Structure of dynein-dynactin on microtubules shows tandem adaptor binding.

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4-6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9-13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.

Molecular architecture of the complete COG tethering complex.

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.

CATCHR, HOPS and CORVET tethering complexes share a similar architecture.

We show here that the Saccharomyces cerevisiae GARP complex and the Cog1-4 subcomplex of the COG complex, both members of the complexes associated with tethering containing helical rods (CATCHR) family of multisubunit tethering complexes, share the same subunit organization. We also show that HOPS, a tethering complex acting in the endolysosomal pathway, shares a similar architecture, thus suggesting that multisubunit tethering complexes use related structural frameworks.

Structural and functional insight into the N-terminal domain of the clathrin adaptor Ent5 from Saccharomyces cerevisiae.

Clathrin-coated vesicles (CCVs) play critical roles in multiple cellular processes, including nutrient uptake, endosome/lysosome biogenesis, pathogen invasion, regulation of signalling receptors, etc. Saccharomyces cerevisiae Ent5 (ScEnt5) is one of the two major adaptors supporting the CCV-mediated TGN/endosome traffic in yeast cells. However, the classification and phosphoinositide binding characteristic of ScEnt5 remain elusive. Here we report the crystal structures of the ScEnt5 N-terminal domain, and find that ScEnt5 contains an insertion α' helix that does not exist in other ENTH or ANTH domains. Furthermore, we investigate the classification of ScEnt5-N(31-191) by evolutionary history analyses and structure comparisons, and find that the ScEnt5 N-terminal domain shows different phosphoinositide binding property from rEpsin1 and rCALM. Above results facilitate the understanding of the ScEnt5-mediated vesicle coat formation process.

Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides.

OBJECTIVE: To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. METHODS: Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. RESULTS: The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. CONCLUSIONS: van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues.

Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Correlative super-resolution fluorescence and metal-replica transmission electron microscopy.

We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.

Characterization of herpes simplex virus type 1 L-particle assembly and egress in hippocampal neurones by electron cryo-tomography.

Visualizing virus-host interactions in situ inside infected cells by electron cryo-tomography provides unperturbed snapshots of the infection process. Here we focus on the assembly and egress pathway of herpesviruses. Cells infected with herpes simplex virus 1 produce and release not only infective virions but also non-infectious light particles (L-particles). L-particles are devoid of viral capsids and genomes. In this study, we analysed L-particle assembly and egress pathways in cultured dissociated hippocampus neurones by electron cryo-tomography. Virion and L-particle formation occurred in close proximity, suggesting shared assembly and exit pathways. Clathrin-like coats were occasionally associated with L-particle and virion assembly sites. Further, we compared the three-dimensional ultrastructure of intracellular and extracellular L-particles and quantified their diameters and the abundance of inclusion bodies contained.

Structural characterization of full-length NSF and 20S particles.

The 20S particle, which is composed of the N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and the SNAP receptor (SNARE) complex, has an essential role in intracellular vesicle fusion events. Using single-particle cryo-EM and negative stain EM, we reconstructed four related three-dimensional structures: Chinese hamster NSF hexamer in the ATPγS, ADP-AlFx and ADP states, and the 20S particle. These structures reveal a parallel arrangement between the D1 and D2 domains of the hexameric NSF and characterize the nucleotide-dependent conformational changes in NSF. The structure of the 20S particle shows that it holds the SNARE complex at two interaction interfaces around the C terminus and N-terminal half of the SNARE complex, respectively. These findings provide insight into the molecular mechanism underlying disassembly of the SNARE complex by NSF.

Molecular organization of the COG vesicle tethering complex.

Multisubunit tethering complexes of the CATCHR (complexes associated with tethering containing helical rods) family are proposed to mediate the initial contact between an intracellular trafficking vesicle and its membrane target. To begin elucidating the molecular architecture of one well-studied example, the conserved oligomeric Golgi (COG) complex, we reconstituted its essential subunits (Cog1, Cog2, Cog3 and Cog4) and used single-particle electron microscopy to reveal a y-shaped structure with three flexible, highly extended legs. Labeling experiments established that the N termini of all four subunits interact along the proximal segment of one leg, whereas three of the four C termini are located at the tips of the legs. Our results suggest that the central region of the Cog1-Cog2-Cog3-Cog4 complex, as well as the distal regions of at least two legs, all participate in interactions with other components of the intracellular trafficking machinery.

Spatial and functional relationship of GGAs and AP-1 in Drosophila and HeLa cells.

The GGAs [Golgi-localised, gamma-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.

Drosophila Past1 is involved in endocytosis and is required for germline development and survival of the adult fly.

Endocytosis, which is a key process in eukaryotic cells, has a central role in maintaining cellular homeostasis, nutrient uptake, development and downregulation of signal transduction. This complex process depends on several protein-protein interactions mediated by specific modules. One such module is the EH domain. The EH-domain-containing proteins comprise a family that includes four vertebrate members (EHD1-EHD4) and one Drosophila ortholog, Past1. We used Drosophila as a model to understand the physiological role of this family of proteins. We observed that the two predicted Past1 transcripts are differentially expressed both temporally and spatially during the life cycle of the fly. Endogenous Past1 as well as Past1A and Past1B, expressed from plasmids, were localized mainly to the membrane of Drosophila-derived cells. We generated mutants in the Past1 gene by excising a P-element inserted in it. The Past1 mutants reached adulthood but died precociously. They were temperature sensitive and infertile because of lesions in the reproductive system. Garland cells that originated from Past1 mutants exhibited a marked decrease in their ability to endocytose fluorescently labeled avidin. Genetic interaction was found between Past1 and members of the Notch signaling pathway, suggesting a role for Past1 in this developmentally crucial signaling pathway.

EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization.

Extended Fer-CIP4 homology (EFC)/FCH-BAR (F-BAR) domains generate and bind to tubular membrane structures of defined diameters that are involved in the formation and fission of endocytotic vesicles. Formin-binding protein 17 (FBP17) and Toca-1 contain EFC/F-BAR domains and bind to neural Wiskott-Aldrich syndrome protein (N-WASP), which links phosphatidylinositol (4,5)-bisphosphate (PIP(2)) and the Rho family GTPase Cdc42 to the Arp2/3 complex. The N-WASP-WASP-interacting protein (WIP) complex, a predominant form of N-WASP in cells, is known to be activated by Toca-1 and Cdc42. Here, we show that N-WASP-WIP complex-mediated actin polymerization is activated by phosphatidylserine-containing membranes depending on membrane curvature in the presence of Toca-1 or FBP17 and in the absence of Cdc42 and PIP(2). Cdc42 further promoted the activation of actin polymerization by N-WASP-WIP. Toca-1 or FBP17 recruited N-WASP-WIP to the membrane. Conserved acidic residues near the SH3 domain of Toca-1 and FBP17 positioned the N-WASP-WIP to be spatially close to the membrane for activation of actin polymerization. Therefore, curvature-dependent actin polymerization is stimulated by spatially appropriate interactions of EFC/F-BAR proteins and the N-WASP-WIP complex with the membrane.

Subcellular localization and function of alternatively spliced Noxo1 isoforms.

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex.

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.

Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein.

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin-interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin-coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin-1-containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP-2 adaptor complex and clathrin. The enrichment of epsin within clathrin-coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non-conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin-sorting endocytic clathrin machinery.

Life of a clathrin coat: insights from clathrin and AP structures.

Membrane sorting between secretory and endocytic organelles is predominantly controlled by small carrier vesicles or tubules that have specific protein coats on their cytoplasmic surfaces. Clathrin-clathrin-adaptor coats function in many steps of intracellular transport and are the most extensively studied of all transport-vesicle coats. In recent years, the determination of structures of clathrin assemblies by electron microscopy, of domains of clathrin and of its adaptors has improved our understanding of the molecular mechanisms of clathrin-coated-vesicle assembly and disassembly.