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ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We fabricated CaCO 3 -coated vesicles as drug carriers that release their cargo under a weakly acidic condition. We designed and synthesized a peptide lipid containing the Val-His-Val-Glu-Val-Ser sequence as the hydrophilic part, and with two palmitoyl groups at the N -terminal as the anchor groups of the lipid bilayer membrane. Vesicles embedded with the peptide lipids were prepared. The CaCO 3 coating of the vesicle surface was performed by the mineralization induced by the embedded peptide lipid. The peptide lipid produced the mineral source, CO 3 2- , for CaCO 3 mineralization through the hydrolysis of urea. We investigated the structure of the obtained CaCO 3 -coated vesicles using transmission electron microscopy (TEM). The vesicles retained the spherical shapes, even in vacuo. Furthermore, the vesicles had inner spaces that acted as the drug cargo, as observed by the TEM tomographic analysis. The thickness of the CaCO 3 shell was estimated as ca. 20 nm. CaCO 3 -coated vesicles containing hydrophobic or hydrophilic drugs were prepared, and the drug release properties were examined under various pH conditions. The mineralized CaCO 3 shell of the vesicle surface was dissolved under a weakly acidic condition, pH 6.0, such as in the neighborhood of cancer tissues. The degradation of the CaCO 3 shell induced an effective release of the drugs. Such behavior suggests potential of the CaCO 3 -coated vesicles as carriers for cancer therapies.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Despite rapid advancements in antitumor drug delivery, insufficient intracellular transport and subcellular drug accumulation are still issues to be addressed. Cancer cell membrane (CCM)-camouflaged nanoparticles (NPs) have shown promising potential in tumor therapy due to their immune escape and homotypic binding capacities. However, their efficacy is still limited due to inefficient tumor penetration and compromised intracellular transportation. Herein, a yolk-shell NP with a mesoporous silica nanoparticle (MSN)-supported PEGylated liposome yolk and CCM coating, CCM@LM, was developed for chemotherapy and exhibited a homologous tumor-targeting effect. The yolk-shell structure endowed CCM@LM with moderate rigidity, which might contribute to the frequent transformation into an ellipsoidal shape during infiltration, leading to facilitated penetration throughout multicellular spheroids in vitro (up to a 23.3-fold increase compared to the penetration of membrane vesicles). CCM@LM also exhibited a cellular invasion profile mimicking an enveloped virus invasion profile. CCM@LM was directly internalized by membrane fusion, and the PEGylated yolk (LM) was subsequently released into the cytosol, indicating the execution of an internalization pathway similar to that of an enveloped virus. The incoming PEGylated LM further underwent efficient trafficking throughout the cytoskeletal filament network, leading to enhanced perinuclear aggregation. Ultimately, CCM@LM, which co-encapsulated low-dose doxorubicin and the poly(ADP-ribose) polymerase inhibitor, mefuparib hydrochloride, exhibited a significantly stronger antitumor effect than the first-line chemotherapeutic drug Doxil. Our findings highlight that NPs that can undergo facilitated tumor penetration and robust intracellular trafficking have a promising future in cancer chemotherapy.

Nuclear pores and coated vesicles are elaborate multi-component protein complexes that oligomerize on membranes, and stabilize or induce membrane curvature. Their components, nucleoporins and coat proteins, respectively, share similar structural folds and some principles of how they interact with membranes. The protocoatomer hypothesis postulates that this is due to divergent evolution from a common ancestor. It therefore has been suggested that nucleoporins and coat proteins have similar higher order architectures. Here, we review recent work that relied on technical advances in cryo-electron microscopy and integrative structural biology to take a fresh look on how these proteins form membrane coats in situ. We discuss the relationship between the architectures of nuclear pores and coated vesicles, and their evolutionary origins., (Copyright © 2018. Published by Elsevier Ltd.)

Vesicular transport was key to the evolution of eukaryotes, and is essential for eukaryotic life today. All modern eukaryotes have a set of vesicle coat proteins, which couple cargo selection to vesicle budding in the secretory and endocytic pathways. Although these coats share common features (e.g. recruitment via small GTPases, β-propeller-α-solenoid proteins acting as scaffolds), the relationships between them are not always clear. Structural studies on the coats themselves, comparative genomics and cell biology in diverse eukaryotes, and the recent discovery of the Asgard archaea and their 'eukaryotic signature proteins' are helping us to piece together how coats may have evolved during the prokaryote-to-eukaryote transition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with β-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.

Protein secretion mediated by the secretory transport pathway is an important cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized proteins pass through several endomembrane compartments en route to their specific destinations. Transport of secretory proteins between different compartments is shuttled by small, membrane-enclosed vesicles. To ensure the fidelity of transport, eukaryotic cells employ elaborate molecular machineries to accurately sort newly synthesized proteins into specific transport vesicles and precisely deliver these transport vesicles to distinct acceptor compartments. In this review, we summarize the molecular machineries that regulate each step of vesicular transport in the secretory transport pathway in yeast and animal cells.

The delivery of proteins to the apoplast or protein secretion is an essential process in plant cells. Proteins are secreted to perform various biological functions such as cell wall modification and defense response. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been demonstrated in plants. In the conventional protein secretion pathway, secretory proteins with an N-terminal signal peptide are transported to the extracellular region via the endoplasmic reticulum-Golgi apparatus and the subsequent endomembrane system. By contrast, multiple unconventional protein secretion pathways are proposed to mediate the secretion of the leaderless secretory proteins. In this review, we summarize the recent findings and provide a comprehensive overview of protein secretion pathways in plant cells.

Lanthanide ions are strong Lewis acids. Their complexation to a variety of ligands can further enhance their Lewis acidity allowing the hydrolysis of phosphoesters and even DNA. We show that the interaction of lanthanide ions with vesicles from zwitterionic phosphatidylcholine lipids gives supramolecular structures in which the metal ion is loosely coordinated to the surface. This assembly provides a high density of Lewis-acidic metal centres, which hydrolyze phosphodiesters with enhanced rates.

A prerequisite for successful oral drug therapy is the drug's ability to cross the gastrointestinal barrier. Considering the increasing number of new chemical entities in modern drug discovery, reliable and fast in vitro models are required for early and efficient prediction of intestinal permeability. To mimic the intestinal environment, use of biorelevant media may provide valuable information on in vivo drug permeation. The present study aims at improving the novel biomimetic phospholipid vesicle-based permeation assay's (PVPAbiomimetic) biorelevance by investigating the applicability of the biorelevant media; fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). The FaSSIF and FeSSIF's influence on the permeability of the model drugs acyclovir, indomethacin, griseofulvin and nadolol was then assessed. The barriers' robustness in terms of storage stability was also evaluated. The barriers were found to maintain their integrity in presence of FaSSIF and FeSSIF. The model drugs showed changes in permeability in presence of the different simulated intestinal fluids that were in agreement with previous reports. Moreover, the barrier showed improved storage stability by maintaining its integrity for 6months. Altogether, this study moves the PVPAbiomimetic an important step towards a better in vitro permeability model for use in drug development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment.

Objectives: Mefloquine (MQ) is an antimalarial drug with high efficacy, often used in the treatment and chemoprophylaxis of malaria. However, it has low solubility in water, a long elimination half-life (4 days), and is neurotoxic, which leads to unwanted side effects., Methods: We investigated a lipid-based drug delivery system, Pheroid vesicles, in combination with MQ (Pheroid MQ), to promote future clinical use. MQ was incorporated into Pheroid vesicles and the formulations characterized. The formulations were evaluated in terms of in-vitro efficacy and toxicity. In-vivo bioavailability studies were conducted in C57 BL6 mice., Key Findings: The vesicles incorporated MQ with ~63% entrapment efficiency. The IC50 values of MQ after 48-h incubation in chloroquine-resistant (RSA11) and chloroquine sensitive (3D7) strains, were reduced by ~50% and ~30% respectively. In-vivo bioavailability study revealed no change in the pharmacokinetic parameters of MQ, and the incorporation of the drug in Pheroid vesicles reduced the in-vitro haemolytic activity by ~75%. Furthermore, the cytotoxicity against human neuroblastoma cells (SH-SY5Y) of the free drug was reduced by ~64% with Pheroid MQ., Conclusions: Pheroid vesicles may therefore decrease the toxicity of MQ and thereby improve its therapeutic index, a strategy that may provide an effective alternative for malaria chemoprophylaxis and treatment., (© 2013 Royal Pharmaceutical Society.)

The transport of proteins and lipids between distinct cellular compartments is conducted by coated vesicles. These vesicles are formed by the self-assembly of coat proteins on a membrane, leading to collection of the vesicle cargo and membrane bending to form a bud. Scission at the bud neck releases the vesicle. X-ray crystallography and electron microscopy (EM) have recently generated models of isolated coat components and assembled coats. Here, we review these data to present a structural overview of the three main coats: clathrin, COPII, and COPI. The three coats have similar function, common ancestry, and structural similarities, but exhibit fundamental differences in structure and assembly. We describe the implications of structural similarities and differences for understanding the function, assembly principles, and evolution of vesicle coats., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The clathrin-associated adaptor protein (AP) complexes AP-1 and AP-2 are two members of a family of heterotetrameric assemblies that connect transmembrane protein cargo to vesicular coats. Cargo binding by AP-1 is activated by the small GTPase Arf1, while AP-2 is activated by the phosphoinositide PI(4,5)P₂. The structures of both AP-1 and AP-2 have been determined in their locked and unlocked conformations. The structures show how different activators use different mechanisms to trigger similar large scale conformational rearrangements. The details of these mechanisms show how membrane docking and allosteric activation of AP complexes are intimately connected., (Copyright © 2013 The Protein Society.)

The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific chain consisting of homopolymeric legionaminic acid. In this chapter we describe a method for the isolation of LPS from L. pneumophila. In the first part we describe the chemical purification, in the second part we outline the application of monoclonal antibody (mAb) in Western blot and immuno-localization by indirect immunofluorescence. This report does not describe physico-chemical methods that analyze the structure of lipopolysaccharide entities.

Eukaryotic cells face a logistical challenge in ensuring prompt and precise delivery of vesicular cargo to specific organelles within the cell. Coat protein complexes select cargo and initiate vesicle formation, while multisubunit tethering complexes participate in the delivery of vesicles to target membranes. Understanding these macromolecular assemblies has greatly benefited from their structural characterization. Recent structural data highlight principles in coat recruitment and uncoating in both the endocytic and retrograde pathways, and studies on the architecture of tethering complexes provide a framework for how they might link vesicles to the respective acceptor compartments and the fusion machinery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In recent years, the interest for proteins that exert key functions in vesicular trafficking through their ability to sense or induce positive membrane curvature has expanded. In this chapter, we first present simple protocols to determine whether a protein targets positively curved membranes with liposomes of well-defined size. Next we describe more sophisticated approaches based on the controlled deformation of giant liposomes. These approaches allow visualization and quantification of protein binding to membrane regions of high curvature by real-time fluorescence microscopy. Last we describe several functional assays to measure how membrane curvature controls the activation state of Arf1 via ArfGAP1 or the asymmetric tethering between flat and curved membranes via the golgin GMAP-210., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nuclear pore complexes (NPCs) are highly selective transport gates that enable the bi-directional traffic of macromolecules across the nuclear envelope (NE). NPCs are located at the fusion pores between the inner and outer membranes of the NE and are built from a common set of ∼30 different proteins, nucleoporins. Remarkably, recent proteomic, bioinformatic, and structural studies have provided firm evidence that key structural nucleoporins share common ancestry with elements of coated vesicles, indicating an evolutionary link between these structures. This has provided novel insight into the origin of NPCs and may help us to better functionally characterize these fundamental components of eukaryotic cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Pegylated liposome formulations of vinorelbine with prolonged circulation half-life (t½) are desirable. However, DSPE-PEG could affect vinorelbine loading into vesicles due to electrostatic interactions. To resolve this problem, chol-PEG was used to prepare pegylated liposomal vinorelbine and the factors affecting drug loading and plasma pharmacokinetics were investigated., Methods: Vinorelbine was loaded into liposomes using a novel triethylamine 5-sulfosalicylate gradient. The effects of cholesterol and chol-PEG on drug loading were investigated. Pharmacokinetic studies were performed in normal KunMing mice treated with different liposomal vinorelbine formulations. To clarify the effects of chol-PEG on membrane permeability, drug release experiments were performed based on the fluorescence dequenching phenomenon of a fluorescence marker., Key Findings: In contrast to DSPE-PEG, even at high PEG grafting density (∼ 8.3 mol%), chol-PEG had no effect on vinorelbine loading into HSPC/cholesterol (3 : 1, mass ratio) vesicles. However, for the formulations with low cholesterol content (HSPC/cholesterol 4 : 1), loading efficiency decreased with increasing chol-PEG content. In vivo, the vinorelbine t½ of low cholesterol formulations decreased with increasing chol-PEG content, but for high cholesterol liposomes, the maximum vinorelbine t½ was achieved at ∼ 3 mol% chol-PEG grafting density. The resulting vinorelbine circulation t½ was ∼ 9.47 h, which was greater than that of non-pegylated liposomes (∼ 5.55 h). Drug release experiments revealed that chol-PEG might induce membrane defects and concomitant release of entrapped marker, especially at high chol-PEG density., Conclusions: Through the investigation of the effects of chol-PEG and cholesterol, an optimum pegylated liposomal vinorelbine formulation with prolonged t½ was achieved. In plasma, the membrane defect induced by chol-PEG may counteract the long circulation characteristics that chol-PEG afforded. When these two opposite effects reached equilibrium, the maximum vinorelbine t½ was achieved., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Vesicle trafficking is a highly regulated process that transports proteins and other cargoes through eukaryotic cells while maintaining cellular organization and compartmental identity. In order for cargo to reach the correct destination, each step of trafficking must impart specificity. During vesicle formation, this is achieved by coat proteins, which selectively incorporate cargo into the nascent vesicle. Classically, vesicle coats are thought to dissociate shortly after budding. However, recent studies suggest that coat proteins can remain on the vesicle en route to their destination, imparting targeting specificity by physically and functionally interacting with Rab-regulated tethering systems. This review focuses on how interactions among Rab GTPases, tethering factors, SNARE proteins, and vesicle coats contribute to vesicle targeting, fusion, and coat dynamics., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The nuclear pore complex (NPC) regulates all traffic between the cytoplasm and the nucleus. It is a large protein assembly composed of multiple copies of ∼30 nucleoporins (nups). Structural studies of the NPC have been limited by its considerable size and complexity. Progress toward understanding the structure of this nanomachine has benefited from its modular nature, which allows for this 40-60 MDa assembly to be broken down into subcomplexes that can be studied individually. While recent work by both crystallographers and electron microscopists has greatly enhanced our model of the NPC, the resolution gap between crystal and EM structures remains too large to confidently place individual proteins within the context of the fully assembled NPC. In an effort to arrive at a veritable model of the NPC, we solved the structure of several scaffold nups and defined the ancestral coatomer element (ACE1) common to a set of nucleoporins and COPII vesicle coat proteins. Subsequently, we proposed a lattice-like model of the NPC, analogous to the COPII lattice, in which ACE1 proteins form the edge elements and β-propellers form the vertex elements. Here, we review our recent studies, speculate on how interactions between subcomplexes of the NPC are mediated, and outline the steps and challenges that lay ahead on the path to understanding this enormous assembly in molecular detail.

Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)*Seh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.

In coated vesicle formation, coat protein recruitment needs to be spatially and temporally controlled. The coating process involves conformational changes of the coat protein complexes that activate them for interaction with cargo or machinery components and coat polymerization. Here we discuss mechanisms that have emerged recently from studies of the clathrin adaptor and the COPI systems.

The major renal Na(+)/phosphate cotransporter, NaPi-IIa, is regulated by a number of factors including parathyroid hormone (PTH), dopamine, and dietary phosphate intake. PTH induces the acute internalization of NaPi-IIa from the brush border membrane (BBM) and its routing to and subsequent degradation in lysosomes. Previous work indicated that megalin, part of the apical receptor-mediated endocytic apparatus, may play a role in the PTH-induced removal of NaPi-IIa. Here we examined in rats the time-dependent internalization route of NaPi-IIa after acute PTH application using immunohistochemistry and markers of several endocytic compartments. NaPi-IIa removal from the BBM was detectable as early as 5 min after PTH injection. After 10-15 min, NaPi-IIa was localized in subapical compartments positive for clathrin. Shortly thereafter, NaPi-IIa appeared in endosomes stained for EEA1 (early endosomal antigen 1). After 45-60 min, NaPi-IIa was found in late endosomes/lysosomes marked with lgp120. In contrast, no change in the subcellular localization of megalin and the Na(+)/H(+) exchanger NHE3 was detected up to 60 min after PTH injection. To further characterize the internalization route, insulin, as a marker for receptor-mediated endocytosis, and horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC)-dextran (10 kDa), as markers for fluid-phase mediated endocytosis, were used. NaPi-IIa colocalized with insulin 5-30 min after PTH injection but did not overlap with HRP or FITC-dextran. These results demonstrate a distinct internalization route of NaPi-IIa in response to acute PTH application that may involve the receptor-mediated endocytic pathway including clathrin-coated vesicles and EEA1-positive early endosomes, and routes NaPi-IIa to lysosomes for degradation.

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.

Clathrin-mediated endocytosis is the major process by which transmembrane proteins are internalized from the cell's limiting membrane into the first compartment of the endosomal system, the early endosome. From here, these transmembrane cargo proteins, which are of widely varying type and function, are trafficked to their required destination. Endocytosis plays, therefore, an important role in cell signalling, nutrient uptake, cellular homoeostasis and the interaction of the cell with its external environment. The formation of clathrin-coated endocytic vesicles requires the complex interplay of many proteins with each other and with the membrane itself. Their formation has served as a paradigm for formation of all types of transport vesicle, which move cargo between the various membrane-bound compartments of the cell. Clathrin-coated vesicles (CCVs) possess three layers: the inner membrane layer, in which the transmembrane cargo is embedded, linked to the outer clathrin lattice by a layer of cargo-binding adaptors and proteins that aid and regulate vesicle formation. Protein X-ray crystallography in combination with biochemical, biophysical and cell biological assays has been used to investigate the structure and function of some of the proteins that make up the middle layer of CCVs. These proteins are diverse in their functions, but are all modular in nature, consisting of folded domains joined by long unstructured linkers. Within these linkers are short motifs that interact with the folded domains of other components of the CCV formation machinery. Many of these folded domains also bind directly to the membrane. These interactions, whose molecular basis we have studied, have affinities in the low micromolar range, making them readily reversible and easily regulated. The mechanism of CCV formation is discussed in the light of this structural and biochemical data.

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.

Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles.

This review includes 16 structures of vesicle coat components and accessory proteins and a description of their roles in vesicle budding or coat disassembly.

One of the possibilities for increasing the penetration rate of drugs through the skin is the use of vesicular systems. Currently, special attention is paid to the elastic properties of liquid-state vesicles, which are supposed to have superior properties compared to gel-state vesicles with respect to skin interactions. In this study, the effects of vesicles on hairless mouse skin, both in vivo and in vitro, were studied in relation to the composition of vesicles. The interactions of elastic vesicles containing the single chain surfactant octaoxyethylene laurate-ester (PEG-8-L) and sucrose laurate-ester (L-595) with hairless mouse skin were studied, in vivo, after non-occlusive application for 1, 3 and 6 h. The skin ultrastructure was examined by ruthenium tetroxide electron microscopy (TEM) and histology. The extent, to which vesicle constituents penetrated into the stratum corneum, was quantified by thin layer chromatography (TLC). The interactions of the elastic vesicles containing PEG-8-L and L-595 surfactants were compared with those observed after treatment with rigid vesicles containing the surfactant sucrose stearate-ester (Wasag-7). Furthermore, skin permeability experiments were carried out to investigate the effect of treatment with PEG-8-L micelles, elastic vesicles (containing PEG-8-L and L-595 surfactants) or rigid Wasag-7 vesicles on the 3H(2)O transport through hairless mouse skin, in vitro, after non-occlusive application. Treatment of hairless mouse skin with the elastic vesicles affected the ultrastructure of the stratum corneum: distinct regions with lamellar stacks derived from the vesicles were observed in intercellular spaces of the stratum corneum. These stacks disrupted the organization of skin bilayers leading to an increased skin permeability, whereas no changes in the ultrastructure of the underlying viable epidermis were observed. Treatment with rigid Wasag-7 vesicles did not affect the skin ultrastructure or skin permeability. TLC measurements showed that after 1 h of non-occlusive application of elastic or rigid vesicles, a six-fold increased amount of elastic vesicle material was present within the stratum corneum compared to rigid vesicle material. After 3 and 6 h of application the amount of PEG-8-L vesicle material in SC decreased to approximately three- and two-fold, respectively, compared to Wasag-7 vesicle material. Pretreatment of the hairless mouse skin with the elastic vesicles containing 70 mol% PEG-8-L increased the diffusion of 3H(2)O with an optimum application dose of 2.5 mg lipids/cm(2) compared to PBS pretreatment. No significant difference in the enhancement of the 3H(2)O-diffusion was observed between PEG-8-L micelles or elastic vesicles containing 30 or 70 mol% PEG-8-L. Pretreatment with the rigid Wasag-7 vesicles decreased the diffusion rate of 3H(2)O, most probably by the formation of a lipid layer on the skin surface. The effect of the elastic vesicles on the skin permeability is supported by the ultrastructural changes observed by TEM in the intercellular lipid domains. The elastic vesicles containing 70 mol% PEG-8-L disorganize the lipid bilayers thereby creating or modifying pathways for possible drug penetration.

Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on (2)H2O-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9+/-10.5 nm and a density of 1.23 g x ml(-1). Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only approximately 5-10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents approximately 70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway. Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of alpha(s1)-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.

Evidence is accumulating that membrane traffic between organelles can be achieved by different types of intermediates. Small (< 100 nm) and short-lived vesicles mediate transport from the plasma membrane or the trans-Golgi network to endosomes, and formation of these vesicles depends on specific adapter complexes. In contrast, transport from early to late endosomes is achieved by relatively large (approximately 0.5 microm), long-lived and multivesicular intermediates, and their biogenesis depends on endosomal COP-I proteins. Here, we review recent work on the formation of these different transport intermediates, and we discuss, in particular, coat proteins, sorting signals contained in cargo molecules and the emerging role of lipid in vesicle biogenesis.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.

The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake. The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane. The V-ATPases are composed of two functional domains. The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases. A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification. Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo. One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity. Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

Clathrin light chain B (LCB) is a major component of clathrin coated vesicles, which are structures involved in intracellular transport. A neuron-specific isoform of LCB is generated by incorporation of a single exon (EN) using an alternative splicing mechanism that reflects the special demands of neurons, such as axonal transport and synaptic neurotransmission. Here, we demonstrate that this neuron-specific exon is developmentally regulated and is excluded in non-neuronal cells because its 5' and 3' splice sites deviate from the mammalian consensus sequences. A gel retardation assay indicated the presence of a developmentally regulated factor in brain that binds to the neuronal exon. In addition, EN usage is repressed by increasing the concentration of htra2-beta1, a splice factor whose isoform expression is influenced by neuronal activity. We propose that a brain-specific factor is involved in EN recognition during development and adulthood. In addition, ubiquitously expressed splicing factors such as htra2-beta1 are involved in regulating EN expression in the adult brain., (Copyright 1999 Elsevier Science B.V.)

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.

Secretion, endocytosis and transport to the lytic compartment are fundamental, highly coordinated features of the eukaryotic cell. These intracellular transport processes are facilitated by vesicles, many of which are small (100 nm or less in diameter) and 'coated' on their cytoplasmic surface. Research into the structure of the coat proteins and how they interact with the components of the vesicle membrane to ensure the selective packaging of the cargo molecules and their correct targeting, has been quite extensive in mammalian and yeast cell biology. By contrast, our knowledge of the corresponding types of transport vesicles in plant cells is limited. Nevertheless, the available data indicate that a considerable homology between plant and non-plant coat polypeptides exists, and it is also suggestive of a certain similarity in the mechanisms underlying targeting in all eukaryotes. In this article we shall concentrate on three major types of transport vesicles: clathrin-coated vesicles, COP-coated vesicles, and 'dense' vesicles, the latter of which are responsible for the transport of vacuolar storage proteins in maturing legume cotyledons. For each we will summarize the current literature on animal and yeast cells, and then present the relevant data derived from work on plant cells. In addition, we briefly review the evidence in support of the 'SNARE' hypothesis, which explains how vesicles find and fuse with their target membrane.

A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.

The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305-598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with gamma adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters.