The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.     

Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.     

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.     

Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.     

Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer

Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.     

Translocation of N-terminal tails across the plasma membrane.

Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli. We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions. We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted. Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein. We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region. Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.     

Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins

Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of α-helical translocases.     

A conserved aromatic residue in the autochaperone domain of the autotransporter Hbp is critical for initiation of outer membrane translocation

Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal β-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The mechanism of passenger translocation and folding is still unclear but involves a conserved region at the C terminus of the passenger domain, the so-called autochaperone domain. This domain functions in the stepwise translocation process and in the folding of the passenger domain after translocation. In the autotransporter hemoglobin protease (Hbp), the autochaperone domain consists of the last rung of the β-helix and a capping domain. To examine the role of this region, we have mutated several conserved aromatic residues that are oriented toward the core of the β-helix. We found that non-conservative mutations affected secretion with Trp(1015) in the cap region as the most critical residue. Substitution at this position yielded a DegP-sensitive intermediate that is located at the periplasmic side of the OM. Further analysis revealed that Trp(1015) is most likely required for initiation of processive folding of the β-helix at the cell surface, which drives sequential translocation of the Hbp passenger across the OM.     

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.     

Tryptophan-dependent sensitized photoinactivation of colicin E1 channels in bilayer lipid membranes

The bacterial toxin colicin E1 is known to induce voltage-gated currents across a planar bilayer lipid membrane. In the present study, it is shown that the colicin-induced current decreased substantially upon illumination of the membrane in the presence of the photosensitizer, aluminum phthalocyanine. This effect was almost completely abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan mutants of colicin E1, Trp495 was identified as the amino acid residue responsible for the sensitized photodamage of the colicin channel activity. Thus, the distinct participation of a specific amino acid residue in the sensitized photoinactivation of a defined protein function was demonstrated. It is suggested that Trp495 is critical for the translocation and/or anchoring of the colicin channel domain in the membrane.     

Membrane association and activity of 15/16-membered peptide antibiotics: zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K(+) transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER>AMP>ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC(16:1)PC, diC(18:1)PC, C(16:0)/C(18:1)PC, diC(20:1)PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Endosomes and toxin translocation

In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells. We studied the translocation process of these toxins using endosomes purified from lymphocytes. This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell. We could show that each of these toxins uses a different strategy to cross the endosome membrane. Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis. DT translocation is directly triggered and energized by the endosome-cytosol pH gradient. Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation. A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.     

Expression level of Sec62 modulates membrane insertion of marginally hydrophobic segments

In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62 interacts with the Sec61 channel and is located on the front side of the Sec61 lateral gate, an entry site for TM domains to the lipid bilayer. Overexpression of Sec62 led to a growth defect in yeast, and we investigated its effects on protein translocation and membrane insertion by pulse labeling of Sec62 client proteins. Our data show that the insertion efficiency of marginally hydrophobic TM segments is reduced upon Sec62 overexpression. This result suggests a potential regulatory role of Sec62 as a gatekeeper of the lateral gate, thereby modulating the insertion threshold of TM segments.     

Membrane interaction and cellular internalization of penetratin peptides.

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.     

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.     

Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.     

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion.     

Chicken liver bile acid-binding protein is in a compact partly folded state at acidic pH. Its relevance to the interaction with lipid membranes

Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B., and Montich, G. (2003) Biochim. Biophys. Acta 1611, 98-106]. To understand the mechanisms of its interactions with membranes, we have investigated the presence of partly folded states in solution. Using fluorescence spectroscopy of the single Trp residue, circular dichroism in the far- and near-UV, Fourier transform infrared spectroscopy, and size-exclusion chromatography, we found that L-BABP was partly unfolded at pH 2.5 and low ionic strength, retaining some of its secondary structure. Addition of 0.1 M NaCl at pH 2.5 or decreasing the pH to 1.5 produced a more compact partly folded state, with a partial increase of secondary structure and none of tertiary structure. Fluorescence emission spectra of this state indicate that the Trp residue is within an environment of low polarity, similar to the native state. This environment is not produced by the insertion of the Trp into soluble aggregates as revealed by size-exclusion chromatography, fluorescence anisotropy, and infrared spectroscopy. The presence of partly folded states under acidic conditions in solution suggests the possibility that membrane binding of L-BABP occurs via this state.