A Chemical Genetic Screen for Modulators of Exocytic Transport Identifies Inhibitors of a Transport Mechanism Linked to GTR2 Function

Membrane and protein traffic to the cell surface is mediated by partially redundant pathways that are difficult to perturb in ways that yield a strong phenotype. Such robustness is expected in a fine-tuned process, regulated by environmental cues, that is required for controlled cell surface growth and cell proliferation. Synthetic genetic interaction screens are especially valuable for investigating complex processes involving partially redundant pathways or mechanisms. In a previous study, we used a triple-synthetic-lethal yeast mutant screen to identify a novel component of the late exocytic transport machinery, Avl9. In a chemical-genetic version of the successful mutant screen, we have now identified small molecules that cause a rapid (within 15 min) accumulation of secretory cargo and abnormal Golgi compartment-like membranes at low concentration (<2 μM), indicating that the compounds likely target the exocytic transport machinery at the Golgi. We screened for genes that, when overexpressed, suppress the drug effects, and found that the Ras-like small GTPase, Gtr2, but not its homolog and binding partner, Gtr1, efficiently suppresses the toxic effects of the compounds. Furthermore, assays for suppression of the secretory defect caused by the compounds suggest that Gtr proteins can regulate a pathway that is perturbed by the compounds. Because avl9Δ and gtr mutants share some phenotypes, our results indicate that the small molecules identified by our chemical-genetic strategy are promising tools for understanding Avl9 function and the mechanisms that control late exocytic transport.Cell growth and proliferation, as well as the regulation of cell surface composition, are achieved by an intracellular transport machinery that delivers proteins and membrane to the cell surface. The transport machinery is regulated by environmental sensing and signaling pathways that are integrated for the fine-tuned control of transport to the cell surface. The mechanisms that regulate cell growth and proliferation are highly robust; therefore, they can function in a wide range of environmental conditions and even when some components of the transport or signaling machinery fail. In eukaryotic cells, this robustness is achieved in part by a complex network of membrane and protein traffic routes to the cell surface (17, 33). Defects in a transport pathway can result in cargo transport by an alternate route, making transport defects difficult to detect in mutant screens (17, 18). Therefore, relatively little is known about the mechanisms by which protein and membrane cargo is transported from late exocytic sorting compartments, the late Golgi compartments and endosomes, and we have yet to identify most of the components that mediate and regulate this process.Complex processes are more readily understood in relatively simple organisms. For this reason, the budding yeast Saccharomyces cerevisiae has become one of the most powerful experimental models for understanding intracellular transport, and most of the conserved components of the exocytic traffic machinery were first discovered by using yeast genetic strategies (27). We used a yeast genetic screen to identify a novel component of the late exocytic transport machinery, Avl9, a member of an ancient eukaryotic protein superfamily (18). Avl9 is essential in a mutant strain lacking Vps1, a dynamin homolog that is thought to function in transport vesicle formation at a late Golgi compartment (26, 34), and also lacking Apl2, a large subunit of the adaptor protein 1 (AP-1) complex, which is required for forming certain classes of clathrin-coated vesicles at late Golgi compartments and endosomes (18, 19, 31, 42). The apl2Δ and vps1Δ mutants have defects in an exocytic pathway(s), but these mutants, as well as an apl2Δ vps1Δ double mutant, grow well because cargo is rerouted into a remaining pathway(s) (18). Mutations such as avl9Δ, which are lethal in an apl2Δ vps1Δ strain but not in a wild-type strain, are expected to cause defects in a branch of the exocytic pathway that remains functional in the apl2Δ vps1Δ strain. Analogous to using mutagenesis to screen for a secretory block in the apl2Δ vps1Δ mutant, we performed a high-throughput screen of a large library of small molecules to identify compounds that inhibit the growth of the vps1Δ apl2Δ mutant but which have relatively little effect on wild-type cells. The targets of these compounds are potential components of the secretory machinery, and some of the compounds may interfere with an Avl9-related function. The biochemical function of Avl9 and related proteins is still unknown, and the inhibitors identified by our screen strategy could be valuable tools in understanding the role of Avl9 in both yeast and mammalian cells.Our high-throughput screen was successful in identifying novel exocytic transport inhibitors, and we describe the phenotypic effects of one structurally similar group of compounds in detail. Furthermore, we show that the toxic effects of this group of compounds are inhibited by highly expressing GTR2, which encodes a Ras-like small GTPase that plays a role in regulating nutrient-responsive TORC1 (target of rapamycin complex 1) kinase signaling, exocytic cargo sorting at endosomes, and epigenetic control of gene expression (7, 11, 14, 25, 37). Therefore, the small molecules identified by our chemical-genetic approach are promising tools for understanding how signaling pathways that respond to environmental conditions regulate the traffic pathways that mediate cell growth and proliferation.