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Plant and Cell Physiology Advance Access originally published online on November 2, 2007
Plant and Cell Physiology 2007 48(12):1815-1820; doi:10.1093/pcp/pcm150
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Short Communication

A Cell-Free Translation and Proteoliposome Reconstitution System for Functional Analysis of Plant Solute Transporters

Akira Nozawa1, Hideaki Nanamiya1, Takuji Miyata2, Nicole Linka3, Yaeta Endo1,2, Andreas P. M. Weber3 and Yuzuru Tozawa1,2,*

1 Cell-Free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, Matsuyama, 790-8577 Japan
2 Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, 790-8577 Japan
3 Institute for Plant Biochemistry, Heinrich-Heine-University, Geb. 26.03.01, Universitätsstraße 1, D-40225 Düsseldorf, Germany

*Corresponding author: E-mail, tozaway{at}ccr.ehime-u.ac.jp; Fax, +81-89-927-8276.


  Abstract

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.

Keywords: Cell-free translation system - Phosphoenolpyruvate - Proteoliposome - Transporter - Wheat germ

Abbreviations: DHAP, dihydroxyacetone phosphate; GFP, green fluorescent protein; PEP, phosphoenolpyruvate; PPT, phosphoenolpyruvate/phosphate translocator; TPT, triose phosphate/phosphate translocator.

(Received September 26, 2007; Accepted October 29, 2007)
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