The SecY complex forms a channel capable of ionic discrimination

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring‐structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion‐conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring‐structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.     

Determining the conductance of the SecY protein translocation channel for small molecules

The channel formed by the SecY complex must maintain the membrane barrier for ions and other small molecules during the translocation of membrane or secretory proteins. We have tested the permeability of the channel by using planar bilayers containing reconstituted purified E. coli SecY complex. Wild-type SecY complex did not show any conductance for ions or water. Deletion of the "plug," a short helix normally located in the center of the SecY complex, or modification of a cysteine introduced into the plug resulted in transient channel openings; a similar effect was seen with a mutation in the pore ring, a constriction in the center of the channel. Permanent channel opening occurred when the plug was moved out of the way by disulfide-bridge formation. These data show that the resting channel on its own forms a barrier for small molecules, with both the pore ring and the plug required for the seal; channel opening requires movement of the plug.     

Bacterial protein translocation requires only one copy of the SecY complex in vivo

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.     

Structural insight into the protein translocation channel

A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time.     

Structural characterization of two pore‐forming peptides: Consequences of introducing a C‐terminal tryptophan

Synthetic channel‐forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22‐residue peptides derived from the second transmembrane segment of the glycine receptor α₁‐subunit (M2GlyR), p22‐S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one‐half maximal short circuit current is reduced to 45 ± 6 μM from that of 210 ± 70 μM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22‐S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane‐anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22‐S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C‐terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR‐derived peptides toward potential peptide‐based channel replacement therapy. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The TpsB translocator HMW1B of haemophilus influenzae forms a large conductance channel

The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane beta-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.     

Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.     

Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation

While engaged in protein transport, the bacterial translocon SecYEG must maintain the membrane barrier to small ions. The preservation of the proton motif force was attributed to (i) cation exclusion, (ii) engulfment of the nascent chain by the hydrophobic pore ring, and (iii) a half-helix partly plugging the channel. In contrast, we show here that preservation of the proton motif force is due to a voltage-driven conformational change. Preprotein or signal peptide binding to the purified and reconstituted SecYEG results in large cation and anion conductivities only when the membrane potential is small. Physiological values of membrane potential close the activated channel. This voltage-dependent closure is not dependent on the presence of the plug domain and is not affected by mutation of 3 of the 6 constriction residues to glycines. Cellular ion homeostasis is not challenged by the small remaining leak conductance.     

Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel

Many proteins are translocated across the bacterial plasma membrane by the interplay of the cytoplasmic ATPase SecA with a protein-conducting channel, formed from the evolutionarily conserved heterotrimeric SecY complex. Here, we have used purified E. coli components to address the mechanism of translocation. Disulfide bridge crosslinking demonstrates that SecA transfers both the signal sequence and the mature region of a secreted substrate into a single SecY molecule. However, protein translocation involves oligomers of the SecY complex, because a SecY molecule defective in translocation can be rescued by linking it covalently with a wild-type SecY copy. SecA interacts through one of its domains with a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy. Oligomeric channels with only one active pore likely mediate protein translocation in all organisms.     

Brownian dynamics investigation into the conductance state of the MscS channel crystal structure

We suggest that the crystal structure of the mechanosensitive channel of small conductance is in a minimally conductive state rather than being fully activated. Performing Brownian dynamics simulations on the crystal structure show that no ions pass through it. When simulations are conducted on just the transmembrane domain (excluding the cytoplasmic residues 128 to 280) ions are seen to pass through the channel, but the conductance of ∼ 30 pS is well below experimentally measured values. The mutation L109S that replaces a pore lining hydrophobic residue with a polar one is found to have little effect on the conductance of the channel. Widening the hydrophobic region of the pore by 2.5 Å however, increases the channel conductance to over 200 pS suggesting that only a minimal conformational change is required to gate the pore.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Study of ionic currents across a model membrane channel using Brownian dynamics.

Brownian dynamics simulations have been carried out to study ionic currents flowing across a model membrane channel under various conditions. The model channel we use has a cylindrical transmembrane segment that is joined to a catenary vestibule at each side. Two cylindrical reservoirs connected to the channel contain a fixed number of sodium and chloride ions. Under a driving force of 100 mV, the channel is virtually impermeable to sodium ions, owing to the repulsive dielectric force presented to ions by the vestibular wall. When two rings of dipoles, with their negative poles facing the pore lumen, are placed just above and below the constricted channel segment, sodium ions cross the channel. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength does not increase the channel conductance further. When only those ions that acquire a kinetic energy large enough to surmount a barrier are allowed to enter the narrow transmembrane segment, the channel conductance decreases monotonically with the barrier height. This barrier represents those interactions between an ion, water molecules, and the protein wall in the transmembrane segment that are not treated explicitly in the simulation. The conductance obtained from simulations closely matches that obtained from ACh channels when a step potential barrier of 2-3 kTr is placed at the channel neck. The current-voltage relationship obtained with symmetrical solutions is ohmic in the absence of a barrier. The current-voltage curve becomes nonlinear when the 3 kTr barrier is in place. With asymmetrical solutions, the relationship approximates the Goldman equation, with the reversal potential close to that predicted by the Nernst equation. The conductance first increases linearly with concentration and then begins to rise at a slower rate with higher ionic concentration. We discuss the implications of these findings for the transport of ions across the membrane and the structure of ion channels.     

On the relationship between anion binding and chloride conductance in the CFTR anion channel

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore – as well as interactions between concurrently bound chloride ions – are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.     

Size, motion, and function of the SecY translocon revealed by molecular dynamics simulations with virtual probes

We report a hybrid, coarse-grained and atomistic, molecular dynamics simulation study of the size, motion, and function of the SecY protein-conducting channel. Growing and pushing virtual soft ball constructs through the pore of SecY, we mimic the push-through of polypeptides, performed cotranslationally by the ribosome and posttranslationally by the SecA ATPase. Forced lateral opening of a "front gate" between transmembrane helices is also induced by the passage of the virtual probes, with implications for the membrane insertion of peptides. We conclude that the SecY channel can stretch to allow passage of peptides with transversal sizes of approximately 16 A. The observed motion of a transmembrane helical "plug" controlling the closed and open states of the channel is consistent with experimental results and confirms previous hypotheses. Additionally, the "hinge" region for front gate opening is observed to be highly mobile as postulated. Both the forced dilation of a "ring" of residues at the middle of the pore and the lateral opening of the front gate are shown to induce plug displacement, but neither accomplish a full-extent motion of the plug to the back of the channel. For probes whose passage does not destroy the resilience of the SecY, both lateral exit and full translocation are observed, despite the fact that applied forces were always in the direction along the pore axis. Lateral exit is accompanied by front gate opening and slight plug displacement, whereas full translocation is accompanied by large plug displacement but no apparent lateral opening. Simulations also reveal that dilating the pore ring is a more effective way to destabilize the plug than intercalation of a cylinder-like probe at the front gate. Based on the simulations, the existence of a family of diverse open states is proposed.     

Chloroplast SecY is complexed to SecE and involved in the translocation of the 33-kDa but not the 23-kDa subunit of the oxygen-evolving complex

SecY is a component of the protein-conducting channel for protein transport across the cytoplasmic membrane of prokaryotes. It is intimately associated with a second integral membrane protein, SecE, and together with SecA forms the minimal core of the preprotein translocase. A chloroplast homologue of SecY (cpSecY) has previously been identified and determined to be localized to the thylakoid membrane. In the present work, we demonstrate that a SecE homologue is localized to the thylakoid membrane, where it forms a complex with cpSecY. Digitonin solubilization of thylakoid membranes releases the SecY/E complex in a 180-kDa form, indicating that other components are present and/or the complex is a higher order oligomer of the cpSecY/E dimer. To test whether cpSecY forms the protein-conducting channel of the thylakoid membrane, translocation assays were conducted with the SecA-dependent substrate OE33 and the SecA-independent substrate OE23, in the presence and absence of antibodies raised against cpSecY. The antibodies inhibited translocation of OE33 but not OE23, indicating that cpSecY comprises the protein-conducting channel used in the SecA-dependent pathway, whereas a distinct protein conducting channel is used to translocate OE23.     

The twisting elevator mechanism of glutamate transporters reveals the structural basis for the dual transport-channel functions

Glutamate transporters facilitate the removal of this excitatory neurotransmitter from the synapse. Increasing evidence indicates that this process is linked to intrinsic chloride channel activity that is thermodynamically uncoupled from substrate transport. A recent cryo-EM structure of Glt_Ph – an archaeal homolog of the glutamate transporters – in an open channel state has shed light on the structural basis for channel opening formed at the interface of two domains within the transporter which is gated by two clusters of hydrophobic residues. These transporters cycle through several conformational states during the transport process, including the chloride conducting state, which appears to be stabilised by protein–membrane interactions and membrane deformation. Several point mutations that perturb the chloride conductance can have detrimental effects and are linked to the pathogenesis of the neurological disorder, episodic ataxia type 6.     

The Ca-activated chloride channel of Ascaris suum conducts volatile fatty acids produced by anaerobic respiration: A patch-clamp study

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of carboxylic acids, a number of which are products of anaerobic respiration, was investigated using the patch-clamp technique and isolated inside-out patches of muscle membrane. The channel has a conductance of 140 pS in symmetrical 140 mm chloride. Replacement of internal chloride with various carboxylic acids (140 mm) caused large hyperpolarizing shifts in the reversal potential. Permeability ratios, relative to chloride, were calculated for each acid. The relationship between permeability ratio and ionic size is inverse and linear predicting a pore diameter of 6.55 Å. This simple relationship was not observed between ionic size and conductance. Calculation of the transition state energy required to transfer a single methyl group from aqueous phase to the binding site afforded a value that was low but favorable, indicating a cationic binding site of low field strength. As the channel is able to open fully at the resting membrane potential of Ascaris and is permeable to fatty acids produced by anaerobic respiration, the possible role of this channel in the removal of metabolic products across the muscle membrane is discussed.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.). M. Valkanov was sponsored by The British Council.