A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters.
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.
Nozawa
Akira
A
Cell-Free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, Matsuyama, Japan.
Nanamiya
Hideaki
H
Miyata
Takuji
T
Linka
Nicole
N
Endo
Yaeta
Y
Weber
Andreas P M
AP
Tozawa
Yuzuru
Y
D017360:Arabidopsis / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014176:Protein Biosynthesis; D011510:Proteolipids
D017931:DNA Primers; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes