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.
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