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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References
Hara-Nishimura I, Hatsugai N (2011) The role of vacuole in plant cell death. Cell Death Differ 18:1298–1304
Marty F (1999) Plant vacuoles. Plant Cell 11:587–599
Tse YC, Mo B, Hillmer S et al (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16:672–693
Lam SK, Tse YC, Robinson DG et al (2007) Tracking down the elusive early endosome. Trends Plant Sci 12:497–505
Mo B, Tse YC, Jiang L (2006) Plant prevacuolar/endosomal compartments. Int Rev Cytol 253:95–129
Robinson DG, Jiang L, Schumacher K (2008) The endosomal system of plants: charting new and familiar territories. Plant Physiol 147:1482–1492
Lam SK, Siu CL, Hillmer S et al (2007) Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells. Plant Cell 19:296–319
Miao Y, Yan PK, Kim H et al (2006) Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells. Plant Physiol 142:945–962
Wang J, Cai Y, Miao Y et al (2009) Wortmannin induces homotypic fusion of plant prevacuolar compartments. J Exp Bot 60:3075–3083
Jia T, Gao C, Cui Y et al (2013) ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies. J Exp Bot 64:2817–2829
Cai Y, Zhuang X, Wang J et al (2012) Vacuolar degradation of two integral plasma membrane proteins, AtLRR84A and OsSCAMP1, is cargo ubiquitination-independent and prevacuolar compartment-mediated in plant cells. Traffic 13:1023–1040
Kotzer AM, Brandizzi F, Neumann U et al (2004) AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells. J Cell Sci 117:6377–6389
Denecke J, Aniento F, Frigerio L et al (2012) Secretory pathway research: the more experimental systems the better. Plant Cell 24:1316–1326
Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2: 2348–2353
Gao C, Yu CK, Qu S et al (2012) The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus. Plant Cell 24:2086–2104
Miao Y, Li KY, Li HY et al (2008) The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells. Plant J 56:824–839
Acknowledgements
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.
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Gao, C., Cai, Y., Zhuang, X., Jiang, L. (2014). Analysis of Prevacuolar Compartment-Mediated Vacuolar Proteins Transport. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 1209. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1420-3_9
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DOI: https://doi.org/10.1007/978-1-4939-1420-3_9
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1419-7
Online ISBN: 978-1-4939-1420-3
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