title: Isolation of Vacuoles and the Tonoplast.
abstract: Isolation of various subcellular compartments followed by a high-coverage proteomic analysis provides an unparalleled foundation for the functional analyses of proteins. Analyses of tonoplast preparations free of major contaminants provide insights into vesicular fusion machinery, solute transport, and the vacuole association with the cytoskeleton, whereas analyses of the vacuolar lumen have yielded numerous soluble glycosidases, proteases, and proteins involved in stress responses. In addition, vacuolar lumen preparations have also allowed characterization of a luminal solute content in response to various abiotic stresses. Here, I revisit and update one of the most successful methodologies for vacuole and tonoplast isolation.
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, I.N.I.A. Parque Científico y Tecnológico, Campus de Montegancedo, 28223, Pozuelo de Alarcón, Spain. jan.zouhar@upm.es.
authors.forename: Jan
authors.initials: J
authors.lastname: Zouhar
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000254:growth & development; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D007425:Intracellular Membranes / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry* / Q000254:growth & development; D011205:Povidone / Q000737:chemistry; D011523:Protoplasts / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D014617:Vacuoles / Q000737:chemistry*
chemical_list: D003470:Culture Media; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
keywords: Arabidopsis; Organelle isolation; Plant vacuole; Tonoplast