, indicative of SecY dimer function. Furthermore, SecA ATPase can be cross-linked to two copies of SecY when the complex contains a translocation intermediate. Collectively, our results suggest that SecYEG dimers are functional units of the translocon.     

SecA functions in vivo as a discrete anti‐parallel dimer to promote protein transport

SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer–dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ‐benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X‐ray structures and assessing their in vivo photo‐crosslinking pattern. A discrete anti‐parallel 1M6N‐like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG‐bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer‐driven translocation mechanism.     

Quaternary structure of SecA in solution and bound to SecYEG probed at the single molecule level

Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.     

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein‐conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross‐) correlation spectroscopy with single‐molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.     

SecA dimer cross-linked at its subunit interface is functional for protein translocation

SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.     

Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

Monitoring the Activity of Single Translocons

Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation.     

Using a low denaturant model to explore the conformational features of translocation-active SecA

The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.     

Covalently dimerized SecA is functional in protein translocation

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.     

The oligomeric state and arrangement of the active bacterial translocon

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.     

Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

The bacterial protein-translocation complex: SecYEG dimers associate with one or two SecA molecules

In bacteria, the Sec-protein transport complex facilitates the passage of most secretory and membrane proteins across and into the plasma membrane. The core complex SecYEG forms the protein channel and engages either ribosomes or the ATPase SecA, which drive translocation of unfolded polypeptide chains through the complex and into the periplasmic space. Escherichia coli SecYEG forms dimers in membranes, but in detergent solution the population of these dimers is low. However, we find that stable dimers can be assembled by the addition of a monoclonal antibody. Normally, a stable SecYEG-SecA complex can only form on isolated membranes or on reconstituted proteo-liposomes. The antibody-stabilised SecYEG dimer binds one SecA molecule in detergent solution. In the presence of AMPPNP, a non-hydrolysable analogue of ATP, a complex forms containing one antibody and two each of SecYEG and SecA. SecYEG monomers or tetramers do not associate to a significant degree with SecA. The observed variability in the stoichiometry of SecYEG and SecA association and its nucleotide modulation may be important and necessary for the protein translocation reaction.     

Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane

The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.     

Escherichia coli translocase: the unravelling of a molecular machine

Protein translocation across the bacterial cytoplasmic membrane has been studied extensively in Escherichia coli. The identification of the components involved and subsequent reconstitution of the purified translocation reaction have defined the minimal constituents that allowed extensive biochemical characterization of the so-called translocase. This functional enzyme complex consists of the SecYEG integral membrane protein complex and a peripherally bound ATPase, SecA. Under translocation conditions, four SecYEG heterotrimers assemble into one large protein complex, forming a putative protein-conducting channel. This tetrameric arrangement of SecYEG complexes and the highly dynamic SecA dimer together form a proton-motive force- and ATP-driven molecular machine that drives the stepwise translocation of targeted polypeptides across the cytoplasmic membrane. Recent findings concerning the translocase structure and mechanism of protein translocation are discussed and shine new light on controversies in the field.