Search

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly., (© 2013 The Authors. Traffic published by John Wiley & Sons Ltd.)

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction (AJ) machinery and invade non-phagocytic cells by a clathrin-dependent mechanism. Here, we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E-cadherin. We demonstrate that non-adherent cells expressing the InlA chimera, as bacteria, can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes., (© 2012 John Wiley & Sons A/S.)

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.

The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues.

Skeletal muscle tissue is made up of highly organized multinuclear cells. The internal organization of the muscle cell is dictated by the necessary regular arrangement of repeated units within the protein myofibrils that mediate muscle contraction. Skeletal muscle cells have the usual membrane traffic pathways for partitioning newly synthesized proteins, internalizing cell surface receptors for hormones and nutrients, and mediating membrane repair. However, in muscle, these pathways must be further specialized to deal with targeting to and organizing muscle-specific membrane structures, satisfying the unique metabolic requirements of muscle and meeting the high demand for membrane repair in a tissue that is constantly under mechanical stress. Specialized membrane traffic pathways in muscle also play a role in the formation of muscle through fusion of myoblast membranes and the development of internal muscle-specific membrane structures during myogenesis and regeneration. It has recently become apparent that muscle-specific isoforms of proteins that are known to mediate ubiquitous membrane traffic pathways, as well as novel muscle-specific proteins, are involved in tissue-specific aspects of muscle membrane traffic. Here we describe the specialized membrane structures of skeletal muscle, how these are developed, maintained and repaired by specialized and generic membrane traffic pathways, and how defects in these pathways result in muscle disease.

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin-Hip1R interaction is involved in the cytoskeletal organization of coated pits.

We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.

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

Clathrin polymerization into a polyhedral basket, surrounding budding membrane vesicles, mediates protein sorting during endocytosis and organelle biogenesis. Adaptor proteins target clathrin assembly to specific membrane sites and sequester receptors into the clathrin coat. We have reconstituted complete clathrin basket formation from recombinantly expressed fragments of clathrin and adaptors. This reconstitution reveals a hierarchy of clathrin self-assembly interactions and demonstrates that adaptors control basket formation by alignment of the distal domains of the clathrin triskelion leg through their binding to the terminal domain.