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Biomedical subjects

B M Pearse

Publications and source records attributed to B M Pearse.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

Adaptor Protein Complex 2↗
PubMed2000

The structure and function of the beta 2-adaptin appendage domain.

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 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 beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.

Adaptor Protein Complex beta Subunits↗
PubMed2000

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.

Adaptor Protein Complex 1↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Clathrin↗
PubMed1998

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

We present a map at 21 A 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.

Adaptor Protein Complex alpha Subunits↗
PubMed1994

A chimera of the cytoplasmic tail of the mannose 6-phosphate/IGF-II receptor and lysozyme localizes to the TGN rather than prelysosomes where the bulk of the endogenous receptor is found.

We fused the cytoplasmic and transmembrane domains of the bovine mannose 6-phosphate/IGF-II receptor (MPR) to lysozyme, a monomeric secretory protein thought to be devoid of sorting information. When the resulting chimera (lys/MPR) was transiently expressed in COS cells or stably expressed in CV1 cells, it had a predominantly intracellular distribution in the trans-Golgi region, with less than 10% present on the surface. In contrast, a similar chimera containing the transmembrane and cytoplasmic domains of the low density lipoprotein receptor (lys/LDLR) was localized to the plasma membrane, even though it endocytoses efficiently. Exchanging domains between the lys/MPR and lys/LDLR chimeras indicated that the MPR cytoplasmic domain contains the information necessary to specify the intracellular localization of the chimeric molecule. This signal must be located in the membrane-proximal third of the tail, as deletion of the last 120 residues of the 163 residue tail has no obvious effect on the distribution of lys/MPR. However, the recycling of the lys/MPR does not completely mimic that of the intact endogenous MPR, as immunofluorescence labelling shows that they are predominantly in different locations, indicating a role for the lumenal domain of the MPR in determining the steady-state distribution of the MPR itself.

Animals↗
PubMed1994

Cloning of Drosophila beta-adaptin and its localization on expression in mammalian cells.

A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit beta-adaptins (beta and beta') has been cloned and sequenced. In its amino-terminal region (residues 1-575), the BAD1 sequence appears intermediate between that of the mammalian beta-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of beta'-adaptin. To test its functional characteristics, a 'myc'-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with alpha-adaptin, although other coated pits were noted which apparently did not contain alpha-adaptin. However, these are probably gamma-adaptin containing pits, as BAD1 was also found colocalized with gamma-adaptin in Golgi coated pits in which, typically, alpha-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike beta-adaptin which complexes with alpha-adaptin and beta'-adaptin which partners gamma-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix alpha-adaptin and gamma-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between beta subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of beta with alpha-adaptin and beta' with gamma-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to beta and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.

Adaptor Protein Complex 1↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Characterization of coated-vesicle adaptors: their reassembly with clathrin and with recycling receptors.

Adaptors sort out those receptors that participate in assembly of coated pits from those that are excluded. Two distinct adaptor units have so far been identified: (1) adaptors restricted to plasma membrane coated pits (HA-II type, named according to their elution position during hydroxylapatite chromatography) and (2) adaptors restricted to Golgi region coated pits (HA-I type). Adaptors contain a heterodimer of two 100-kDa polypeptides, a beta-adaptin (possibly carrying an essentially common clathrin-binding domain) and a distinct alpha- or gamma-adaptin characteristic of the type of adaptor and its specific location. Each adaptor in constructed from four different polypeptides. Thus HA-II adaptors contain a beta-adaptin and an alpha-adaptin in combination with a 50-kDa protein and a 16-kDa polypeptide. The HA-I adaptors contain a beta-adaptin and a gamma-adaptin in combination with a 47-kDa protein and a 19-kDa polypeptide. Both types of adaptors and also a 180-kDa polypeptide will promote the assembly of clathrin to form coats, the size range of which appears to be relatively restricted compared to cages made from clathrin alone. The HA-II adaptors, characteristic of plasma membrane coated pits, bind to the cytoplasmic tail of the LDL receptor. They also assemble with the mannose 6-phosphate receptor in vitro in the absence of membrane. When clathrin is included, the adaptors promote the assembly of coats containing bound receptor.

Animals↗
PubMed1988

Receptors compete for adaptors found in plasma membrane coated pits.

An affinity matrix of LDL receptor cytoplasmic tails binds the HA-II 100/50/16 kd complexes found in plasma membrane coated pits. Other receptors (or their cytoplasmic domains), which are localized in coated pits during endocytosis, inhibit this binding. This includes an 8 residue peptide containing tyrosine, corresponding to the cytoplasmic portion of a mutant influenza haemagglutinin. In contrast, the equivalent peptide lacking tyrosine (like the tail of the native haemagglutinin, a protein excluded from coated pits) does not compete. These results imply that the HA-II complex has a recognition site for a common signal, probably involving a tyrosine residue, carried by the LDL receptor and competing receptors also found in plasma membrane coated pits. The HA-II complex therefore fulfils the role of an 'adaptor', the name proposed for the structural units which mediate the binding of clathrin to receptors in coated vesicles. Another related complex, the HA-I adaptor, which is restricted to Golgi coated pits, probably does not recognize the 'tyrosine signal' on the LDL receptor tail. The HA-I adaptor is likely to contain a recognition site for a different signal carried by receptors, e.g. the mannose-6-phosphate receptor, which are found in Golgi coated pits.

Coated Pits, Cell-Membrane↗
PubMed1986

Three-dimensional structure of clathrin cages in ice.

We have collected tilt series of electron micrographs from unstained clathrin cages embedded in vitreous ice. From these micrographs we have generated three-dimensional reconstructions of individual hexagonal barrels, which show details of the internal structure. Four types of preparation have been examined: (i) coated vesicles; (ii) cages reassembled from clathrin heavy and light chains; (iii) reassembled cages treated with elastase to remove the light chains; and (iv) reassembled cages treated with trypsin to remove the light chains and the terminal domains of the clathrin heavy chains. In the intact and elastase-treated cages, the clathrin extends from the vertices into the interior of the polyhedron and forms an inner shell of material. Limited digestion with trypsin removes the inner shell, which indicates that this material corresponds to the terminal domains of the clathrin heavy chains.

Animals↗
PubMed1986

Location of the 100 kd-50 kd accessory proteins in clathrin coats.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.

Clathrin↗
PubMed1986

Immunofluorescent localization of 100K coated vesicle proteins.

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

Animals↗
PubMed1986

Clathrin cubes: an extreme variant of the normal cage.

Clathrin triskelions form polyhedral cages with hexagonal and pentagonal faces when dialyzed against suitable assembly buffers. However, when the buffer is made 12% saturated in ammonium sulfate and the dialysis is performed at 4 degrees C, clathrin polymerizes into cubes. The cube is constructed from eight triskelions with one at each corner. The edge length of the cube is approximately 45 nm, equivalent to the length of the leg of a triskelion. Thus, each edge of the cube is composed of two antiparallel legs overlapping over their whole length. The interactions between the legs in the cube are a subset of those postulated to occur in cages. Indeed, the cube can be derived from a pentagonal dodecahedron by removing 12 of the 20 triskelions with only slight adjustment of the legs of the remaining triskelions. The cube forms regular arrays and appears to be a favorable species for crystallization of clathrin.

Clathrin↗