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Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation
Published on: August 21, 2017
Analysis of prevacuolar compartment-mediated vacuolar proteins transport
Caiji Gao1, Yi Cai, Xiaohong Zhuang
1School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Room 384, Science Centre, Shatin, New Territories, Hong Kong, China.
Abstract:
Transient expression using protoplasts is a quick and powerful tool for studying protein trafficking and subcellular localization in plant cells. Prevacuolar compartments (PVCs) or multivesicular bodies (MVBs) are intermediate compartments that mediate protein transport between late Golgi or trans-Golgi network (TGN) and vacuole. Both wortmannin treatment and ARA7(Q69L) expression can induce PVC homotypic fusion and PVC enlargement in plant cells. Here, we describe detailed protocols to use transient expression of protoplasts derived from Arabidopsis suspension culture cells for studying protein trafficking and localization. Using three GFP-tagged vacuolar cargo proteins and RFP-tagged PVC membrane marker as examples, we illustrate the major tools and methods, including wortmannin treatment, ARA7(Q69L) expression and immunoblot analysis, to analyze PVC-mediated vacuolar protein transport in plant cells.
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