Yvonne Vallis

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P2 in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

FCHo Proteins are Nucleators of Clathrin-Mediated Endocytosis

Initiator for Coated Pit Assembly During clathrin-mediated endocytosis, it has been thought that the sensing and binding of the clathrin adaptor protein AP2 to cargo and lipids leads to the recruitment of clathrin, nucleating the formation of a clathrin-coated pit. Henne et al. (p. 1281, published online 6 May) have now found that this process of AP2 binding may not in fact represent either the first or the nucleation event of endocytosis. Instead, ubiquitous proteins called FCHo1/2 (F-BAR proteins) bind to the plasma membrane and define the sites of endocytosis independently of AP2. The F-BAR protein can generate very low curvature and, at higher concentration, generates higher curvature like those required at the neck of budding vesicles. The C terminus of the protein has a mu-homology domain (with homology to the mu domain of the AP2 complex) that interacts with Eps15 and intersectin, and via these proteins recruits AP2, which further recruits clathrin. Thus, a curvature-inducing protein can act to nucleate clathrin-coated pit assembly during endocytosis. Membrane-sculpting proteins nucleate clathrin‐coated pits during endocytosis. Clathrin-mediated endocytosis, the major pathway for ligand internalization into eukaryotic cells, is thought to be initiated by the clustering of clathrin and adaptors around receptors destined for internalization. However, here we report that the membrane-sculpting F-BAR domain–containing Fer/Cip4 homology domain-only proteins 1 and 2 (FCHo1/2) were required for plasma membrane clathrin-coated vesicle (CCV) budding and marked sites of CCV formation. Changes in FCHo1/2 expression levels correlated directly with numbers of CCV budding events, ligand endocytosis, and synaptic vesicle marker recycling. FCHo1/2 proteins bound specifically to the plasma membrane and recruited the scaffold proteins eps15 and intersectin, which in turn engaged the adaptor complex AP2. The FCHo F-BAR membrane-bending activity was required, leading to the proposal that FCHo1/2 sculpt the initial bud site and recruit the clathrin machinery for CCV formation.

Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling

The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

Endophilin marks and controls a clathrin-independent endocytic pathway

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and β1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME).

Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain

BACKGROUND Receptor-mediated endocytosis appears to require the GTP-binding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several proline-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate protein-protein interactions. Amphiphysin, a protein that is highly expressed in the brain, interacts with dynamin in vitro, as do Grb2 and many other SH3 domain-containing proteins. In this study, we examined the role of amphiphysin in receptor-mediated endocytosis in vivo. RESULTS To address the importance of the amphiphysin SH3 domain in dynamin recruitment, we used a transferrin and epidermal growth factor (EGF) uptake assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence revealed that cells transfected with the amphiphysin SH3 domain showed a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin could associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis. CONCLUSIONS Our results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. We propose that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis.

Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis

The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamins function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamins interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.

Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis

Clathrin‐mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on α‐appendages, called ‘top’ and side’ sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high‐affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered α(appendage)‐hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.

The GTPase-Activating Protein GRAF1 Regulates the CLIC/GEEC Endocytic Pathway

Summary Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation

The amphiphysins are brain‐enriched proteins, implicated in clathrin‐mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin‐2 (Amph2) SH3 domain to 2.2 Å. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline‐rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self‐assembly into rings. Deletion of a unique insert in the n‐Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n‐Src loop of the N‐terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n‐Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.

The structure and function of the β2-adaptin appendage domain

The heterotetrameric AP2 adaptor (α, β2, μ2 and σ2 subunits) plays a central role in clathrin‐mediated endocytosis. We present the protein recruitment function and 1.7 Å resolution structure of its β2‐appendage domain to complement those previously determined for the μ2 subunit and α appendage. Using structure‐directed mutagenesis, we demonstrate the ability of the β2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the β2‐appendage site and to a second site on the adjacent β2 hinge. This results in the displacement of the other ligands from the β2 appendage. Thus clathrin binding to an AP2–accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.