Analysis of Prevacuolar Compartment-Mediated Vacuolar Proteins Transport
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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.
Key wordsArabidopsis suspension culture cells Protoplast Prevacuolar compartment Transient expression Vacuolar transport Wortmannin treatment ARA7(Q69L) expression
We are grateful to the current and previous members, especially Dr. Yansong Miao of Prof. Jiang’s Laboratory and our long-term research collaborator, Dr. Peter Pimpl (University of Tübingen) for their contributions in setting up and improving the protoplasts transient expression system using suspension culture cells. This work was partially supported by grants from the Research Grants Council of Hong Kong (CUHK466610, 466011, 465112, 466313, CUHK2/CRF/11G, HKUST10/CRF/12R, and AoE/M-05/12), NSFC/RGC (N_CUHK406/12), NSFC (31270226), Shenzhen Peacock Project (KQTD201101), and CUHK to L.J.