Identifying Targets/Pathways of Chemical Probes for the Plant Endomembrane System

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The vacuole is prominent in plant cells and is required for viability. This compartment is responsible for storing sugars, pigments, ions, proteins, and volatile compounds necessary for the flavor and nutrition of fruits and vegetables, and it maintains cellular homeostasis by regulating cytosolic pH. Plant vacuoles breakdown and recycle cellular components, and are involved in detoxification, as do yeast and mammalian lysosomes. However, plant vacuoles perform additional functions in defense. The vacuole is part of the endomembrane system, which includes the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, pre-vacuolar compartments, endosomes and the plasma membrane. Transport between these compartments occurs via vesicle trafficking. Beyond a role in cargo delivery, the plant endomembrane system is essential for development and signal transduction. A large proportion of knockout mutations in the Arabidopsis endomembrane system are either lethal or provide no visible phenotype due to complete or partial redundancy. However, the use of diverse chemicals to interrogate molecular processes provides a novel avenue for rapid and effective dissection of mechanisms and gene networks in ways not feasible with mutation-based approaches. Three novel compounds have been identified that specifically disrupt the trafficking of membrane or soluble proteins to the vacuole. Sortin1 affects the morphology of the vacuole and the delivery of the vacuolar lumen proteins CPY and invertase. However, it does not affect the delivery of tonoplast or other membrane proteins or the morphology of Golgi, ER or endosomes. In contrast, Gravacin interferes with protein trafficking to the tonoplast (and the trafficking of at least one plasma membrane protein) but does not affect the delivery of lumen proteins. Another chemical, known as 050, affects the localization of proteins that traffic through the ER and induces the fusion of multiple compartments. This combination of pharmacological reagents is a powerful resource to discover new components of the trafficking machinery within these pathways. The challenge now is to: identify target(s) or/and pathway(s) of each compound by genetic approaches. The intellectual merit of this award is that novel components of the protein trafficking machinery will be identified using our reagents. Furthermore, this diverse set of chemical probes will permit scientists to understand the relationships between protein trafficking pathways to the vacuole and other pathways within the cell, a challenge for classical mutational approaches. More broadly, the chemical genomics approach can be easily translated to any eukaryotic system, as well as plants of economic importance and human disease and nutrition. A truly interdisciplinary approach to understand the mechanisms of endomembrane trafficking has produced a new cohort of scientists. During the last several years, students and postdocs in the Raikhel laboratory have become well-versed and experienced in cell and molecular biology, genetics, chemistry and computational sciences, all necessary to ensure the success of the proposal and to become competitive in the workforce. The broad impact of this award will continue by producing, in addition to scientific discoveries, a new generation of diverse scientists who can form complementary teams required for 21st century research and education.