Abstract
Turnover of membrane proteins or soluble proteins associated to plasma membrane involves clathrin-mediated endocytosis (CME), endosomal trafficking, and vacuolar degradation. Thus, endocytic and endosomal trafficking regulate numerous physiological processes, including mineral transport, hormone signaling, and pathogen response. Abscisic acid (ABA) signaling is triggered upon ABA perception by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), which are soluble proteins that can associate to membrane by interaction with members of the C2-domain ABA-related (CAR) protein family and the RING finger of seed longevity (RSL1) E3 ubiquitin ligase. Half-life of PYR/PYL/RCAR ABA receptors is regulated by ubiquitination and degradation in different subcellular compartments. In particular, pharmacological, genetic, and cell biology approaches have been used to study the different steps that encompass from CME to receptor degradation in the vacuole. In this chapter, we will focus on (1) coimmunoprecipitation (co-IP) assays of clathrin heavy chain (CHC) subunits together with HA-tagged PYL4 ABA receptor and (2) analysis of PYL4 delivery to the vacuole using the TMD23-Ub marker.
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Acknowledgments
We acknowledge Dr. Peter Pimpl for the vacuolar marker RFP-TMD23-Ub. Work in Dr. Rodriguez’s laboratory was supported by the Ministerio de Ciencia, Innovacion y Universidades, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (grant BIO2017-82503-R to P.L.R.). B.B-P. was funded by Programa VALi+d GVA APOSTD/2017/039.
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Belda-Palazón, B., Rodriguez, P.L. (2020). Degradation of Abscisic Acid Receptors Through the Endosomal Pathway. 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_4
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DOI: https://doi.org/10.1007/978-1-0716-0767-1_4
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