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

植物 H 偶联糖和氨基酸转运蛋白的表征

• 批准号: 9420599
• 负责人: Donald Loo
• 金额: $ 22.5万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-15 至 1998-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9420599&HistoricalAwards=false
• 关键词: Characterization; Plant; H+; Coupled; Sugar

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. ***

MCB-9420599肉毒杆菌 克隆的拟南芥H+/己糖转运蛋白（STP）、菠菜H+/蔗糖转运蛋白（SoSUC 1）和马铃薯H +/蔗糖转运蛋白（StSUC 1）以及拟南芥H+/氨基酸转运蛋白（NAT 2）将在非洲爪蟾卵母细胞中表达。 将使用2电极电压钳和切开卵母细胞凡士林间隙方法研究它们的转运动力学，以确定转运蛋白的外部和内部转运反应步骤。 将确定最大转运速率、imax和配体的表观亲和力K0.5对电压和配体浓度的依赖性。 反转电位的测量和标记底物和H+电流的通量的同时测量将用于确定运输的化学计量。 稳态前动力学的分析将在运输反应循环的部分反应步骤和膜中转运蛋白的数量方面提供独特的见解。 该动力学信息将用于制定和检查运输模型，以描述糖和氨基酸转运蛋白的动力学特性。 已通过补充酵母氨基酸转运突变体检测功能的突变转运蛋白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。这将使运输模型得到测试。 对于SoSUC 1和STSUC 1之间的具体相互作用的运输基板和运输抑制剂将进行调查和最佳的3D结构识别的运输确定的能量最小化和分子模型的建立。 从长远来看，转运类似物的一致结构可以用于靶向蔗糖转运蛋白，以设计新的除草剂和农药。 此外，突变体转运蛋白将用于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作物植物中。 本研究的目的是了解氨基酸和糖进出植物细胞的分子机制。氨基酸和糖是植物细胞必需的营养物质，通过H+-偶联共转运蛋白转运到植物细胞中，H +-偶联共转运蛋白是一种整合的膜蛋白，它将底物的吸收与H+泵ATP酶产生的H+的电化学梯度偶联。 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，这是作物产量的主要决定因素。 该方法将表达克隆的H+依赖的己糖，蔗糖和氨基酸转运蛋白在非洲爪蟾卵母细胞和使用电生理和放射性示踪剂通量技术来表征这些cotorporters的生物物理特性和动力学（配体特异性，配体化学计量，电压依赖性，可逆性）。 这些研究将结合分子生物学技术来表征突变转运蛋白，这将允许鉴定参与H+和底物结合和转运机制的特定氨基酸残基。 ***