Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End?

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.     

Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia

Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed to specific recognition of the A chain by diphtheria toxin's translocation pathway because the translocation pathway also accommodates folded myoglobin.     

Major transmembrane movement associated with colicin Ia channel gating

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin- containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

Structure and dynamics of the colicin E1 channel

The toxin-like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage-gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel-forming domain into the inner membrane involves a translocation-competent intermediate state and a membrane potential-dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage-gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site-directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly alpha-helix, have been determined by physico-chemical techniques. Hypothesis for the identity of the trans-membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

Membrane protein spanning segments as export signals.

Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and membrane-spanning segments adjacent to each of the periplasmic domains acted as export signals for an attached sensor protein (alkaline phosphatase). However, the export activity of one of the spanning segments was considerably lower than that of the others, and was limited by the presence of a positively charged residue (Arg302). These observations are compatible with models of membrane protein insertion in which hydrophilic domains are translocated independently. However, the results suggest that efficient translocation may sometimes require interaction between individual spanning segments.     

A carboxy-terminal fragment of colicin Ia forms ion channels

A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers. This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein. The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec. In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min. Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states. Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.     

Membrane topography of ColE1 gene products: the immunity protein.

The topography of the colicin E1 immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin E1 Imm protein folds in the membrane as three trans-membrane alpha-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to Ser-28, Ile-43 to Phe-62, and Leu-84 to Leu-104. An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin E1 Imm protein for interaction with the translocation apparatus and the colicin E1 ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight-residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side.     

Requirements for translocation of periplasmic domains in polytopic membrane proteins.

Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.     

In vitro depolarization of Escherichia coli membrane vesicles by colicin Ia.

Conditions are reported under which membrane vesicles prepared from Escherichia coli K12 are depolarized by colicin Ia. Although incubation of membrane vesicles with active colicin Ia affects neither transport activity nor the ability of such vesicles to generate a deltapH or deltapsi, a single freeze-thaw cycle of such vesicles in the presence of colicin Ia leads to 1) retention of the colicin by the vesicles, 2) inactivation of transport activity, and 3) membrane depolarization, with a concomitant increase in the transmembrane deltapH. These effects are dependent upon the presence of active colicin Ia during the freeze-thaw cycle. These findings are consistent with our previous results showing that Ia-treated whole cells or membrane vesicles prepared from such cells are defective in their ability to generate a deltapsi, yet generate an increased deltapH (Tokuda, H., and Konisky, J. (1978) Proc. Natl. Acad. Sci. U. S. A., 75, 2579--2583). In addition to its effect on vesicles prepared from sensitive cells, we show that vesicles prepared from both colicin Ia-resistant and -tolerant cells are depolarized by colicin treatment with a concomitant increase in deltapH. It is concluded that the final target of colicin Ia is the cytoplasmic membrane. A model for the mechanism of colicin Ia action is presented in which colicin Ia binds to the specific colicin Ia outer membrane receptor and is subsequently translocated to the cytoplasmic membrane where its integration leads to the formation of ion channels.     

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane

A stop transfer sequence derived from the extreme carboxyl terminus of membrane IgM heavy chain has been shown to confer predictable transmembrane orientation to secretory proteins by aborting translocation of subsequently synthesized protein domains. Here we demonstrate that, in certain peptide sequence contexts, the same stop transfer sequence is also capable of initiating domain translocation across the endoplasmic reticulum (ER) membrane. Translocation directed by a stop transfer sequence is similar to, but distinguishable from, the action of a conventional signal sequence. Translocation is dependent on participation of the ribosome and protein receptors both in the cytoplasm and in the ER membrane. Moreover, both amino- and carboxy-terminal flanking protein domains can be translocated. Unlike a signal sequence, the stop transfer sequence is not itself translocated across the membrane. These results have implications for the nature of signal sequences, stop transfer sequences, and their receptor interactions.     

Topography of diphtheria Toxin's T domain in the open channel state

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a "double dagger" model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed "daggers," TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of approximately 70 residues, is translocated across the membrane to the trans side.     

The Diphtheria Toxin Channel-forming T Domain Translocates Its Own NH2-Terminal Region Across Planar Bilayers

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH₂ terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH₂-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH₂ terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH₂-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH₂ terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH₂ terminus be translocated across the membrane from the cis to the trans side, even though the NH₂ terminus is >100 residues removed from the channel-forming part of the molecule.     

Topological characterization of the essential Escherichia coli cell division protein FtsN.

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.