Intraluminal Vesicle Research Articles

Biomolecular condensates mediate bending and scission of endosome membranes

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins1–3. The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles4,5. Here we report that the plant ESCRT component FREE16 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization.

Interaction between ESCRT-III proteins and the yeast SERINC homolog Tms1.

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes.

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

Clathrin assemblies at a glance.

Clathrin assembles into honeycomb-like lattices at the plasma membrane but also on internal membranes, such as at the Golgi and tubular endosomes. Clathrin assemblies primarily regulate the intracellular trafficking of different cargoes, but clathrin also has non-endocytic functions in cell adhesion through interactions with specific integrins, contributes to intraluminal vesicle formation by forming flat bilayered coats on endosomes and even assembles on kinetochore k-fibers during mitosis. In this Cell Science at a Glance article and the accompanying poster, we review our current knowledge on the different types of canonical and non-canonical membrane-associated clathrin assemblies in mammalian cells, as observed by thin-section or platinum replica electron microscopy in various cell types, and discuss how the structural plasticity of clathrin contributes to its functional diversity.

Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication

BackgroundSmall extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored.MethodsVacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months.ResultsVMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation.ConclusionsOur study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome.

Mitochondrial DNA (mtDNA) leakage into the cytoplasm can occur when cells are exposed to noxious stimuli. Specific sensors recognize cytoplasmic mtDNA to promote cytokine production. Cytoplasmic mtDNA can also be secreted extracellularly, leading to sterile inflammation. However, the mode of secretion of mtDNA out of cells upon noxious stimuli and its relevance to human disease remain unclear. Here, we show that pyroptotic cells secrete mtDNA encapsulated within exosomes. Activation of caspase-1 leads to mtDNA leakage from the mitochondria into the cytoplasm via gasdermin-D. Caspase-1 also induces intraluminal membrane vesicle formation, allowing for cellular mtDNA to be taken up and secreted as exosomes. Encapsulation of mtDNA within exosomes promotes a strong inflammatory response that is ameliorated upon exosome biosynthesis inhibition in vivo. We further show that monocytes derived from patients with Behçet's syndrome (BS), a chronic systemic inflammatory disorder, show enhanced caspase-1 activation, leading to exosome-mediated mtDNA secretion and similar inflammation pathology as seen in BS patients. Collectively, our findings support that mtDNA-containing exosomes promote inflammation, providing new insights into the propagation and exacerbation of inflammation in human inflammatory diseases.

The plant-unique protein DRIF1 coordinates with sorting nexin 1 to regulate membrane protein homeostasis.

Membrane protein homeostasis is fine-tuned by the cellular pathways for vacuolar degradation and recycling, which ultimately facilitate plant growth and cell-environment interactions. The endosomal sorting complex required for transport (ESCRT) machinery plays important roles in regulating intraluminal vesicle (ILV) formation and membrane protein sorting to vacuoles. We previously showed that the plant-specific ESCRT component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) performs multiple functions in plants, although the underlying mechanisms remain elusive. In this study, we performed a suppressor screen of the FREE1-RNAi mutant and identified and characterized 2 suppressor of free1 (sof) mutants in Arabidopsis (Arabidopsis thaliana). These mutants, sof10 and sof641, result in a premature stop codon or a missense mutation in AT5G10370, respectively. This gene was named DEAH and RING domain-containing protein as FREE1 suppressor 1 (DRIF1). DRIF1 has a homologous gene, DRIF2, in the Arabidopsis genome with 95% identity to DRIF1. The embryos of drif1 drif2 mutants arrested at the globular stage and formed enlarged multivesicular bodies (MVBs) with an increased number of ILVs. DRIF1 is a membrane-associated protein that coordinates with retromer component sorting nexin 1 to regulate PIN-FORMED2 recycling to the plasma membrane. Altogether, our data demonstrate that DRIF1 is a unique retromer interactor that orchestrates FREE1-mediated ILV formation of MVBs and vacuolar sorting of membrane proteins for degradation in plants.

Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation.

Membrane lipids play vital roles in small extracellular vesicle (sEV) biogenesis. However, the function of various lipids in the biogenesis of sEVs is still poorly understood. Phosphoinositolphosphates (PIPs), a group of the most critical lipids in vesicle transport, can undergo rapid conversion in response to a variety of cell signals, which in turn influence the generation of vesicles. Due to the challenge in detecting the low amount of PIP content in biological samples, the function of PIPs in sEVs has been insufficiently investigated. Here, we employed an LC-MS/MS method to detect the levels of PIPs in sEVs. We revealed phosphatidylinositol-4-phosphate (PI4P) was the main PI-monophosphate in macrophage-derived sEVs. The release of sEVs was regulated in a time-dependent manner and correlated with the PI4P level during the lipopolysaccharide (LPS) stimulation. In terms of mechanism, within 10h of LPS treatment, the LPS-induced production of type I interferon inhibited the expression of PIP-5-kinase-1-gamma, which increased the PI4P content on multivesicular bodies (MVBs) and recruited RAB10, member RAS oncogene family, to promote sEV generation. When LPS stimulation was extended to 24h, the heat shock protein family A member 5 (HSPA5) expression level was elevated. PI4P interacted with HSPA5 on the Golgi or endoplasmic reticulum away from MVBs, which disrupted the continuous fast sEV release. In conclusion, the present study demonstrated an inducible sEV release model response to LPS treatment. The inducible release may be due to PI4P regulating the generation of intraluminal vesicles secreted as sEVs.

Insights into the function of ESCRT complex and LBPA in ASFV infection.

The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca2+-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.

Vps68 cooperates with ESCRT-III in intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the yeast ESCRT-III-associated protein Mos10 (also known as Vps60) in ILV formation, we screened for new interaction partners by using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to that of the SNF7 deletion strain when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the VPS68 deletion strain. Our data further indicate that at some point in the functional cycle of ESCRT-III, Snf7 could be replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices that localize to the luminal side of the endosomal membrane. Based on this membrane topology, we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

Expedition into Exosome Biology: A Perspective of Progress from Discovery to Therapeutic Development.

Simple SummaryExosomes symbolize membrane-enclosed entities of endocytic origin. They play an important role in the intracellular communication by shuttling proteins, nucleic acids, etc., between cells of different tissues and organs. Recent studies have revealed an interplay between cell and exosomes; thereby highlighted their importance in disease diagnosis and possible implication for use in therapeutics. They are currently been explored for the strategic development of platforms towards their employment in achieving the target specific delivery of therapeutics. This review summarizes the composition, biogenesis and trafficking of exosomes in different cellular backgrounds and explores their multifarious role as drug delivery vehicles towards achieving correct functionality and efficacy of the therapeutic molecules. Additionally, it discusses genetic engineering platforms for employment in the designing of optimal delivery modules for their application in the delivery of drugs as part of anticancer therapy.Exosomes are membrane-enclosed distinct cellular entities of endocytic origin that shuttle proteins and RNA molecules intercellularly for communication purposes. Their surface is embossed by a huge variety of proteins, some of which are used as diagnostic markers. Exosomes are being explored for potential drug delivery, although their therapeutic utilities are impeded by gaps in knowledge regarding their formation and function under physiological condition and by lack of methods capable of shedding light on intraluminal vesicle release at the target site. Nonetheless, exosomes offer a promising means of developing systems that enable the specific delivery of therapeutics in diseases like cancer. This review summarizes information on donor cell types, cargoes, cargo loading, routes of administration, and the engineering of exosomal surfaces for specific peptides that increase target specificity and as such, therapeutic delivery.

In Vitro Membrane Remodeling by ESCRT is Regulated by Negative Feedback from Membrane Tension

SummaryArtificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles to create complex “vesicles-within-a-vesicle” architectures resembling the compartmentalization in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III remodeling activity. Intraluminal vesicle (ILV) formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions.

Src in endosomal membranes promotes exosome secretion and tumor progression

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src–transformed cells. Our results indicate that activated c-Src in the endosomal membrane promoted the secretion of exosomes, in which c-Src was encapsulated. In addition, the ESCRT-interacting molecule, Alix was identified as a c-Src–interacting protein in exosomes. We revealed that the interaction between the SH3 domain of c-Src and the proline-rich region of Alix activates ESCRT–mediated intra-luminal vesicle (ILV) formation, resulting in the upregulation of exosome secretion in c-Src–transformed cells. We observed also a correlation between malignant phenotypes and Alix–dependent aberrant exosome secretion in Src–upregulated cancer cells. Collectively, our findings provide a unique mechanism for the upregulation of exosomes in cancer cells, as well as new insights into the significance of exosome secretion in cancer progression.

Effect of Late Endosomal Dobmp Lipid and Traditional Model Lipids of Electrophysiology on the Anthrax Toxin Channel Activity

Abstract Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.

Exosomes and Their Role in Cancer Progression.

Exosomes are a subset of extracellular vesicles and their size is approximately 100 nm in diameter. They are surrounded by a lipid bilayer membrane and secreted from almost all of cells. Exosomes are generated within the endocytic system as ILV (intraluminal membrane vesicle) and secreted during the fusion of MVB (multivesicular body) with the cell membrane. Recently it has been reported that exosomes modulate cell-cell communication contributing to the maintenance of tissue homeostasis by molecules including exosomes. Moreover, exosomes released from cancer cells are involved in cancer progression. Thus, data regarding the role of the exosomes in malignant cancer will lead to development of novel diagnostic and therapeutic methods.

Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation.

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

A plant Bro1 domain protein BRAF regulates multivesicular body biogenesis and membrane protein homeostasis

Plant development, defense, and many physiological processes rely on the endosomal sorting complex required for transport (ESCRT) machinery to control the homeostasis of membrane proteins by selective vacuolar degradation. Although ESCRT core components are conserved among higher eukaryotes, the regulators that control the function of the ESCRT machinery remain elusive. We recently identified a plant-specific ESCRT component, FREE1, that is essential for multivesicular body/prevacuolar compartment (MVB/PVC) biogenesis and vacuolar sorting of membrane proteins. Here we identify a plant-specific Bro1-domain protein BRAF, which regulates FREE1 recruitment to the MVB/PVC membrane by competitively binding to the ESCRT-I component Vps23. Altogether, we have successfully identified a role for BRAF, whose function as a unique evolutionary ESCRT regulator in orchestrating intraluminal vesicle formation in MVB/PVCs and the sorting of membrane proteins for degradation in plants makes it an important regulatory mechanism underlying the ESCRT machinery in higher eukaryotes.

Effect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the anthrax toxin channel activity

Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.