Structure and transport cycle of H+/sucrose symporter ZmSUT1

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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.