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.
Vacuoles are organelles – specialized subunits within cells – that plants depend on for many crucial physiological processes. Natasha Raikhel's laboratory has been involved for several years in understanding the mechanisms of protein trafficking to vacuoles. Transport to the vacuole is complex, requiring multiple cellular elements (vesicles called endosomes) and players (proteins such as transporters ions and metabolites). Chemical genomics is an excellent tool that allows researchers to uncover previously unknown connections within and between cellular pathways. This approach uses small synthetic or natural compounds to perturb a particular biological function in a tunable and reversible manner. By combining chemical genomics, cell biology and genetics approaches, this project characterized several exciting small molecules that affect and identify connections and pathways within the cell. Sortin1 is valuable probe for dissecting the relationships between antioxidant status (free radicals), vacuolar integrity and the vacuolar accumulation of proteins and flavonoids in plants. It is important to understand protein and flavonoid transport to the vacuole because proteins are key components of nutritional value whereas flavonoids, in addition to traits like color and flavor, are strong antioxidants that protect cells and their DNA from damage. Consequently, results from this project have implications for age-related diseases such as dementia and cancer. At the cellular level, the Raikhel laboratory discovered that Sortin1 controls the endogenous (internal) amounts of the plant antioxidant molecule, gluthatione, and demonstrated that this endogenous metabolite is essential for the accumulation of flavonoids and proteins in plants. This demonstrates how chemical genomics can uncover unexpected connections within metabolic networks inside the plant. A third molecule, Endocidin1, was discovered to block a protein transport from a particular endosome. This discovery revealed that this endosome is involved in signaling that regulates steroid-responsive genes. Finally, a small molecule, Gravasin, was identified as a potent inhibitor of a plant’s ability to sense gravity. One of the multi-drug resistance transporters (ABC transporters) is a cognate target of Gravacin. ABC transporters have been implicated in multidrug resistance in cancer and the development of several diseases such as cystic fibrosis. This suggests that Gravasin could help in the search for drugs to enhance the effectiveness of cancer therapeutics. This project also provided an excellent interdisciplinary experience in science to graduate students, postdocs and professional researchers. These scientists are now well-versed and experienced in cell biology, chemistry and computational sciences, will become adept practitioners in this new field of chemical genomics and will be very competitive in the job market; in fact, several involved in this project have obtained positions within the country and abroad. Since the Center for Plant Cell Biology at the University of California, Riverside is an NSF REU (Research Experiences for Undergraduates) site, the project was an excellent and very successful venue for integrating undergraduate students from underrepresented institutions into chemical genomics research.
