Coated Vesicle Formation Research Articles

An Update on Coat Protein Complexes for Vesicle Formation in Plant Post-Golgi Trafficking.

Endomembrane trafficking is an evolutionarily conserved process for all eukaryotic organisms. It is a fundamental and essential process for the transportation of proteins, lipids, or cellular metabolites. The aforementioned cellular components are sorted across multiple membrane-bounded organelles. In plant cells, the endomembrane mainly consists of the nuclear envelope, endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network or early endosome (TGN/EE), prevacuolar compartments or multivesicular bodies (PVCs/MVBs), and vacuole. Among them, Golgi apparatus and TGN represent two central sorting intermediates for cargo secretion and recycling from other compartments by anterograde or retrograde trafficking. Several protein sorting machineries have been identified to function in these pathways for cargo recognition and vesicle assembly. Exciting progress has been made in recent years to provide novel insights into the sorting complexes and also the underlying sorting mechanisms in plants. Here, we will highlight the recent findings for the adaptor protein (AP) complexes, retromer, and retriever complexes, and also their functions in the related coated vesicle formation in post-Golgi trafficking.

Clathrin: the molecular shape shifter.

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised within different cellular processes including synaptic vesicle recycling, hormone desensitisation, spermiogenesis, cell migration and mitosis, and how clathrin's remarkable 'shapeshifting' ability to form diverse lattice structures might contribute to its multiple cellular functions.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

The AP2 adaptor enhances clathrin coat stiffness.

Deformation of the plasma membrane into clathrin‐coated vesicles is a critical step in clathrin‐mediated endocytosis and requires the orchestrated assembly of clathrin and endocytic adaptors into a membrane‐associated protein coat. The individual role of these membrane‐bending and curvature‐stabilizing factors is subject to current debate. As such, it is unclear whether the clathrin coat itself is stiff enough to impose curvature and if so, whether this could be effectively transferred to the membrane by the linking adaptor proteins. We have recently demonstrated that clathrin alone is sufficient to form membrane buds in vitro. Here, we use atomic force microscopy to assess the contributions of clathrin and its membrane adaptor protein 2 (AP2) to clathrin coat stiffness, which determines the mechanics of vesicle formation. We found that clathrin coats are less than 10‐fold stiffer than the membrane they enclose, suggesting a delicate balance between the forces harnessed from clathrin coat formation and those required for membrane bending. We observed that clathrin adaptor protein AP2 increased the stiffness of coats formed from native clathrin, but did not affect less‐flexible coats formed from clathrin lacking the light chain subunits. We thus propose that clathrin light chains are important for clathrin coat flexibility and that AP2 facilitates efficient cargo sequestration during coated vesicle formation by modulating clathrin coat stiffness.

Structure and Assembly of Clathrin Cages.

The unusual structure of clathrin, combined with its ability to assemble and disassemble rapidly in cells provides a model system for us to learn about the ways in which proteins can contribute mechanically to a functioning cell. In this article, we discuss the structural properties of clathrin cages and the triskelions which assemble to form them. The function of clathrin depends on the structure of these triskelions and the interactions they make both with each other during assembly and with the adaptor protein network that drives coated vesicle formation. The atomic resolution structure of clathrin domains has been revealed by X-ray crystallography while scattering studies have enabled the shape of a triskelion in solution to be deduced. Cryo-electron microscopy maps have shown the secondary structure of entire cages, how individual triskelion legs are arranged to form a cage and enabled some bound adaptor proteins to be located. Cage formation itself is energetically finely balanced and requires specific interactions between triskelion legs to be productive, as biochemical studies and in silico modeling have shown. Theoretical, structural and cell biological investigations over many years have contributed to our knowledge of clathrin structure and assembly. It now remains to determine the precise nature of the interactions which occur between clathrin triskelions, light chain and heavy chain and the adaptor protein network.

Helping hands for budding prospects: ENTH/ANTH/VHS accessory proteins in endocytosis, vacuolar transport, and secretion.

Coated vesicles provide a major mechanism for the transport of proteins through the endomembrane system of plants. Transport between the endoplasmic reticulum and the Golgi involves vesicles with COPI and COPII coats, whereas clathrin is the predominant coat in endocytosis and post-Golgi trafficking. Sorting of cargo, coat assembly, budding, and fission are all complex and tightly regulated processes that involve many proteins. The mechanisms and responsible factors are largely conserved in eukaryotes, and increasing organismal complexity tends to be associated with a greater numbers of individual family members. Among the key factors is the class of ENTH/ANTH/VHS domain-containing proteins, which link membrane subdomains, clathrin, and other adapter proteins involved in early steps of clathrin coated vesicle formation. More than 30 Arabidopsis thaliana proteins contain this domain, but their generally low sequence conservation has made functional classification difficult. Reports from the last two years have greatly expanded our knowledge of these proteins and suggest that ENTH/ANTH/VHS domain proteins are involved in various instances of clathrin-related endomembrane trafficking in plants. This review aims to summarize these new findings and discuss the broader context of clathrin-dependent plant vesicular transport.

Molecular mechanisms of Sar/Arf GTPases in vesicular trafficking in yeast and plants.

Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants.

Molecular structure, function, and dynamics of clathrin-mediated membrane traffic.

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.

Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes

Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62.

Mechanisms for differential endocytosis of CD44 involved in turnover and signaling in chondrocytes

In cartilage, hyaluronan (HA) is anchored to chondrocytes via binding to CD44. We have shown that CD44‐mediated endocytosis and lysosomal degradation of HA requires CD44 palmitoylation and subsequent transit into lipid raft microdomains. Interestingly, raft‐CD44 is also a substrate for MMP cleavage generating a 20kD C‐terminal fragment. In chondrocytes, CD44‐HA complexes are thought to be present in different membrane pools, some destined for endocytosis and others involved in signaling, migration or anchorage of matrix. Differentiating these pools is challenging due in part to the constant replenishment of CD44. Trypsin treatment of chondrocytes generates a unique 25kD fragment representative of CD44 present in all membrane pools prior to typsinization; the 20kD fragment only the lipid raft pool. Decay of both 20 and 25kD CD44 fragments is completely blocked by chloroquine suggesting lysosomal degradation as a common endpoint. No 20kD domains were derived from 25kD fragments suggesting the 25kD cannot subsequently move into lipid rafts and serve as a substrate for MMP and γ‐secretase. Chlorpromazine, 350 mM sucrose and Dynasore, inhibitors of clathrin coated vesicle formation, partially block the decay of the 25kD fragment. We hypothesize that raft‐CD44 is responsible for HA endocytosis; whereas CD44 involved in signaling may be downregulated by clathrin‐associated endocytosis. Supported by NIH R01‐AR39507 and R01‐AR43384.Grant Funding Source: NIH R01‐AR39507 and R01‐AR43384

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked β-N-acetylglucosaminyl-6-phosphate modification

A novel post-translational modification of threonine, β-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.

Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits

Recently, it has become clear that the actin cytoskeleton is involved in clathrin-mediated endocytosis. During clathrin-mediated endocytosis, clathrin triskelions and adaptor proteins assemble into lattices, forming clathrin-coated pits. These coated pits invaginate and detach from the membrane, a process that requires dynamic actin polymerization. We found an unexpected role for the clathrin adaptor epsin in regulating actin dynamics during this late stage of coated vesicle formation. In Dictyostelium cells, epsin is required for both the membrane recruitment and phosphorylation of the actin- and clathrin-binding protein Hip1r. Epsin-null and Hip1r-null cells exhibit deficiencies in the timing and organization of actin filaments at clathrin-coated pits. Consequently, clathrin structures persist on the membranes of epsin and Hip1r mutants and the internalization of clathrin structures is delayed. We conclude that epsin works with Hip1r to regulate actin dynamics by controlling the spatial and temporal coupling of actin filaments to clathrin-coated pits. Specific residues in the ENTH domain of epsin that are required for the membrane recruitment and phosphorylation of Hip1r are also required for normal actin and clathrin dynamics at the plasma membrane. We propose that epsin promotes the membrane recruitment and phosphorylation of Hip1r, which in turn regulates actin polymerization at clathrin-coated pits.

Roles of AP-2 in Clathrin-Mediated Endocytosis

BackgroundThe notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.Methodology/Principal FindingsWe report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37°C. If cells are first incubated with LDL at 4°C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37°C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway.Conclusions/SignificanceThese results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.

Dynasore, a Dynamin Inhibitor, Inhibits Trypanosoma cruzi Entry into Peritoneal Macrophages

Background Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process.Methodology/Principal FindingsWe used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages) when we used 100 µM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane.Conclusions/SignificanceTaken together our results demonstrate that dynamin is an essential molecule necessary for cell invasion and specifically parasitophorous vacuole formation by host cells during interaction with Trypanosoma cruzi.

Conserved Functions of Membrane Active GTPases in Coated Vesicle Formation

Coated vesicles concentrate and package cargo molecules to mediate their efficient transport between intracellular compartments. Cytosolic coat proteins such as clathrin and adaptor complexes and coat protein complex I (COPI) and COPII self-assemble to deform the membrane and interact directly with cargo molecules to capture them in nascent buds. The guanosine triphosphatases (GTPases) Arf, Sar1, and dynamin are core components of the coated vesicle machinery. These GTPases, which associate with and dissociate from donor membranes in a guanosine triphosphate-dependent manner, can also actively remodel membranes. Recent evidence suggests that, although structurally diverse, Arf family GTPases and dynamin may play mechanistically similar roles as fidelity monitors that govern cargo packaging and coated vesicle maturation and as components of the fission machinery to mediate vesicle release.

Coats, COPS, and GTPases

Within cells, vesicles coated with specific proteins, such as clathrin and adaptor complexes, concentrate and package cargo molecules to mediate transport between compartments. Pucadyil and Schmid (p. [1217][1]) review the role of membrane-active GTPases in coated vesicle formation and propose that

Conformational changes of coat proteins during vesicle formation

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.

Clathrin-Mediated Constitutive Endocytosis of PIN Auxin Efflux Carriers in Arabidopsis

Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.

Novel Binding Sites on Clathrin and Adaptors Regulate Distinct Aspects of Coat Assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.

Bending a membrane: How clathrin affects budding

Receptor-mediated endocytosis of ligands, such as transferrin and LDL, is suppressed when clathrin synthesis is blocked by RNA interference in HeLa cells. We have found that domains containing the adapter complex 2 (AP2)-coated vesicle adapter and the endocytic accessory proteins CALM (clathrin assembly lymphoid myeloid leukemia protein), epsin, and eps15/eps15R (EGF receptor pathway substrate 15-related) nevertheless persist at the plasma membrane. They are similar in size and number to those seen in clathrin-expressing cells. Here we characterize these membrane domains by fluorescence and electron microscopy in detail. Fluorescence recovery after photobleaching measurements suggest that the exchange between membrane-bound and free cytosolic AP2 molecules is not significantly influenced by the depletion of clathrin. The AP2 membrane domains are dispersed upon interfering with protein-protein interactions that involve the alpha appendage domain of AP2. Electron microscopy of cellular cortices revealed that the AP2 membrane domains lack any curvature, suggesting that clathrin is essential for driving coated pit invagination. A model for coated vesicle formation, incorporating a mechanism commonly referred to as a "Brownian ratchet," is consistent with our observations.