Structure and transport cycle of H+/sucrose symporter ZmSUT1

大多数高等植物以蔗糖作为其主要的可移动碳水化合物。质子驱动的蔗糖转运蛋白在蔗糖在植物细胞间和远距离分布中起着至关重要的作用。非常负的植物膜电位和蔗糖转运蛋白积累蔗糖浓度超过1M的能力，表明植物进化出具有独特结构和功能特征的转运蛋白。该项目将使用电生理和生物物理方法来阐明玉米蔗糖载体ZmSUT1的运输机制(I)和结构特征(II)。I)为了剖析ZmSUT1的运输周期并量化个别步骤，将使用复杂的双电极电压钳和膜片钳技术。此外，伴随着传输机制的构象变化将通过电压钳荧光法和单分子FRET测量来可视化。对蔗糖或H+转运受损的突变体以及转运蛋白胞外和胞内面的蔗糖和H+的表观亲和力进行研究，以建立植物蔗糖运输的机制模型。Ii)ZmSUT1的Lacy模型代表了寻找与蔗糖结合有关的氨基酸残基的结构基础。突变体的电生理特征与定点烷基化技术相结合将有助于揭示植物蔗糖载体的结构。

The majority of higher plants use sucrose as their main mobile carbohydrate. Proton-driven sucrose transporter play a crucial role in cell-to-cell and long-distance distribution of sucrose throughout the plant. A very negative plant membrane potential and the ability of sucrose transporters to accumulate sucrose concentrations of more than 1 M, indicate that plants evolved transporters with unique structural and functional features. The proposed project will employ electrophysiological and biophysical methods to elucidate the transport mechanism (I) and structural features (II) of the maize sucrose carrier ZmSUT1. I) To dissect the ZmSUT1 transport cycle and to quantify individual steps, sophisticated two-electrode voltage clamp and patch clamp techniques will be utilized. Furthermore conformational changes accompanying the transport mechanism will be visualized by voltage clamp fluorometry and single molecule FRET measurements. Mutants impaired in sucrose or H+ translocation and apparent sucrose and H+ affinities at the extra- and intra-cellular face of transporter will be studied to build a mechanistic model for plant sucrose transport. II) A LacY-based model of ZmSUT1 represents the structural basis for the search of amino acid residues involved in sucrose binding. The electrophysiological characterization of mutants in combination with site-directed alkylation techniques will shed light on the structure of plant sucrose carriers.