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.Young meristematic plant cells contain a large number of small vacuoles known to fuse during cell expansion and finally form a single large central vacuole, which occupies 80% to 90% of the cell volume (Marty, 1999). Cell expansion is, therefore, closely linked with increase of vacuolar volume. To reach the full cell size without expending too much energy, large amounts of inorganic ions are transported into the vacuole during vacuolar growth, concomitantly followed by a substantial water influx (Maeshima, 2001;Martinoia et al., 2007). In adult cells the vacuole plays a central role in cytosolic homeostasis. The concentration of metabolites in the cytosol and other metabolic active compartments has to be tightly regulated to facilitate the optimal functioning of a large number of metabolic pathways. Uptake, storage, and release of solutes into and o...

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance

“…The latter study also identified hexokinase in the tonoplast fraction (Carter et al, 2004). Glyceraldehyde 3 phosphate dehydrogenase was present in a proteomic study of vacuoles purified from cauliflower (Brassica oleracea) buds (Schmidt et al, 2007) and also in a barley tonoplast phosphoproteomic study (Endler et al, 2009). Enolase was identified as a protein in highly purified vacuoles from Arabidopsis cell suspensions (Shimaoka et al, 2004).…”

“…Evidence for tonoplast localization of glycolytic enzymes in plants has come from a number of independent proteomic studies, although it has been widely ignored as having no functional significance, being attributed to contaminating fractions (Carter et al, 2004) or characterized as soluble cytoplasmic proteins that have no role at the tonoplast (Schmidt et al, 2007;Endler et al, 2009). Triosephosphate isomerase was identified in a proteomic study of barley (Hordeum vulgare) tonoplast (Endler et al, 2006), as well as in the vegetative vacuole proteome of Arabidopsis of both whole vacuoles and tonoplast (Carter et al, 2004).…”

“…Enolase was identified as a protein in highly purified vacuoles from Arabidopsis cell suspensions (Shimaoka et al, 2004). Aldolase has been identified in several studies, including a quantitative proteomics analysis of rice (Oryza sativa) root tonoplast proteins induced by gibberellin treatment (Tanaka et al, 2004), in the proteome of vacuoles from cauliflower buds (Schmidt et al, 2007), and as a phosphopeptide in barley tonoplast (Endler et al, 2009). Jaquinod et al (2007), in a proteomic study of vacuoles from Arabidopsis cell suspensions, identified five glycolytic enzymes associated with the chaotropetreated membrane fraction, namely, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, aldolase, and triosephosphate isomerase.…”

“…Jaquinod et al (2007), in a proteomic study of vacuoles from Arabidopsis cell suspensions, identified five glycolytic enzymes associated with the chaotropetreated membrane fraction, namely, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, aldolase, and triosephosphate isomerase. In specific studies, the glycolytic proteins were associated with the membrane fractions following chaotrope treatment (Schmidt et al, 2007).…”

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

“…Such studies have also been performed with isolated vacuoles [20][21][22][23] and allowed the identification of new vacuolar membrane proteins. However, these studies do not give any information about changes of the vacuolar membrane proteome occurring in response to changes in environmental conditions.…”