Abstract
To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker alpha-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 proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (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, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies 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 known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs.
Highlights
Plant cell vacuoles are multifunctional organelles that play a key role in plant physiology
Our membrane protein data was much more complete because the transporters identified in the previous study were well overlapped and our analysis identified a larger number of transporters with better coverage rates
We have shown the strategy developed in this work to be very efficient to get a broader view of the vacuole membrane proteome
Summary
Plant cell vacuoles are multifunctional organelles that play a key role in plant physiology. Most of the compounds present in vacuoles have to be transported in a passive or active manner across the tonoplast (the vacuolar membrane) for storage or degradation, but they need to be exported in response to plant cell demands. A proteomic approach was developed in order to identify the protein components present in the membrane and the soluble fractions of the Arabidopsis cell vacuoles. This approach includes: a mild oxidation step leading to the transformation of the cysteinyl residues into cysteic acid and methionine into methionine sulfoxide, which facilitates peptide assignment; an in-solution proteolytic digestion of membrane proteins; and/or a pre-fractionation of proteins by SDS-PAGE. The sub-cellular localization of several putative vacuolar proteins was confirmed by transient expression in Arabidopsis protoplasts overexpressing GFP-fusion proteins
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