A large number of proteins in the vacuolar membrane (VM; tonoplast), including transporters and receptors, support the various functions of the vacuole. Molecular analysis of membrane proteins is an essential step in understanding how the vacuole operates but so far only a small number of tonoplast proteins have been identified at the molecular level. Accordingly, mutant lines with altered level of tonoplast proteins for characterizing their physiological roles have been developed sparsely. Also, detecting activities of tonoplast proteins remains difficult as it requires a certain degree of enrichment of this organelle fraction. In order to extend our understanding of the vacuole, several groups have turned to proteomic analysis of tonoplast membrane proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high so that its composition can be accurately analyzed with mass spectrometry. In this chapter, we describe a simple method for the isolation of intact vacuoles and subsequent proteome analysis of the VM fraction of cells from Arabidopsis suspension cultures.
