The general objective is to study the structure of clathrin and associated proteins as a way of understanding the molecular basis of the formation and function of coated pits and coated vesicles in the cell. The proposed experiments involve an integration of structural, biochemical and molecular biological approaches that take advantage of the unusual special features of clathrin. Two parts of the long range project are presented in this proposal. The first part is the determination of the primary structure of calf brain clathrin heavy chain. This work involves cloning the heavy chain cDNA and requires protein chemical and recombinant DNA methodologies. The sequence information will be then used to study the domain organization and its relation to the topological map of the heavy chain. Analysis of domain organization will be carried out at three levels: 1) computer analysis of the primary structure; 2) amino terminal sequence determination of selected proteolytic fragments along the heavy chain; and 3) immunoelectron microscopy mapping of antibodies raised against selected synthetic peptides of the heavy chain. The second part of the project is the analysis of interactions between clathrin and associated proteins that participate in the regulation of coated vesicle formation. We will characterize the oligomeric structure of 100 Kd/50 Kd complex and map its binding site along a clathrin arm. The approach involves chemical crosslinking and single molecule electron microscopy. We will also attempt to establish which subunits of 100 Kd/50 Kd complex are in contact with clathrin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM036548-01
Application #
3290732
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1986-04-01
Project End
1989-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
1
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Böcking, Till; Aguet, François; Rapoport, Iris et al. (2014) Key interactions for clathrin coat stability. Structure 22:819-29
Ivanovic, Tijana; Boulant, Steeve; Ehrlich, Marcelo et al. (2011) Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking. Traffic 12:1179-95
Böcking, Till; Aguet, François; Harrison, Stephen C et al. (2011) Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18:295-301
Yu, Anan; Xing, Yi; Harrison, Stephen C et al. (2010) Structural analysis of the interaction between Dishevelled2 and clathrin AP-2 adaptor, a critical step in noncanonical Wnt signaling. Structure 18:1311-20
Xing, Yi; Bocking, Till; Wolf, Matthias et al. (2010) Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly. EMBO J 29:655-65
Guan, Rong; Dai, Han; Han, Dai et al. (2010) Structure of the PTEN-like region of auxilin, a detector of clathrin-coated vesicle budding. Structure 18:1191-8
Rapoport, Iris; Boll, Werner; Yu, Anan et al. (2008) A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction. Mol Biol Cell 19:405-13
Yu, Anan; Rual, Jean-Francois; Tamai, Keiko et al. (2007) Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling. Dev Cell 12:129-41
Ma, Yu May; Boucrot, Emmanuel; Villen, Judit et al. (2007) Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation. J Biol Chem 282:9805-12
Cheng, Yifan; Boll, Werner; Kirchhausen, Tomas et al. (2007) Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly. J Mol Biol 365:892-9

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