Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles

Methods Mol Biol. 2020:2177:15-21. doi: 10.1007/978-1-0716-0767-1_2.

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.

Keywords: FM dye; FREE1; Multivesicular body; Plasma membrane protein; Prevacuolar compartment; Vacuolar degradation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / growth & development*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism*
  • Cell Membrane / metabolism
  • DNA, Bacterial / pharmacology
  • Dexamethasone / pharmacology
  • Endosomes / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mutation*
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / growth & development
  • Plants, Genetically Modified / metabolism
  • Protein Transport
  • Proteolysis
  • Vesicular Transport Proteins / genetics*

Substances

  • Arabidopsis Proteins
  • DNA, Bacterial
  • FREE1 protein, Arabidopsis
  • PIN2 protein, Arabidopsis
  • T-DNA
  • Vesicular Transport Proteins
  • Green Fluorescent Proteins
  • Dexamethasone