Novel tonoplast transporters identified using a proteomic approach with vacuoles isolated from cauliflower buds

Abstract

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane. These results demonstrate that proteomic analysis of highly purified vacuoles from specific tissues allows the identification of new vacuolar proteins and provides an additional view of tonoplastic proteins.

Figure 1.

Evaluation of vacuolar membrane purity by western blot analysis (A) and fractionation strategy for cauliflower tonoplast proteins (B). A, Purified vacuoles were treated with five different extraction methods: KI and alkaline (NaOH) washing, acetone and C/M extraction, and treatment with sodium phosphate pH 6 followed by C/M pH 6 extraction, subjected to one-dimensional SDS-PAGE followed by in gel digestion and LC/MS/MS. B, For analysis of purification 10 μg of total membrane proteins (totM) and tonoplast proteins (vacM) were loaded on each lane. Compartment specific antibodies were used for western-blot analysis: chloroplastic ATPase α-subunit (ATP-α; 55 kD), luminal binding protein (BIP; 73 kD), plasma membrane aquaporin (PAQ1; 30 kD), and γ-TIP (25 kD).

Figure 2.

Organellar distribution of membrane protein isolates. Classification according to the total number of peptides. Tono, Tonoplast (excluding the V₁ domain of the V-ATPase); unknown > 1 TMD, unknown proteins with two or more transmembrane domains; unknown 1 TMD, unknown proteins with one transmembrane domain; PM, plasma membrane; Chl, chloroplast; MT, mitochondria; Endo, endosomes; Per, peroxisomes. TMDs were predicted using ARAMEMNON database accessible at www.aramemnon.botanik.uni-koeln.de.

Figure 3.

Identified proteins from the V-ATPase. Identified proteins are depicted in gray and missing components in white. The V₁ catalytic domain consists of subunits A and B in an A₃B₃ arrangement, C, D, E, F, G, and H. The V₀ membrane sector is composed of a and c subunits in an ac₆ complex. The detailed position of subunit e is not clear. Except for subunits c″ and e, all known components of the V-ATPase were identified.

Figure 4.

Subcellular localization of five novel identified proteins. Tonoplast localization of At5g45370 (A–D) and At1g16390 (E–H) in Arabidopsis protoplasts; bright-field image of an Arabidopsis protoplast (A and E), fluorescence of At5g45370∷GFP (B), fluorescence of KCO1∷DsRed (C), colocalization of the KCO1∷DsRed and At5g45370∷GFP fluorescence (D), fluorescence of At1g16390∷GFP (F), overlay of transmission and At1g16390∷GFP fluorescence (G), and lysed protoplast expressing At1g16390∷GFP showing the intact vacuole surrounded by cytosolic material from the broken cell (H). Vacuolar membrane localization of At4g28770 (I–K) and At1g06470 (N and O) in onion epidermal cells; fluorescence of At4g28770∷GFP (I), bright-field image (J and N), fluorescence of KCO1∷DsRed (K), colocalization of KCO1∷DsRed and At4g28770∷GFP (L), fluorescence of At1g06470∷GFP (M), and overlay of transmission and At1g06470∷GFP fluorescence (O). Localization of At1g17840 in the plasma membrane; overlay of transmission and At1g17840∷GFP fluorescence (P).

Figure 5.

Overlap between different proteomic analyses of the tonoplast. The Venn diagram compares membrane proteins identified in cauliflower with membrane proteins identified in the three largest Arabidopsis tonoplast proteomic analyses (Carter et al., 2004; Shimaoka et al., 2004; Jaquinod et al., 2007). Numbers in parentheses indicate the total number of identified proteins with at least one transmembrane domain in the particular study, excluding contaminating proteins of the proteomic analyses. The prediction of TMDs by TMHMM was considered for membrane proteins identified by Carter et al. (2004).