Characterization of Plant H+-Coupled Sugar and Amino Acid Transporters

Mcb-9420599克隆的拟南芥H+/己糖转运蛋白(STP)、菠菜H+/蔗糖转运蛋白(SoSU_1)、马铃薯H~+/蔗糖转运蛋白(S_SU_1)和拟南芥H~+/氨基酸转运蛋白(NAT_2)将在非洲爪哇卵母细胞中表达。它们的转运动力学将使用双电极电压钳和切开卵母细胞凡士林间隙的方法来研究，以确定转运体的外部和内部转运反应步骤。最大转运速率IMAX和配体的表观亲和力K0.5与电压和配体浓度的依赖关系将被确定。反转电位的测量以及标记底物的通量和H+电流的同时测量将用于确定运输的化学计量比。对稳态前动力学的分析将为运输反应循环的部分反应步骤和膜中转运体的数量提供独特的见解。这些动力学信息将被用来制定和检查运输模型，以描述糖和氨基酸转运体的动力学特性。突变的转运蛋白已经通过补充酵母氨基酸转运体进行了功能测试，将在卵母细胞中表达，并将对转运蛋白进行详细的动力学表征。这将允许对运输模型进行测试。对于SoSUC1和STSUC1，将研究转运底物和转运抑制剂之间的特定相互作用，并通过能量最小化和分子模型建立来确定用于转运体识别的最佳3D结构。从长远来看，在设计新型除草剂和杀虫剂时，可以使用转运类似物的连接结构来靶向蔗糖转运体。此外，突变的转运蛋白将被用来改变转基因植物中碳和氮的分配，这反过来将导致将有利的农艺性状引入农作物。这项研究的目标是了解氨基酸和糖进出植物细胞的分子机制。氨基酸和糖是必需的营养物质，通过H+偶联共转运蛋白进入植物细胞，转运蛋白是将底物的吸收与H+泵ATPase产生的H+的电化学梯度连接起来的膜蛋白。这些转运蛋白在同化物在源和库组织之间的分配中起着至关重要的作用，而源和库组织是作物产量的主要决定因素。该方法将在非洲爪哇卵母细胞中表达克隆的H+依赖的己糖、蔗糖和氨基酸转运体，并使用电生理和放射性示踪通量技术来表征这些共转运体的生物物理性质和动力学(配体专一性、配基化学计量比、电压依赖性、可逆性)。这些研究将与分子生物学技术相结合，以表征突变的转运蛋白，从而能够识别与H+有关的特定氨基酸残基以及底物结合和运输机制。***

MCB-9420599 Boorer The cloned H+/hexose transporters from Arabidopsis (STP), H+/sucrose transporters from spinach (SoSUC1) and potato (StSUC1) and a H+/amino acid transporter from Arabidopsis (NAT2) will be expressed in Xenopus oocytes. Their transport kinetics will be investigated using the 2- electrode voltage-clamp and the cut-open oocyte vaseline gap methods to determine the external and internal transport reaction steps of the transporters. The dependence of maximal rate of transport, imax, and the apparent affinity K0.5 for ligands on voltage and the concentration of ligands will be determined. Measurement of reversal potentials and the simultaneous measurement of the flux of labelled substrates and H+ currents will be used to determine the stoichiometry of transport. Analysis of the presteady- state kinetics will provide unique insights in both partial reaction steps of the transport reaction cycle and the number of transporters in the membrane. This kinetic information will be used to formulate and examine transport models to describe the kinetic properties of the sugar and amino acid transporters. Mutant transporters which have been tested for function by complementing yeast amino acid transport mutants will be expressed in oocytes and a detailed kinetic characterization of the transporters will be undertaken. This will allow transport models to be tested. For SoSUC1 and STSUC1 specific interactions between transported substrates and inhibitors of transport will be investigated and the optimal 3D structure for recognition by the transporter determined by energy minimization and molecular model building. In the long term, a concensus structure for transported analogues may be used to target the sucrose transporter in the design of novel herbicides and pesticides. Also, the mutants transtporters will be used to alter the partitioning of carbon and nitrogen in transgenic plants which will, in turn, lead to the introduction of favorable agron omic traits into crop plants. %%% The goal of this study is to understand the molecular mechanisms underlying the transport of amino acids and sugars into and out of plant cells. Amino acids and sugars are essential nutrients and are transported into plant cells via H+-coupled cotransporters which are integral membrane protiens that couple the uptake of substrates to the electrochemical gradient for H+ generated by the H+- pumping ATPase. These transporters play a vital role in the partitioning of assimilates between source and sink tissues which is a major determinant of crop yield. The approach will be to express the cloned H+-dependent hexose, sucrose and amino acid transporters in Xenopus oocytes and to use electrophysiological and radiotracer flux techniques to characterize the biophysical properties and kinetics (ligand specificity, ligand stoichiometry, voltage-dependence, reversiblity) of these cotransporters. These studies will be combined with molecular biological techniques to characterize mutant transporters which will allow the identification of specific amino acid residues involved in H+ and substrate-binding and transport mechanisms. ***