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1.  Chemical dissection of endosomal pathways 
Plant Signaling & Behavior  2009;4(1):57-62.
Membrane trafficking and associated signal transduction pathways are critical for plant development and responses to environment. These transduction pathways, including those for brassinosteroids and auxins, require endocytosis to endosomes and recycling back to the plasma membrane. A major challenge toward understanding these processes and their biological roles has been the highly dynamic nature of endomembrane trafficking. To effectively study endocytosis and recycling, which occur in a time frame of minutes, bioactive chemicals provide a powerful and exacting tool. Pharmacological inhibitors such as Brefeldin A (BFA) and the newly identified Endosidin 1 (ES1) have been used to define endosome compartments. ES1 is a clear example of the ability of chemicals to dissect even distinct subpopulations of endosomes involved in trafficking and signal transduction. The ability to characterize and dissect such highly dynamic pathways in a temporal and spatial manner is possible only using pharmacological reagents which can act rapidly and reversibly.
PMCID: PMC2634075  PMID: 19704710
endocytosis; endosome; endosidin 1; brassinosteroid; brefeldin A; ARF-GEF; SYP61; VHA-a1
2.  A Small Molecule Inhibitor Partitions Two Distinct Pathways for Trafficking of Tonoplast Intrinsic Proteins in Arabidopsis 
PLoS ONE  2012;7(9):e44735.
Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle–mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.
PMCID: PMC3434187  PMID: 22957103
3.  Powerful Partners: Arabidopsis and Chemical Genomics 
Chemical genomics (i.e. genomics scale chemical genetics) approaches capitalize on the ability of low molecular mass molecules to modify biological processes. Such molecules are used to modify the activity of a protein or a pathway in a manner that it is tunable and reversible. Bioactive chemicals resulting from forward or reverse chemical screens can be useful in understanding and dissecting complex biological processes due to the essentially limitless variation in structure and activities inherent in chemical space. A major advantage of this approach as a powerful addition to conventional plant genetics is the fact that chemical genomics can address loss-of-function lethality and redundancy. Furthermore, the ability of chemicals to be added at will and to act quickly can permit the study of processes that are highly dynamic such as endomembrane trafficking. An important aspect of utilizing small molecules effectively is to characterize bioactive chemicals in detail including an understanding of structure-activity relationships and the identification of active and inactive analogs. Bioactive chemicals can be useful as reagents to probe biological pathways directly. However, the identification of cognate targets and their pathways is also informative and can be achieved by screens for genetic resistance or hypersensitivity in Arabidopsis thaliana or other organisms from which the results can be translated to plants. In addition, there are approaches utilizing “tagged” chemical libraries that possess reactive moieties permitting the immobilization of active compounds. This opens the possibility for biochemical purification of putative cognate targets. We will review approaches to screen for bioactive chemicals that affect biological processes in Arabidopsis and provide several examples of the power and challenges inherent in this new approach in plant biology.
PMCID: PMC3243329  PMID: 22303245
4.  Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae 
Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics [1]. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.
In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.
Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.
PMCID: PMC2265672  PMID: 18179719

Results 1-4 (4)