Abstract
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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Acknowledgments
We are grateful to the current and previous members of Prof. Jiang’s Laboratory for their contributions in improving methods of endocytic protein study. This work was supported by grants from the Research Grants Council of Hong Kong (CUHK14130716, 14102417, 14100818, R4005-18F, C4012-16E, C4002-17G and AoE/M-05/12), the National Natural Science Foundation of China (31670179 and 91854201) and Research Committee of CUHK to LJ.
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Luo, M., Zhu, Y., Liu, Z., Jiang, L. (2020). Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_2
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DOI: https://doi.org/10.1007/978-1-0716-0767-1_2
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