Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

Structural basis of membrane bending by the N-BAR protein endophilin

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the?N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through?a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.     

Mechanism of endophilin N-BAR domain-mediated membrane curvature

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

The Bin/Amphiphysin/Rvs (BAR) Domain Protein Endophilin B2 Interacts with Plectin and Controls Perinuclear Cytoskeletal Architecture

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.     

Dual Role of BAR Domain-containing Proteins in Regulating Vesicle Release Catalyzed by the GTPase,Dynamin-2

Dynamin-2 (Dyn2) is ubiquitously expressed and catalyzes membrane fission during clathrin-mediated endocytosis in nonneuronal cells. We have previously shown that Dyn2 inefficiently generates membrane curvature and only mediates fission of highly curved membranes. This led to the hypothesis that other endocytic accessory proteins (EAPs) generate curvature needed to sculpt a sufficiently narrow neck to trigger Dyn2 assembly and fission. Candidates for this activity are EAPs that bind to the dynamin proline/arginine-rich domain (PRD) through their SH3 (src homology-3) domains and also encode curvature-generating BAR (Bin/Amphiphysin/Rvs) domains. We show that at low concentrations, amphiphysin and endophilin, but not SNX9 or the curvature-generating epsin N-terminal homology (ENTH) domain, are able to generate tubules from planar membrane templates and to synergize with Dyn2ΔPRD to catalyze vesicle release. Unexpectedly, SH3-PRD interactions were inhibitory and reciprocally regulate scaffold assembly. Of the three proteins studied, only full-length amphiphysin functions synergistically with full-length Dyn2 to catalyze vesicle release. The differential activity of these proteins correlates with the relative potency of their positive, curvature-generating activity, and the negative regulatory effects mediated by SH3 domain interactions. Our findings reveal opportunities for the spatio-temporal coordination of membrane curvature generation, dynamin assembly, and fission during clathrin-mediated endocytosis.     

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses

Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.     

RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes

RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events.     

The HECT domain ligase itch ubiquitinates endophilin and localizes to the trans-Golgi network and endosomal system

Endophilin A1 is an SH3 domain-containing protein functioning in membrane trafficking on the endocytic pathway. We have identified the E3 ubiquitin ligase itch/AIP4 as an endophilin A1-binding partner. Itch belongs to the Nedd4/Rsp5p family of proteins and contains an N-terminal C2 domain, four WW domains and a catalytic HECT domain. Unlike other Nedd4/Rsp5p family members, itch possesses a short proline-rich domain that mediates its binding to the SH3 domain of endophilin A1. Itch ubiquitinates endophilin A1 and the SH3/proline-rich domain interaction facilitates this activity. Interestingly, itch co-localizes with markers of the endosomal system in a C2 domain-dependent manner and upon EGF stimulation, endophilin A1 translocates to an EGF-positive endosomal compartment where it colocalizes with itch. Moreover, EGF treatment of cells stimulates endophilin A1 ubiquitination. We have thus identified endophilin A1 as a substrate for the endosome-localized ubiquitin ligase itch. This interaction may be involved in ubiquitin-mediated sorting mechanisms operating at the level of endosomes.     

Multiple Modes of Endophilin-mediated Conversion of Lipid Vesicles into Coated Tubes: IMPLICATIONS FOR SYNAPTIC ENDOCYTOSIS*

Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. Three-dimensional reconstructions of quasicylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic helices whose folding and membrane insertion were attested by EPR. We also observed the compression of bulbous coated tubes into 70-Å-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Our findings suggest that the adaptability of endophilin-lipid interactions underlies dynamic changes of endocytic membranes.     

BAR domain proteins—a linkage between cellular membranes,signaling pathways,and the actin cytoskeleton

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.     

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.     

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

Autoinhibition of Endophilin in Solution via Interdomain Interactions

Endophilin is a key protein involved in clathrin-mediated endocytosis. Previous computational and experimental work suggested that the N-terminal helix is embedded into the membrane to induce curvature; however, the role of the SH3 domain remains controversial. To address this issue, we performed computer simulations of the endophilin dimer in solution to understand the interaction between the N-BAR and SH3 domains and its effect on biological function. We predict that the helix binds to the SH3 domain through hydrophobic and salt-bridge interactions. This protects the hydrophobic residues on both domains and keeps the SH3 domain near the end of the N-BAR domain, in agreement with previous experimental results. The complex has a binding strength similar to a few hydrogen bonds (13.0 ± 0.6 kcal/mol), and the SH3 domain stabilizes the structure of the N-terminal helix in solution. Electrostatic calculations show a large region of strongly positive electrostatic potential near the N-terminal that can orient the helix toward the membrane and likely embed the helix into the membrane surface. This predicted mechanism suggests that endophilin can select for both curvature and electrostatic potential when interacting with membranes, highlighting the importance of the SH3 domain in regulating the function of endophilin.     

Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase

Membrane-sculpting BAR (Bin/Amphiphysin/Rvs) domains form a crescent-shaped homodimer that can sense and induce membrane curvature through its positively charged concave face. We have recently shown that Arfaptin-2, which was originally identified as a binding partner for the Arf and Rac1 GTPases, binds to Arl1 through its BAR domain and is recruited onto Golgi membranes. There, Arfaptin-2 induces membrane tubules. Here, we report the crystal structure of the Arfaptin-2 BAR homodimer in complex with two Arl1 molecules bound symmetrically to each side, leaving the concave face open for membrane association. The overall structure of the Arl1·Arfaptin-2 BAR complex closely resembles that of the PX-BAR domain of sorting nexin 9, suggesting similar mechanisms underlying BAR domain targeting to specific organellar membranes. The Arl1·Arfaptin-2 BAR structure suggests that one of the two Arl1 molecules competes with Rac1, which binds to the concave face of the Arfaptin-2 BAR homodimer and may hinder its membrane association.     

Barfly: sculpting membranes at the Drosophila neuromuscular junction

The ability of a cell to change the shape of its membranes is intrinsic to many cellular functions. Proteins that can alter or recognize curved membrane structures and those that can act to recruit other proteins which stabilize the membrane curvature are likely to be essential in cell functions. The BAR (Bin, amphiphysin, RVS167 homology) domain is a protein domain that can either induce lipidic membranes to curve or can sense curved membranes. BAR domains are found in several proteins at neuronal synapses. We will review BAR domain structure and the role that BAR domain containing proteins play in regulating the morphology and function of the Drosophila neuromuscular junction. In flies the BAR domain containing proteins, endophilin and syndapin affect synaptic vesicle endocytosis, whereas CIP4, dRich, nervous wreck and syndapin affect synaptic morphology. We will review the growing evidence implicating mutations in BAR domain containing proteins being the cause of human pathologies.     

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P₂ specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H₀) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P₂-dependent membrane penetration of H₀ is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P₂-independent manner. Depletion of PtdIns(4,5)P₂ from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P₂ concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.     

BAR domains and membrane curvature: bringing your curves to the BAR

BAR (bin, amphiphysin and Rvs161/167) domains are a unique class of dimerization domains, whose dimerization interface is edged by a membrane-binding surface. In its dimeric form, the membrane-binding interface is concave, and this gives the ability to bind better to curved membranes, i.e. to sense membrane curvature. When present at higher concentrations, the domain can stabilize membrane curvature, generating lipid tubules. This domain is found in many contexts in a wide variety of proteins, where the dimerization and membrane-binding function of this domain is likely to have a profound effect on protein activity. If these proteins function as predicted, then there will be membrane subdomains based on curvature, and thus there is an additional layer of compartmentalization on membranes. These and other possible functions of the BAR domain are discussed.     

A Mutational Analysis of the Endophilin-A N-BAR Domain Performed in Living Flies

Background

Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo.

Methodology/Principal Findings

The study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature was functional, another mutation (P143A, ΔLEN) designed to decrease the curvature was not.

Conclusions/Significance

Our results provide novel insight into the structure/function relationship of the endophilin-A BAR domain in vivo, especially with relation to synaptic function.