Endocytosis and endosomal trafficking to vacuoles play important roles in regulating the homeostasis of plasma membrane (PM) proteins in plant cells. FREE1 (FYVE domain protein required for endosomal sorting 1) is a plant-unique component of the ESCRT (endosomal sorting complex required for transport) machinery. In free1 mutant plants, PIN-FORMED 2 (PIN2)-GFP was found to mislocalize from the PM to the tonoplast. In this chapter, we describe a detailed protocol for studying vacuolar sorting and degradation of PIN2-GFP by using T-DNA insertional mutants, dexamethasone (DEX) inducible RNAi lines, and other tools, including Fei-Mao (FM) dye staining and dark treatment. By using these methods, we illustrate the endosomal trafficking and vacuolar degradation of PIN2-GFP in plants.
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