Mechanism Of Membrane Insertion Research Articles

The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions to nucleate and organize spindle microtubules, and it is embedded in the nuclear envelope throughout the yeast life cycle. However, the mechanism of membrane insertion of the SPB has not been elucidated. Ndc1p is an integral membrane protein that localizes to SPBs, and it is required for insertion of the SPB into the nuclear envelope during SPB duplication. To better understand the function of Ndc1p, we performed a dosage suppressor screen using the ndc1-39 temperature-sensitive allele. We identified an essential SPB component, Nbp1p. NBP1 shows genetic interactions with several SPB genes in addition to NDC1, and two-hybrid analysis revealed that Nbp1p binds to Ndc1p. Furthermore, Nbp1p is in the Mps2p-Bbp1p complex in the SPB. Immunoelectron microscopy confirmed that Nbp1p localizes to the SPB, suggesting a function at this location. Consistent with this hypothesis, nbp1-td (a degron allele) cells fail in SPB duplication upon depletion of Nbp1p. Importantly, these cells exhibit a "dead" SPB phenotype, similar to cells mutant in MPS2, NDC1, or BBP1. These results demonstrate that Nbp1p is a SPB component that acts in SPB duplication at the point of SPB insertion into the nuclear envelope.

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Interfacial folding and membrane insertion of designed peptides studied by molecular dynamics simulations.

The mechanism of interfacial folding and membrane insertion of designed peptides is explored by using an implicit membrane generalized Born model and replica-exchange molecular dynamics. Folding/insertion simulations initiated from fully extended peptide conformations in the aqueous phase, at least 28 A away from the membrane interface, demonstrate a general mechanism for structure formation and insertion (when it occurs). The predominately hydrophobic peptides from the synthetic WALP and TMX series first become localized at the membrane-solvent interface where they form significant helical secondary structure via a helix-turn-helix motif that inserts the central hydrophobic residues into the membrane interior, and then fluctuations occur that provide a persistent helical structure throughout the peptide and it inserts with its N-terminal end moving across the membrane. More specifically, we observed that: (i) the WALP peptides (WALP16, WALP19, and WALP23) spontaneously insert in the membrane as just noted; (ii) TMX-1 also inserts spontaneously after a similar mechanism and forms a transmembrane helix with a population of approximately 50% at 300 K; and (iii) TMX-3 does not insert, but exists in a fluctuating membrane interface-bound form. These findings are in excellent agreement with available experimental data and demonstrate the potential for new implicit solvent/membrane models together with advanced simulation protocols to guide experimental programs in exploring the nature and mechanism of membrane-associated folding and insertion of biologically important peptides.

Bcl-2 family members: intracellular targeting, membrane-insertion, and changes in subcellular localization

The members of the Bcl-2 family of proteins are crucial regulators of apoptosis. In order to determine cell fate, these proteins must be targeted to distinct intracellular membranes, including the mitochondrial outer membrane (MOM), the membrane of the endoplasmic reticulum (ER) and its associated nuclear envelope. The targeting sequences and mechanisms that mediate the specificity of these proteins for a particular cellular membrane remain poorly defined. Several Bcl-2 family members have been reported to be tail-anchored via their predicted hydrophobic COOH-terminal transmembrane domains (TMDs). Tail-anchoring imposes a posttranslational mechanism of membrane insertion on the already folded protein, suggesting that the transient binding of cytosolic chaperone proteins to the hydrophobic TMD may be an important regulatory event in the targeting process. The TMD of certain family members is initially concealed and only becomes available for targeting and membrane insertion in response to apoptotic stimuli. These proteins either undergo a conformational change, posttranslational modification or a combination of these events enabling them to translocate to sites at which they are functional. Some Bcl-2 family members lack a TMD, but nevertheless localize to the MOM or the ER membrane during apoptosis where they execute their functions. In this review, we will focus on the intracellular targeting of Bcl-2 family members and the mechanisms by which they translocate to their sites of action. Furthermore, we will discuss the posttranslational modifications which regulate these events.

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.

Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis.

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

In Vitro Synthesis of Lactose Permease to Probe the Mechanism of Membrane Insertion and Folding

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.

Activation of Delta Opioid Receptors Induces Receptor Insertion and Neuropeptide Secretion

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca 2+ release from IP 3-sensitive stores and Ca 2+ entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y 1 receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca 2+-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

Secondary and Tertiary Structure Formation of the β-Barrel Membrane Protein OmpA is Synchronized and Depends on Membrane Thickness

The mechanism of membrane insertion and folding of a β-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded β-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of β-sheet secondary structure and closure of the β-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other β-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of α-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.

Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition.

In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition.

An Amino-terminal Amphipathic α-Helix Mediates Membrane Association of the Hepatitis C Virus Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein.

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.

Mechanism of Membrane Insertion of a Multimeric β-Barrel Protein: Perfringolysin O Creates a Pore Using Ordered and Coupled Conformational Changes

Perfringolysin O, a bacterial cytolytic toxin, forms unusually large pores in cholesterol-containing membranes by the spontaneous insertion of two of its four domains into the bilayer. By monitoring the kinetics of domain-specific conformational changes and pore formation using fluorescence spectroscopy, the temporal sequence of domain-membrane interactions has been established. One membrane-exposed domain does not penetrate deeply into the bilayer and is not part of the actual pore, but is responsible for membrane recognition. This domain must bind to the membrane before insertion of the other domain into the bilayer is initiated. The two domains are conformationally coupled, even though they are spatially separated. Thus, cytolytic pore formation is accomplished by a novel mechanism of ordered conformational changes and interdomain communication.

The membrane insertion of trichosanthin is membrane-surface-pH dependent.

Trichosanthin (TCS) is the active component extracted from Tianhuafen, a traditional herbal medicine that has been used for abortion in China for centuries. It belongs to the type-I ribosome-inactivating protein (RIP) family and can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. Recent studies have shown TCS to be multifunctional, its pharmacological properties including immunomodulatory, anti-tumour and anti-HIV activities. The membrane-insertion property of TCS is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In this paper, the membrane-insertion mechanism of TCS was studied. The monolayer experiment revealed that TCS's membrane-insertion ability was dependent on low pH. Fluorescence spectroscopy using 1-anilinonaphthalene-8-sulphonic acid as a probe showed that low pH may induce the conformational change of TCS that leads to the hydrophobic-site exposure, and the CD result showed that this conformational change did not alter its secondary structure. Such conformational change leads to an intermediate state, called the 'molten globular state' by previous investigators. The pH-dependent membrane insertion and conformational change were related by the fact that the optimal membrane-surface pH needed was the same for the two events. From these and other results, a membrane-insertion model was proposed.

Thiol-activated cytolysins: structure, function and role in pathogenesis

Members of the thiol-activated family of cytolysins are involved in the mechanism of pathogenesis of a number of Gram-positive species. While they are pore-forming toxins, their major pathogenic effects may be more subtle than simple lysis of host cells, and may include interference with immune cell function and cytokine induction. Crystal structure, electron microscopy, mutagenesis and antibody binding studies have led to the modeling of a novel mechanism of pore formation, encompassing membrane-binding, membrane insertion and oligomerization. Despite their designation as thiol-activated cytolysins, it is now clear that thiol activation is not an important property of this group of toxins.

Thiol-activated cytolysins: structure, function and role in pathogenesis

Members of the thiol-activated family of cytolysins are involved in the mechanism of pathogenesis of a number of Gram-positive species. While they are pore-forming toxins, their major pathogenic effects may be more subtle than simple lysis of host cells, and may include interference with immune cell function and cytokine induction. Crystal structure, electron microscopy, mutagenesis and antibody binding studies have led to the modeling of a novel mechanism of pore formation, encompassing membrane-binding, membrane insertion and oligomerization. Despite their designation as thiol-activated cytolysins, it is now clear that thiol activation is not an important property of this group of toxins.

The Mechanism of Membrane Insertion for a Cholesterol-Dependent Cytolysin: A Novel Paradigm for Pore-Forming Toxins

Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic β hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic β hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six α helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin.

Cultured glial cells express the SNAP-25 analogue SNAP-23

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP-25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP-23, an analogue of SNAP-25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP-23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t-SNAREs SNAP-23 and syntaxin 1 and the v-SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis.

Phylogenetic transfer of organelle genes to the nucleus can lead to new mechanisms of protein integration into membranes.

Subunits CFo-I and CFo-II of ATP synthase in chloroplast thylakoid membranes are two structurally and functionally closely related proteins of bitopic membrane topology which evolved from a common ancestral gene. In higher plants, CFo-I still originates in plastid chromosomes (gene: atpF), while the gene for CFo-II (atpG) was phylogenetically transferred to the nucleus. This gene transfer was accompanied by the reorganization of the topogenic signals and the mechanism of membrane insertion. CFo-I is capable of integrating correctly as the mature protein into the thylakoid membrane, whereas membrane insertion of CFo-II strictly depends on a hydrophobic targeting signal in the transit peptide. This requirement is caused by three negatively charged residues at the N-terminus of mature CFo-II which are lacking from CFo-I and which have apparently been added to the protein only after gene transfer has occurred. Accordingly, the CFo-II transit peptide is structurally and functionally equivalent to typical bipartite transit peptides, capable of also translocating hydrophilic lumenal proteins across the thylakoid membrane. In this case, transport takes place by the Sec-dependent pathway, despite the fact that membrane integration of CFo-II is a Sec-independent, and presumably spontaneous, process.

The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis delta-endotoxin are consistent with an "umbrella-like" structure of the pore.

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis delta-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices alpha4 and alpha5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the "umbrella model"). Our results also support the suggestion that alpha7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.