Gregory S. Payne - US grants

Clathrin-coated membranes have been implicated in a variety of intracellular protein transport processes in eukaryotic cells. Yeast contain both heavy and light chain clathrin subunits and display a secretory pathway analogous to that found in animal cells. In both cell types, newly synthesized cell surface glycoproteins are processed during transport through the endoplasmic reticulum, the Golgi body then secretory vesicles. To study the role of clathrin during intracellular protein traffic, the yeast clathrin heavy chain gene (CHC1) has been cloned and used to disrupt the chromosomal gene in vivo. Clathrin-deficient yeast are viable but grow more slowly than wild-type cells. Transport of two secreted proteins is anomalous in mutant cells and appears to reflect abnormalities in Golgi body or nascent secretory vesicle transport steps. A more precise definition of clathrin's function will be pursued by using in vitro mutagenesis of cloned DNA to generate temperature or cold-sensitive mutations of CHC1. Conditionally- defective alleles will be inserted into the chromosomal CHC1 locus. The immediate effects of conditional mutations will be determined by incubating cells at the nonpermissive temperature and monitoring the rate and fidelity of secreted and vacuolar protein transport and rates endocytosis. Three additional approaches are proposed to identify genes whose products affect stages of transport involving clathrin-coated membranes. First, a clathrin-deficient cell defect in mating pheromone precursors processing forms the basis for a screen to obtain other mutations which cause the same defect. Second, clathrin-coated vesicles will be extensively purified to characterize associated proteins. From the proteins, probes for the respective genes will be generated and used to obtain molecular clones. Third, genes capable of suppressing the growth properties of clathrin-deficient cells will be sought. Genes isolated by these approaches will be used in a combined genetic, molecular cloning and biochemical investigation of clathrin- coated membrane function.

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): A major sorting station in the secretory pathway is the trans Golgi network (TGN), where proteins are directed to the plasma membrane, endosomes, and lysosomes. Defects in sorting at the TGN can lead to inherited human diseases such as I-cell disease and Hermansky-Pudlak syndrome, and likely to contribute to more common, multigenic diseases such as cancer and heart disease. The long-term goal of this project is to define the molecular basis of protein sorting into vesicle-mediated pathways between the TGN and endosomes. Studies of Saccharomyces cerevisiae indicate that clathrin coated vesicles (ccv) participate in evolutionarily conserved protein transport between the TGN and endosomes. Two types of clathrin adaptors are implicated in these pathways, the AP-1 complex and monomeric Gga proteins. Analyses of the yeast adaptors has opened unique avenues to address ccv formation at the TGN/endosomes. A combination of genetic, molecular, biochemical, and cell biological strategies will be applied to achieve three specific aims. First, roles of Gga proteins and AP-1 in TGN/endosome clathrin-mediated trafficking pathways will be determined. Towards this end, roles for each adaptor in clathrin recruitment to membranes will be evaluated. Also, the relative distribution and function of each adaptor in different trafficking pathways between the TGN and endosomes will be defined. Second, the functions of two novel Gga2p-interacting proteins, Ent3p and Ent5p, will be determined. These proteins, which are required for clathrin-mediated traffic between the TGN and endosomes, are distinguished by the presence of ENTH-related domains. Function of these proteins in clathrin coat assembly and clathrin-mediated transport will be characterized in vivo using mutant cells and in vitro by development of a coat assembly assay using pure proteins and liposomes. Emphasis will be directed towards understanding the role of phosphoinositide binding by Ent3p and Ent5p ENTH-related domains. Additional TGN/endosome clathrin coat components will be identified using interaction and genetic approaches. Third, we will initiate a chemical genetic strategy to study AP-1 and Gga-mediated traffic by identifying and characterizing small molecule inhibitors. Together these studies are expected to provide significant advances in our understanding of the fundamental process of ccv formation and protein sorting at the TGN and endosomes.

clathrin; intermolecular interaction; vesicle /vacuole; biomedical resource;

binding sites; ubiquitin; intermolecular interaction; biomedical resource;

DESCRIPTION (provided by applicant): A major sorting station in the secretory pathway is the trans Golgi network (TGN), where proteins are directed to the plasma membrane, endosomes, and lysosomes. Defects in sorting at the TGN can lead to inherited human diseases such as I-cell disease and Hermansky-Pudlak syndrome, and likely to contribute to more common, multigenic diseases such as cancer and heart disease. The long-term goal of this project is to define the molecular basis of protein sorting into vesicle-mediated pathways between the TGN and endosomes. Studies of Saccharomyces cerevisiae indicate that clathrin coated vesicles (ccv) participate in evolutionarily conserved protein transport between the TGN and endosomes. Two types of clathrin adaptors are implicated in these pathways, the AP-1 complex and monomeric Gga proteins. Analyses of the yeast adaptors has opened unique avenues to address ccv formation at the TGN/endosomes. A combination of genetic, molecular, biochemical, and cell biological strategies will be applied to achieve three specific aims. First, roles of Gga proteins and AP-1 in TGN/endosome clathrin-mediated trafficking pathways will be determined. Towards this end, roles for each adaptor in clathrin recruitment to membranes will be evaluated. Also, the relative distribution and function of each adaptor in different trafficking pathways between the TGN and endosomes will be defined. Second, the functions of two novel Gga2p-interacting proteins, Ent3p and Ent5p, will be determined. These proteins, which are required for clathrin-mediated traffic between the TGN and endosomes, are distinguished by the presence of ENTH-related domains. Function of these proteins in clathrin coat assembly and clathrin-mediated transport will be characterized in vivo using mutant cells and in vitro by development of a coat assembly assay using pure proteins and liposomes. Emphasis will be directed towards understanding the role of phosphoinositide binding by Ent3p and Ent5p ENTH-related domains. Additional TGN/endosome clathrin coat components will be identified using interaction and genetic approaches. Third, we will initiate a chemical genetic strategy to study AP-1 and Gga-mediated traffic by identifying and characterizing small molecule inhibitors. Together these studies are expected to provide significant advances in our understanding of the fundamental process of ccv formation and protein sorting at the TGN and endosomes.