Yeast Chemical Genetics For Identifying Regulators of Late Secretory Traffic Pathways

Abstract

Abstract The intracellular transport of proteins and membrane lipids to the cell surface or between organelles is a fundamental process in eukaryotic cells. This process is required for the biogenesis and maintenance of organelles, as well as for traffic to the cell surface for cell growth and proliferation. The transport routes in the late secretory pathways are branched and complex, and their regulation requires sensing and responding to environmental conditions for proper control of cell growth. Both the transport and regulatory mechanisms are robust, so that defects can be overcome by alternate mechanisms. This complexity has made it difficult to identify the late exocytic transport machinery and its regulators. The goal of my thesis work was to use a yeast chemical genetic strategy to identify components of the exocytic transport machinery, and to generate useful chemical tools that will help us to understand how the machinery functions. I analyzed the effects of small molecules that we obtained in two similar high-throughput screens of large libraries of drug-like compounds, in order to identify compounds that cause a block in the late exocytic pathway. Several of our new compounds cause exocytic defects and are selectively toxic to yeast mutants in which one of numerous transport pathways are blocked. The design of the highthroughput screen strategy was based on that of an earlier mutant screen that led to the discovery of a novel component of the transport machinery, Avl9, a conserved eukaryotic protein that has not yet been well characterized. Some of our new compounds are expected to target Avl9 or proteins with functions related to that of Avl9. In order to identify proteins and processes affected by our compounds, I screened for genes which, when overexpressed, can suppress the toxic effects of our compounds. I found that highly-expressed GTR2, which encodes a Ras-family small GTPase, can suppress the effects of one of our compounds. Gtr2 and its paralog and binding partner, Gtr1, as well as their metazoan orthologs, signal nutrient availability to regulate both traffic and the activity of TOR (target of rapamycin) kinase, a master regulator of growth. Furthermore, the gtr1Δ and gtr2Δ mutants share some phenotypes with the avl9Δ mutant. Our results indicate that our new compounds will serve as tools to help us understand how Avl9 and Gtr proteins function in cellular response to environmental conditions for proper regulation of protein and membrane transport in the late exocytic pathway.