The compartmental organization of eukaryotic cells relies on accurate distribution of proteins to different compartments and on retention of resident proteins within each compartment. The ability to sort proteins with different destinations is a key feature of the transport machinery which governs protein distribution and retention. The molecular basis of this selectivity is the focus of our proposal. Studies of yeast strains carrying mutations (chc1) in the clathrin heavy chain subunit revealed that clathrin plays an important role in several selective protein transport steps, including selective retention of Golgi membrane proteins, receptor-mediated endocytosis of the mating peptide alpha-factor, and sorting of proteins from the Golgi apparatus to the lysosome-like vacuole. Based on clathrin's function in Golgi membrane protein retention, a genetic screen was employed to isolate two additional mutants (lam mutants) which are defective in Golgi membrane protein retention. These studies provide the basis for a combined genetic and biochemical approach to investigate three aspects of selective protein transport: 1) the mechanism of Golgi membrane protein retention; 2) the role played by clathrin-associated proteins in each clathrin-dependent transport process; 3) the mechanism of clathrin- mediated alpha-factor endocytosis. Clathrin's interaction with Golgi membrane proteins will be investigated biochemically by fractionation of clathrin-coated vesicles, and genetically by determining the localization of mutant Golgi membrane proteins lacking cytoplasmic retention signals in chc1 mutants. Additional lam mutants defective in Golgi membrane protein retention will be identified and used to clone wild-type versions of LAM genes. Antibodies raised against LAM gene products will be used to characterize the Lam proteins. To further define the function of clathrin coats in selective transport, genes encoding clathrin-associated proteins (APs) will be cloned and used to generate gene disruptions. Clathrin-dependent transport pathways will be monitored in mutant strains. Different ap and chc1 mutations will be combined to explore genetic interrelationships. Biochemical fractionations will be used to assess physical interactions between different APs and between APs and clathrin. Finally, an in vitro assay for clathrin-dependent selective transport will be developed by reconstituting alpha-factor endocytosis in permeabilized cells. The functional components of the in vitro reaction will be identified and characterized by complementation of permeabilized mutant cells or fractionation of cytosol. Together, these studies will identify previously unrecognized components of the selective protein transport machinery and address the specific roles played by each identified participant.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM039040-06
Application #
3295823
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-02-01
Project End
1997-01-31
Budget Start
1993-02-01
Budget End
1994-01-31
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Daboussi, Lydia; Costaguta, Giancarlo; Ghukasyan, Razmik et al. (2017) Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans-Golgi network. Proc Natl Acad Sci U S A 114:3433-3438
Myers, Margaret D; Payne, Gregory S (2017) Vps13 and Cdc31/centrin: Puzzling partners in membrane traffic. J Cell Biol 216:299-301
Myers, Margaret D; Ryazantsev, Sergey; Hicke, Linda et al. (2016) Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis. Dev Cell 37:162-73
Myers, Margaret D; Payne, Gregory S (2013) Clathrin, adaptors and disease: insights from the yeast Saccharomyces cerevisiae. Front Biosci (Landmark Ed) 18:862-91
Daboussi, Lydia; Costaguta, Giancarlo; Payne, Gregory S (2012) Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network. Nat Cell Biol 14:239-48
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
Gorynia, Sabine; Lorenz, Todd C; Costaguta, Giancarlo et al. (2012) Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins. Mol Biol Cell 23:4416-29
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
van der Bliek, Alexander M; Payne, Gregory S (2010) Dynamin subunit interactions revealed. Dev Cell 18:687-8
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

Showing the most recent 10 out of 39 publications