Identification and Characterization of the Endosomal Recycling Inhibitor Endosidin2
Protein trafficking through the endomembrane system of plants is a highly dynamic and transient process. Protein trafficking plays a role in several signal transduction and developmental pathways such as gravitropic response, plant pathogen resistance, cell pattern formation and autophagy. One of the most dynamic compartments of the endomembrane system is the endosome. The endosome is comprised on proteins that transit to and from the plasma membrane, the trans-Golgi network, and the prevacuolar compartment. Given the highly dynamic nature of this compartment, chemical genomics is a useful approach to slow down trafficking of proteins or inhibit trafficking through specific compartments. Since high-throughput cellular phenotyping is not available in Arabidopsis thaliana, tobacco pollen was used as a model system for membrane cycling to identify putative trafficking inhibitors for Arabidopsis. Tobacco pollen displays polarized growth that is dependent on the delivery and recycling of protein to the apical tip, and tobacco pollen is highly amenable to high-throughput screening. Utilizing tobacco pollen, over 46,000 natural and synthetic compounds were assayed and 378 were found to inhibit pollen germination and/or morphology. Of these 378, 365 were novel compounds. These 378 were then screened on three highly characterized Arabidopsis plasma membrane protein known to cycle through endosomal compartments, the brassinosteroid receptor BRI1:BRI1:GFP, and two PINFORMED auxin efflux proteins PIN2:PIN2:GFP and PIN2:PIN1:GFP. With just these three markers, 129 compounds from the pollen screen disrupted the normal localization patterns of at least one of these proteins. To examine a specific physiological response, these 129 were screened for effects on root gravitropic response. Sixteen compounds were identified as strongly disrupting gravity response. From these, the compound Endosidin2 (ES2) was characterized. ES2 disrupted several specific plasma membrane markers without affecting any internal protein markers, with the exception of the auxin homeostasis protein PINFORMED5, which localizes to the endoplasmic reticulum. ES2 specifically inhibits endosomal recycling to the plasma membrane without affecting endocytosis. ES2 allows for specific interrogation into the effects of endosomal recycling without affecting any other internal compartments as Brefeldin A does, the only other compound known to inhibit endosomal recycling.