Expression and transport characterisation of the wheat low-affinity cation transporter (LCT1) in the methylotrophic yeast Pichia pastoris.
The low-affinity cation transporter (LCT1) from wheat (Triticum aestivum) was expressed in the methylotrophic yeast Pichia pastoris and its transport characteristics studied employing Ca(45) and Cd(109). A clone (LCT1#3) with the highest uptake of 14pmol of Ca/10(6)cells/10min when exposed to 100microM Ca(45) was chosen for further Ca(45) and Cd(109) transport characteristics. We report for the first time a K(m) for Ca by LCT1 of 0.43+/-0.15mM Ca activity which confirms LCT1 to be a low affinity transporter. Interestingly, the expression of LCT1 in Pichia resulted in reduced Cd(109) uptake compared to wild type cells, when cells were exposed to >or=60microM Cd. This is the first report of the ability of a heterologously expressed transporter to reduce the activity of endogenous transporter proteins to transport Cd. To our knowledge, this is the first demonstration of functional expression of a plant ion transporter using P. pastoris.
Diatloff
Eugene
E
Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK. e.diatloff@lancaster.ac.uk
Forde
Brian G
BG
Roberts
Stephen K
SK
D001693:Biological Transport, Active / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D014908:Triticum / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; C108683:LCT1 protein, Triticum aestivum; D010940:Plant Proteins; D011994:Recombinant Proteins; D002118:Calcium