分析从内体隔室向液泡转运的膜蛋白。

Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles.

机构信息

Centre for Cell & Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

出版信息

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

DOI:10.1007/978-1-0716-0767-1_2

PMID:32632801

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.

摘要

内吞作用和内体运输到液泡在调节植物细胞质膜（PM）蛋白的动态平衡中起着重要作用。FREE1（内体分选必需的 FYVE 结构域蛋白 1）是植物中独特的内体分选复合物（ESCRT）机器的组成部分。在 free1 突变体植物中，发现 PIN-FORMED 2（PIN2）-GFP 从质膜错误定位到液泡膜。在本章中，我们描述了一种详细的研究方法，用于研究 T-DNA 插入突变体、地塞米松（DEX）诱导的 RNAi 系和其他工具（包括 Fei-Mao（FM）染料染色和暗处理）中 PIN2-GFP 的液泡分选和降解。通过使用这些方法，我们说明了植物中 PIN2-GFP 的内体运输和液泡降解。