Probing Plant Endomembrane Pathways with Sortin 1 and Tagged Triazines

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Compared to other eukaryotes, Arabidopsis contains a large number of genes predicted to be involved in trafficking processes. Unlike yeast, many trafficking genes are essential for plants and a large proportion of knockout mutations in components of the endomembrane system are lethal. Analysis of the Arabidopsis endomembrane system has shown that beyond proper delivery of cargoes, it is essential for aspects of plant development and signal transduction. To gain new insights into the sub-cellular interactions of the endomembrane system, it is critical to examine the function(s) of these essential genes. Chemical genomics (i.e. genomics-scale chemical genetics) is the use of small molecules to modify or disrupt the function of specific proteins. By contrast, classical genetics utilizes mutations to disrupt gene function. The concept of chemical genomics is that protein structure or can be altered by chemical binding. Given the immense structural complexities of proteins compared to genes, the number of potential effectors is vast. At the practical level, chemical genomics entails screening diverse chemical libraries for effectors of a process or a gene product of interest. This approach overcomes the limitations of mutant lethality because chemicals can be applied in a spaciotemporal manner, are tunable and their effects are frequently reversible. Chemical genomics provides an alternative to classical genetics, which is often hampered by an inability to identify loss-of-function mutations associated with essential functions such as endomembrane trafficking. A chemical-genomics screen in the model plant Arabidopsis thaliana identified a chemical (Sortin 1) that induces secretion of carboxypeptidase Y (CPY), a protein normally targeted to vacuoles via the N-terminal propeptide (NTPP) pathway. Sortin1 causes reversible defects in vacuole biogenesis and root development in seedlings. It leads to aberrant secretion of a C-terminal propeptide (CTPP) marker protein but not of a protein that is targeted via precursor vesicles that bypass the Golgi. Experiments to be performed in this project utilize biochemistry, proteomics, cell biology, and combinatorial chemistry to investigate Sortin 1 effects on protein targeting pathway(s). The molecular targets of the compound will be identified via hypersensitive and resistant mutant screens; two will be characterized in detail. An approach applicable to nonmodel organisms will also be pursued in which a tagged triazine library of compounds will be screened for new chemicals that interfere with vacuole targeting. These chemicals can also be immobilized for rapid affinity based target purification. The major goal of this project is to identify and analyze new components of trafficking pathways and their interaction with related signal transduction and developmental networks. It will also provide a suite of new approaches for investigating any essential pathway of interest. The bioactive chemicals to be identified will be useful for probing conserved processes such as endomembrane trafficking in other organisms. Targets identified in Arabidopsis may be used in non-model species to improve agronomic traits and increase crop value. Young scientists involved in this project will have a broad integrated experience within interdisciplinary chemical genomics research and will be well trained and prepared for independent academic careers.