Diatloff Eugene, Forde Brian G, Roberts Stephen K
Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK.
Biochem Biophys Res Commun. 2006 Jun 9;344(3):807-13. doi: 10.1016/j.bbrc.2006.03.212. Epub 2006 Apr 19.
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.
从小麦（Triticum aestivum）中提取的低亲和力阳离子转运蛋白（LCT1）在甲基营养型酵母毕赤酵母中表达，并利用Ca(45)和Cd(109)研究其转运特性。选择一个克隆（LCT1#3），当暴露于100μM Ca(45)时，其对Ca的摄取量最高，为14pmol/10(6)个细胞/10分钟，用于进一步研究Ca(45)和Cd(109)的转运特性。我们首次报道LCT1对Ca的米氏常数（K(m)）为0.43±0.15mM Ca活性，这证实LCT1是一种低亲和力转运蛋白。有趣的是，当细胞暴露于≥60μM Cd时，与野生型细胞相比，毕赤酵母中LCT1的表达导致Cd(109)摄取减少。这是关于异源表达转运蛋白降低内源性转运蛋白运输Cd活性能力的首次报道。据我们所知，这是首次利用毕赤酵母证明植物离子转运蛋白的功能表达。
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