A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture

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.

Figure 1. Major protein constituents and evaluation of purity of vacuoles isolated from Arabidopsis cell culture

(A) SDS-PAGE analysis of proteins (15 μg) isolated from protoplasts (P) and purified vacuoles (V) separated on 12% acrylamide gel stained with Coomassie blue (R250). The identification of the proteins present in the most intensely stained bands was determined by LC-MS/MS analysis. Numbers, on the left, represent the size of the molecular weight markers (MW) in kDa. (B) Enrichment and purity of vacuole samples were estimated by western blots. The vacuolar markers, Tonoplast Intrinsic Protein (TIP) (α and γ isoforms), were revealed using specific antibodies; cross-contaminations were evaluated using antibodies raised against: the outer envelop protein 21 (OEP 21) and the light harvesting complex b (LHC) of the chloroplast; the preprotein translocase of the mitochondrial outer membrane (TOM 40); the HDEL domain of the endoplasmic reticulum proteins and the plasma membrane P-type H⁺-ATPase.

Figure 2. Strategy used for the identification of vacuolar proteins purified from Arabidopsis cell culture

Vacuoles were purified from Arabidopsis thaliana suspension cultures. After vacuole purification, proteins were either separated by 12% SDS-PAGE and stained with Coomassie blue (A) or subjected to centrifugation to obtain 2 different enriched fractions: membranes (B, C) and vacuolar sap (D). Major bands from the whole vacuole SDS-PAGE migration were cut out and subjected to in-gel trypsin digestion (A, Fig. 1A). Proteins from the membrane fraction were either subjected to in-solution trypsin digestion (B) (see text and experimental procedures) or separated by a short SDS-PAGE migration and digested in-gel (C). Proteins from the soluble fraction were digested in-gel following a short SDS-PAGE migration (D). Peptides from (A) to (D) were separated by liquid chromatography prior to MS/MS analysis.

Figure 3. Cross-correlation of the different proteome analyses of the vacuolar membrane system

(A) This Venn diagram presents the proteins identified in our study with those presented in the different published studies. The combined data from Shimaoka et al., Szponarski et al. and Carter et al. and our dataset identified 815 non redundant proteins. Overlap of the different protein sets is shown. Numbers in parentheses indicate the total number of proteins found by a particular study. (B) This Venn diagram presents the overlap of transporters and H⁺-ATPase subunits identified in our study with those presented in the different published studies. The combined data from Shimaoka et al., Szponarski et al. and Carter et al. and our dataset identified 123 non redundant transporters and related proteins.

Figure 4. Sub-cellular localisation of selected proteins by transient expression of GFP-fusion proteins

GFP-fusion proteins were expressed either in tobacco leaf cells (A to H) or in Arabidopsis protoplasts (I to K) (1 represents the GFP fluorescence alone and 2 the overlay of transmission and GFP fluorescence). Nramp3-GFP (A1 & A2) and TIP2.1-GFP constructions (B1) were used as vacuolar protein controls and GFP-GUS protein (C1) as a cytosolic protein control. The tested fusion proteins were: (D1) Dwarf1, At1g19820; (E1) a band 7 family protein, At1g69840; (F1) putative sugar transporter, At1g19450; (G1) lipocalin, At5g58070 and (H1) CCD1, At3g63520. I–K: transient expression in Arabidopsis protoplasts, lipocalin (I & J), putative sugar transporter (K). The bar corresponds to 15 μm.