Clathrin-coated vesicles mediate selective movement of receptors between cellular membranes. This process contributes to efficient endocytosis and subsequent recycling of receptors for nutrients and for proteins which need to be degraded. It is critical for rapid down-regulation of hormone and signalling receptors. Clathrin-coated vesicles also participate in transport of proteins from the trans-Golgi to lysosomes and secretory clathrin from the cytoplasm. Cytoplasmic tails of receptors are trapped by this polymerization process which also contributes to membrane vesiculation. Incorporation of receptors into a clathrin-coated vesicle is selective. This selectivity depends both on the signals provided by the receptor tails and the characteristics of the coated vesicles proteins that recognize the tails and nucleate clathrin assembly. The recognition- nucleation function has been attributed to the adaptor proteins which together with clathrin comprise the major protein constituents of the coated vesicle. The goal of this proposal is to define the molecular basis for control of clathrin-coated vesicle formation within the cell. This will involve characterizing the dynamics of known coated vesicle components (Aim 1), establishing whether any additional molecules are required for receptor binding and nucleation of clathrin assembly (Aim 2) and analyzing the recognition sites which influence the interactions of the coated vesicle components (Aim 3). Two experimental systems will be used. An in vitro assay for assembly of clathrin onto fragments of plasma membrane has been established. Assembly can be achieved using purified coat proteins from coated vesicles or cytosol. Monoclonal antibodies specific for coated vesicle components, purified components themselves, and soluble proteins containing receptor tail sequences will be used to define the molecular requirements for clathrin assembly. The second experimental approach will analyze the binding between coated vesicle components, independent of cell membranes. Assays for interaction between soluble receptor tails and adaptors and between adaptors and clathrin will be used to determine whether these interactions are direct or whether additional proteins are required. These assays will also be used to identify the subunits and domains of adaptors and clathrin involved in recognizing each other and receptor tails. Analysis of the molecular processes which control clathrin-mediated endocytosis will undoubtedly lead to a better understanding of how to manipulate this important pathway for macromolecule uptake. This will ultimately contribute to design of receptor-mediated therapeutics and to new approaches to vaccination, since this pathway is utilized for the uptake and processing of foreign antigens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038093-05
Application #
3294124
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-02-01
Project End
1995-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Pharmacy
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Wu, Shuang; Majeed, Sophia R; Evans, Timothy M et al. (2016) Clathrin light chains' role in selective endocytosis influences antibody isotype switching. Proc Natl Acad Sci U S A 113:9816-21
Goyos, Ana; Guethlein, Lisbeth A; Horowitz, Amir et al. (2015) A Distinctive Cytoplasmic Tail Contributes to Low Surface Expression and Intracellular Retention of the Patr-AL MHC Class I Molecule. J Immunol 195:3725-36
Brodsky, Frances M; Sosa, R Thomas; Ybe, Joel A et al. (2014) Unconventional functions for clathrin, ESCRTs, and other endocytic regulators in the cytoskeleton, cell cycle, nucleus, and beyond: links to human disease. Cold Spring Harb Perspect Biol 6:a017004
Sullivan, Chelsea S; Scheib, Jami L; Ma, Zhong et al. (2014) The adaptor protein GULP promotes Jedi-1-mediated phagocytosis through a clathrin-dependent mechanism. Mol Biol Cell 25:1925-36
Vassilopoulos, Stéphane; Gentil, Christel; Lainé, Jeanne et al. (2014) Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization. J Cell Biol 205:377-93
Majeed, Sophia R; Vasudevan, Lavanya; Chen, Chih-Ying et al. (2014) Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration. Nat Commun 5:3891
Holmes, Brandon B; DeVos, Sarah L; Kfoury, Najla et al. (2013) Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A 110:E3138-47
Asensio, Cedric S; Sirkis, Daniel W; Maas Jr, James W et al. (2013) Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway. Dev Cell 27:425-37
Stachowiak, Jeanne C; Brodsky, Frances M; Miller, Elizabeth A (2013) A cost-benefit analysis of the physical mechanisms of membrane curvature. Nat Cell Biol 15:1019-27
Young, Anna; Stoilova-McPhie, Svetla; Rothnie, Alice et al. (2013) Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly. Traffic 14:987-96

Showing the most recent 10 out of 67 publications