Structure and transport cycle of H+/sucrose symporter ZmSUT1

大多数高等植物以蔗糖作为主要的可移动碳水化合物。质子驱动的蔗糖转运体在蔗糖在植物体内的细胞间转运和长距离转运中起着至关重要的作用。极负的植物膜电位和蔗糖转运体积累超过1 M蔗糖浓度的能力表明，植物进化出具有独特结构和功能特征的转运体。拟建项目将采用电生理和生物物理方法阐明玉米蔗糖载体ZmSUT1的转运机制(I)和结构特征（II）。I)为了剖析ZmSUT1传输周期并量化单个步骤，将使用复杂的双电极电压钳和膜片钳技术。此外，伴随传递机制的构象变化将通过电压箝位荧光法和单分子FRET测量可视化。我们将研究蔗糖或H+转运受损的突变体，以及转运体细胞外和细胞内表面蔗糖和H+的明显亲和力，以建立植物蔗糖转运的机制模型。II)基于lase的ZmSUT1模型为寻找参与蔗糖结合的氨基酸残基提供了结构基础。结合位点定向烷基化技术对突变体进行电生理表征，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.