Barbara M. F. Pearse

Coated vesicles from pig brain: Purification and biochemical characterization

Electron microscopy of various tissues has revealed the existence of intracellular vesicles with “coats” on their cytoplasmic surfaces. Such vesicles have been seen associated with micropinocytosis. A simple and rapid procedure for the purification of coated vesicles from pig brain has been developed. The structures obtained have an external diameter of about 600 A and a remarkable network-like appearance. The most striking observation about these vesicles, reported here, is that they contain essentially just one protein species, having an apparent molecular weight on sodium dodecyl sulphate gels of about 180,000. Digestion of the coats with trypsin or Pronase shows that most of this protein is located on the external surface (i.e. facing the cytoplasm) of these structures. The lipid vesicles thus liberated appear to have an external diameter of about 350 A. An estimate of the number of protein molecules per vesicle is consistent with the coat having an icosahedral arrangement of subunits.

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA‐II adaptors (found in plasma membrane coated pits) and the HA‐I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6‐phosphate (M6P) receptor‐a receptor which is concentrated in coated pits on both the plasma membrane and in the trans‐Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA‐II plasma membrane adaptor, which recognizes a ‘tyrosine’ signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA‐I adaptors. This suggests that there is an HA‐I recognition signal in another region of the M6P receptor tail, C‐terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans‐Golgi network, thus mediating efficient transfer of these enzymes to lysosomes.

On the structure of coated vesicles

Abstract Electron micrographs of tilted specimens of coated vesicles show that their coats are based on polyhedral lattices constructed from 12 pentagons plus a variable number of hexagons. We have identified three such structures among the smaller particles, two containing 108 molecules of clathrin and a third containing 84. The coats of larger particles are believed to be constructed on similar principles. This polymorphism enables a variety of vesicles to be accommodated in an economical manner.

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.

Clathrin coats at 21 Å resolution: a cellular assembly designed to recycle multiple membrane receptors

We present a map at 21 Å resolution of clathrin assembled into cages with the endocytic adaptor complex, AP‐2. The map was obtained by cryo‐electron microscopy and single‐particle reconstruction. It reveals details of the packing of entire clathrin molecules as they interact to form a cage with two nested polyhedral layers. The proximal domains of each triskelion leg depart from a cage vertex in a skewed orientation, forming a slightly twisted bundle with three other leg domains. Thus, each triskelion contributes to two connecting edges of the polyhedral cage. The clathrin heavy chains continue inwards under the vertices with local 3‐fold symmetry, the terminal domains contributing to ‘hook‐like’ features which form an intermediate network making possible contacts with the surface presented by the inner adaptor shell. A node of density projecting inwards from the vertex may correspond to the C‐termini of clathrin heavy chains which form a protrusion on free triskelions at the vertex. The inter‐subunit interactions visible in this map provide a structural basis for considering the assembly of clathrin coats on a membrane and show the contacts which will need to be disrupted during disassembly.

Clathrin coat construction in endocytosis

Electron cryomicroscopy of the clathrin coat and X-ray crystallography of parts of the clathrin heavy chain combine to give a detailed picture of the clathrin molecule, assembled as a cage. Recently determined domain structures of other components of the endocytic machinery, particularly the mu2 subunit and the alpha-appendage domain of the AP2 adaptor complex, provide important information on the sequence of recognition events involved in the dynamic process of clathrin coat assembly.

Functional Organization of Clathrin in Coats: Combining Electron Cryomicroscopy and X-Ray Crystallography

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.

The structure and function of the beta 2-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.

Clathrin: anatomy of a coat protein

Clathrin is a vesicle coat protein involved in the assembly of membrane and cargo into transport vesicles at the plasma membrane and on certain intracellular organelles. Recently, crystal structures of two separate parts of the clathrin heavy chain, a fragment of the proximal leg and the N-terminal domain, have been analysed, providing the first high-resolution data for a vesicle coat protein. Viewing these structures in the context of a hexagonal barrel coat, recently determined to 21 A by cryo-electron microscopy, provides new insights into the assembly of clathrin coats.

The Structure and Function of the Beta2-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.