Isolation of Vacuoles and the Tonoplast

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.