Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells;pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer, heart disease, and Alzheimer's disease. Also, pathogens such as HIV take advantage of these pathways to infect cells and avoid immune surveillance. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. During the previous funding period a network of three types of clathrin adaptors that function in transport between the TGN and endosomes has been defined, consisting of the AP-1 complex, Gga proteins, and epsin-related proteins. Analysis of these adaptors in yeast has opened unique avenues to address the mechanism of ccv formation at the TGN and endosomes. Additionally, a novel role for ubiquitin binding by an endocytic BAR domain protein, Rvs167p, has been uncovered. This finding provides an opportunity to define functions for ubiquitin binding in endocytosis other than cargo recognition. A combination of genetic, chemical genetic, biochemical, and live cell imaging strategies will be applied to achieve four specific aims. First, the mechanism of clathrin coat assembly at the TGN/endosomes will be characterized in wild-type and mutant yeast strains using time-lapse live cell imaging of endogenously expressed fluorescent adaptors and clathrin. Second, complementary biochemical strategies will be directed at defining roles for particular adaptors in coat assembly, membrane binding and deformation, and cargo selection. Third, approaches in the first two aims will be extended to determine the functions of conserved TGN/endosome accessory factors in ccv formation, and new accessory factors will be identified. Fourth, the mechanism and function of ubiquitin binding by Rvs167p during endocytosis will be determined. Together these studies are expected to provide significant insights into the fundamental process of ccv formation and protein sorting in pathways between the TGN and endosomes, and during endocytosis, thereby helping to establish a foundation for understanding the roles of these processes in human disease.

Public Health Relevance

A fundamental aspect of animal cell structure and function involves protein transport between compartments within the cell. This project will employ yeast as a model eukaryotic cell to address the mechanism of transport mediated by a specific type of transport carrier, clathrin coated vesicles. Insights provided by this project will help to understand how defects in clathrin-mediated transport contribute to disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039040-25
Application #
8212359
Study Section
Cell Structure and Function (CSF)
Program Officer
Ainsztein, Alexandra M
Project Start
1988-02-01
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
25
Fiscal Year
2012
Total Cost
$369,792
Indirect Cost
$129,667
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Hung, Chao-Wei; Aoh, Quyen L; Joglekar, Ajit P et al. (2012) Adaptor autoregulation promotes coordinated binding within clathrin coats. J Biol Chem 287:17398-407
Di Pietro, Santiago M; Cascio, Duilio; Feliciano, Daniel et al. (2010) Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain. EMBO J 29:1033-44
Anand, Vikram C; Daboussi, Lydia; Lorenz, Todd C et al. (2009) Genome-wide analysis of AP-3-dependent protein transport in yeast. Mol Biol Cell 20:1592-604
Lorenz, Todd C; Anand, Vikram C; Payne, Gregory S (2008) High-throughput protein extraction and immunoblotting analysis in Saccharomyces cerevisiae. Methods Mol Biol 457:13-27
Mahadev, Ravi K; Di Pietro, Santiago M; Olson, John M et al. (2007) Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif. EMBO J 26:1963-71
Piao, Hai Lan; Machado, Iara M P; Payne, Gregory S (2007) NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor. Mol Biol Cell 18:57-65
Zegzouti, Hicham; Li, Wei; Lorenz, Todd C et al. (2006) Structural and functional insights into the regulation of Arabidopsis AGC VIIIa kinases. J Biol Chem 281:35520-30
Fernandez, G Esteban; Payne, Gregory S (2006) Laa1p, a conserved AP-1 accessory protein important for AP-1 localization in yeast. Mol Biol Cell 17:3304-17
Costaguta, Giancarlo; Duncan, Mara C; Fernandez, G Esteban et al. (2006) Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p. Mol Biol Cell 17:3907-20

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