Based on the specific activity of the vacuolar marker ß-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42 fold with an average yield of 2.1 %. Absence of significant contamination by other cellular compartments was validated by western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic study. Therefore, a proteomic approach was developed in order to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles.
This approach includes:
(i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide,
(ii) an in-solution proteolytic digestion of very hydrophobic proteins,
(iii) a pre-fractionation of proteins by short migration on SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry.
This procedure allowed the identification of more than 650 proteins, 2/3 of which copurify with the membrane hydrophobic fraction and 1/3 with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20 % of the proteins identified were previously known to be associated with vacuolar activities. The proteins identified are involved in: ion and metabolite transport (26 %), stress response (9 %), signal transduction (7 %), metabolism (6 %) or have been described to be involved in typical vacuolar activities, such as protein- and sugar-hydrolysis. The sub-cellular localization of several putative vacuolar proteins was confirmed by transient expression of GFP-fusion constructs.