title: Analysis of prevacuolar compartment-mediated vacuolar proteins transport.
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.
authors.affiliation: School 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.
authors.forename: Caiji
authors.initials: C
authors.lastname: Gao
authors.forename: Yi
authors.initials: Y
authors.lastname: Cai
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhuang
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
mesh_terms: D000730:Androstadienes / Q000008:administration & dosage; D017360:Arabidopsis / Q000187:drug effects; D029681:Arabidopsis Proteins / Q000096:biosynthesis*; D011992:Endosomes / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D049452:Green Fluorescent Proteins; D008967:Molecular Biology / Q000379:methods*; D021381:Protein Transport; D011523:Protoplasts / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis*; D021601:trans-Golgi Network / Q000187:drug effects
chemical_list: D000730:Androstadienes; D029681:Arabidopsis Proteins; D049452:Green Fluorescent Proteins; C495672:Ara7 protein, Arabidopsis; D020691:rab GTP-Binding Proteins; D000077191:Wortmannin