Stereochemical Analysis of Interaction of Signal Peptide with Phospholipids at the Initiation of Protein Translocation Across the Membrane

Abstract

Stereochemical analysis of signal peptide interaction with E. coli membrane phospholipids revealed the structural complementarity of N-terminus of signal peptide α-helix and acid phospholipids. The formation of their complex leads to neutralization of charges and decrease in hydrophilicity of both components, and promotes insertion of peptide and phospholipid into the membrane, not separately but as a complex. Interaction of acid phospholipids with the E. coli alkaline phosphatase (AP) signal peptide was thoroughly analyzed, and it was shown that in this case a complex of signal peptide α-helix with phosphatidylglycerol is inserted into the membrane with the lowest energy expense. On the basis of the results of stereochemical analysis and the available experimental data, a molecular mechanism of protein translocation initiation across the membrane has been proposed, in which the key events are the formation of the complex “signal peptide α-helix-acid phospholipid”, the coupled insertion of hydrophobic peptide-lipid complex into a nonpolar membrane interior and translocation across the membranes.     

Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli

The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments. The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide. PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix. The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane. We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet. We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A. Although expression of the E. coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis. EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane. A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo. Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro. These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet.     

Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli

The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.     

Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(–20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.     

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.     

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.     

Purification and characterization of a signal peptide, a product of protein secretion across the cytoplasmic membrane of Escherichia coli

A signal peptide, a processing product of the precursor of the lipoprotein in the cytoplasmic membrane of Escherichia coli, has been purified through extractions with butanol and ethyl ether and chromatographies with a Sephadex LH-60 column and Sep-pak C18. Analysis of the amino acid composition and sequencing of the N- and C-termini indicate that the signal peptide was intact, suggesting that the first step of the signal peptide catabolism in the cytoplasmic membrane is the cleavage of the intact signal peptide. During the purification, the signal peptide exhibited unique features, including strong interaction with phospholipids. The possible importance of such features in the process of protein translocation across membranes is discussed.     

Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane

The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane alpha-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500-10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane.     

Disruption of Escherichia coli outer membranes by EM 49. A new membrane active peptide.

A new peptide antibiotic, EM 49, is shown to disrupt the structure of Escherichia coli outer membranes and release outer membrane fragments into the surrounding media. Evidence supporting this conclusion indludes EM 49 stimulated release of outer membrane phospholipids, lipopolysaccharide, and membrane fragments having a phospholipid and polypeptide composition similar to outer membranes. The density of the membrane fragments released by EM 49 was 1.22 g/cm3, which was identical to isolated outer membranes. Approximately 10 to 15% of the E. coli lipopolysaccharide was released upon treatment with EM 49. Both scanning and transmission electron microscopy revealed that the antibiotic caused the formation of numerous protrusions or blebs on the surface of E. coli with apparent release of membrane vesicles from the cells. Direct interaction between EM 49 and outer membranes was demonstrated using outer membranes labeled with the fluorescent dye diphenylhexatriene. Treatment of the fluorescent-labeled outer membranes with EM 49 increased fluorescence intensity and decreased polarization, indicating that the peptide perturbed outer-membrane structure. In addition, strong interactions between EM 49 and purified E. coli phospholipids were detected using the Hummel and Dreyer technique. Association constants between the peptide and phospholipids were approximately 10(5) M-1. A model for the disruptive effect of EM 49 on outer-membrane structure is proposed in which the fatty acid chain of the antibiotic is inserted into the hydrophobic core of the membrane. This orientation would allow the polycationic, peptide portion of the antibiotic to disrupt the antibiotic to disrupt the normal electrostatic interactions between divalent cations and components of the outer membrane. Evidence supporting this conclusion includes specific protection of E. coli from EM 49 by Mg2+ and Ca2+ and inhibition of EM 49 stimulated phospholipid release by these cations. Disruption of the antibiotic to penetrate to the inner membrane, which is probably the primary killing site of EM 49.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

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.     

Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.     

Destabilization of the colicin E9 Endonuclease domain by interaction with negatively charged phospholipids: implications for colicin translocation into bacteria

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lema?tre, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (lambda(max)) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in lambda(max), from 354 to 342 nm. Changes in lambda(max) were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.     

Membrane protein insertion of variant MscL proteins occurs at YidC and SecYEG of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously.     

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.     

Requirement for phospholipids of the translocation of the trimethylamine N-oxide reductase through the Tat pathway in Escherichia coli

Trimethylamine N-oxide reductase (TorA) is an anaerobically synthesized molybdoenzyme. It is translocated across the cytoplasmic membrane in a folded conformation via the Tat pathway of Escherichia coli. The requirement for phospholipids for the export of this enzyme was analyzed in the pgsA and pss mutants lacking anionic phospholipids and phosphatidylethanolamine, respectively. Anaerobic growth did not influence phospholipid composition of the pgsA and pss mutants. Interestingly, both pgsA and pss mutations severely retarded the translocation of TorA into the periplasm. Therefore, translocation of proteins through the Tat pathway is dependent on the anionic phospholipids and on lipid polymorphism.     

The phase property of membrane phospholipids is affected by the functionality of signal peptides from the Escherichia coli ribose-binding protein

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wild-type and revertant signal peptides predominantly formed alpha-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the alpha-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.     

Interactions of ovalbumin and of its putative signal sequence with phospholipid monolayers. Possible importance of differing lateral stabilities in protein translocation.

Surface properties of ovalbumin and of its putative signal sequence, and their interactions with phospholipids at an air-water interface, have been studied. The mature protein can form an interfacial film spontaneously from its bulk solution, whereas the signal sequence cannot. Mature ovalbumin also penetrates phospholipid monolayers from the subphase (independently of the type of phospholipid present), whereas its signal sequence does not. The surface stability of a spread film of the signal sequence is, however, higher than that of a film of mature ovalbumin. Above specific threshold concentrations of signal peptide and of mature ovalbumin in mixed films with phospholipids, two separate phases are formed. In such immiscible films, the signal sequence peptide is also able to support a higher lateral surface pressure than mature ovalbumin, at corresponding areas of peptide and mature protein in the mixed monolayers. It is suggested that the differing lateral stabilities of ovalbumin and of its putative signal sequence may be relevant to the translocation of ovalbumin across the membrane of the endoplasmic reticulum, and a scheme for its translocation is proposed that is based on these properties.