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Published on July 27, 2007; 10.1104/pp.107.096917

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Received January 30, 2007
Accepted July 6, 2007

Novel Tonoplast Transporters Identified using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds

Ulrike G. Schmidt , Anne Endler , Silvia Schelbert , Arco Brunner , Magali Schnell , H. Ekkehard Neuhaus , Daniéle Marty-Mazars , Francis Marty , Sacha Baginsky *, and Enrico Martinoia *

University of Zürich, Institute of Plant Biology, CH-8008 Zürich, Switzerland; Swiss Federal Institute of Technology, Institute of Plant Science and Functional Genomics Center Zürich, CH-8092 Zürich, Switzerland; Zürich-Basel Plant Science Center, CH-8092 Zürich, Switzerland; Technical University of Kaiserslautern, Plant Physiology, Postfach 3049, D-67653 Kaiserslautern, Germany and UMR Plante-Microbe-Environnement CNRS/INRA/University of Bourgogne BP 47870, F-21078 Dijon cédex, France

* Corresponding author; email: sacha.baginsky{at}ipw.biol.ethz.ch.

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. In order to analyze vacuoles from growing tissues we isolated vacuoles from cauliflower buds, which is constituted by a large amount of small cells but also contains cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as KI, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis 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 eleven 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, whilst a white-brown ABC transporter homologue 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.




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S. Schneider, D. Beyhl, R. Hedrich, and N. Sauer
Functional and Physiological Characterization of Arabidopsis INOSITOL TRANSPORTER1, a Novel Tonoplast-Localized Transporter for myo-Inositol
PLANT CELL, April 1, 2008; 20(4): 1073 - 1087.
[Abstract] [Full Text] [PDF]


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