Biomedical subjects

Brett M Collins

Publications and source records attributed to Brett M Collins.

7 recordsLinked to original sources

PubMed2004

Gamma-COP appendage domain - structure and function.

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the beta-, gamma-, delta- and zeta-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the alpha-, beta'- and epsilon-COP subunits. Here, we present the structure of the appendage domain of gamma-COP and show that it has a similar overall fold as the alpha-appendage of AP2. Again, like the alpha-appendage the gamma-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian gamma-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of gamma-COP, Sec21p, a second binding site is proposed to exist on the gamma-COP appendage that interacts with the alpha,beta',epsilon COPI subcomplex.

ADP-Ribosylation Factors↗

PubMed2004

Adaptors for clathrin coats: structure and function.

Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrin forms a scaffold around these vesicles that is linked to membranes by clathrin adaptors. The adaptors simultaneously bind to clathrin and to transmembrane proteins and/or phospholipids and can also interact with each other and with other components of the CCV formation machinery. The result is a collection of proteins that can make multiple, moderate strength (microM Kd) interactions and thereby establish the dynamic regulatable networks to drive vesicle genesis at the correct time and place in the cell. This review focuses on the structure of clathrin adaptors and how these structures provide functional information on the mechanism of CCV formation.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin.

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.

ADP-Ribosylation Factors↗

PubMed2003

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF.

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Binding Sites↗

PubMed2003

The structure of the GGA1-GAT domain reveals the molecular basis for ARF binding and membrane association of GGAs.

The GGAs are a family of clathrin adaptor proteins involved in vesicular transport between the trans-Golgi network and endosomal system. Here we confirm reports that GGAs are targeted to the Golgi via interaction between the GGA-GAT domain and ARF-GTP, and we present the structure of the GAT domain of human GGA1, completing the structural description of the folded domains of GGA proteins. The GGA-GAT domain possesses an all alpha-helical fold with a "paper clip" topology comprising two independent subdomains. Structure-based mutagenesis demonstrates that ARF1-GTP binding by GGAs is exclusively governed by the N-terminal "hook" subdomain, and, using an in vitro recruitment assay, we show that ARF-GTP binding by this small structure is required and sufficient for Golgi targeting of GGAs.

ADP-Ribosylation Factor 1↗

PubMed2003

Structural basis for binding of accessory proteins by the appendage domain of GGAs.

The Golgi-associated, gamma-adaptin-related, ADP-ribosylation-factor binding proteins (GGAs) and adaptor protein (AP)-1 are adaptors involved in clathrin-mediated transport between the trans-Golgi network and endosomal system. The appendage domains of GGAs and the AP-1 gamma-adaptin subunit are structurally homologous and have been proposed to bind to accessory proteins via interaction with short sequences containing phenylalanines and acidic residues. Here we present the structure of the human GGA1 appendage in complex with its cognate binding peptide from the p56 accessory protein (DDDDFGGFEAAETFD) as determined by X-ray crystallography. The interaction is governed predominantly by packing of the first two phenylalanine residues of the peptide with conserved basic and hydrophobic residues from GGA1. Additionally, several main chain hydrogen bonds cause the peptide to form an additional beta-strand on the edge of the preexisting beta-sheet of the protein. Isothermal titration calorimetry was used to assess the affinities of different peptides for the GGA and gamma-appendage domains.

ADP-Ribosylation Factors↗

PubMed2002

Molecular architecture and functional model of the endocytic AP2 complex.

AP2 is the best-characterized member of the family of heterotetrameric clathrin adaptor complexes that play pivotal roles in many vesicle trafficking pathways within the cell. AP2 functions in clathrin-mediated endocytosis, the process whereby cargo enters the endosomal system from the plasma membrane. We describe the structure of the 200 kDa AP2 "core" (alpha trunk, beta2 trunk, mu2, and sigma2) complexed with the polyphosphatidylinositol headgroup mimic inositolhexakisphosphate at 2.6 A resolution. Two potential polyphosphatidylinositide binding sites are observed, one on alpha and one on mu2. The binding site for Yxxphi endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered mu2 linker, is necessary to allow Yxxphi motif binding. A model for AP2 recruitment and activation is proposed.

Adaptor Protein Complex 1↗