Molecular Analysis of Plant Mitochondrial Substrate Transporters

针对豌豆内线粒体膜的二羧酸、单羧酸和谷氨酸/天冬氨酸转运蛋白的一组独特的单抗将被用于筛选这些转运蛋白的cDNA克隆的lambda gt11文库。二羧酸转运蛋白的部分克隆已经被分离出来，最初的工作将集中在这个蛋白质上。一旦全长克隆可用，将对它们进行测序，并使用分子建模技术来评估它们在膜内的结构。当获得结构模型后，将使用抗体和化学修饰剂进行测试，两者都针对合成肽，通过评估部分蛋白质暴露在线粒体膜的胞浆和基质面上来确认模型。这些克隆将用于通过体外转录/翻译和在细菌中的异源表达来生产蛋白质的前体形式。这些未成熟的蛋白质形式将被用来研究转运蛋白与线粒体膜的结合。它们还将被用来试图分离和重建负责识别运输蛋白并引导其并入膜的受体蛋白。线粒体是活细胞的能量工厂。分子进出线粒体的运动在细胞的能量代谢中起着不可或缺的作用。线粒体有一个非常复杂的膜结构，这是生化分析所不能修正的。分子生物学的新技术正被用来理解在其中产生能量和运输分子的膜。

A unique group of monoclonal antibodies that are specific for the dicarboxylate, monocarboxylate, and glutamate/aspartate transporters of the inner mitochondrial membrane of pea plants will be used to screen lambda gt11 libraries for cDNA clones of these transport proteins. A partial clone for the dicarboxylate transporter has already been isolated and the work will initially concentrate on this protein. Once full length clones are available, they will be sequenced and molecular modelling techniques used to evaluate their structures within the membrane. When a structural model has been obtained, it will be tested by using antibodies and chemical modifiers, both targeted to synthetic peptides, to confirm the model by evaluating that portions of the proteins are exposed to the cytosolic and matrix face of the mitochondrial membrane. The cDNA clones will be used to produce the precursor form of the protein by in vitro transcription/translation and heterologous expression in bacteria. These immature forms of the protein will be used to study the incorporation of the transporter into the mitochondrial membrane. They also will be used in an attempt to isolate and reconstitute the receptor protein that is responsible for recognizing the transport protein and guiding its incorporation into the membrane. Mitochondria are the living cell's energy factory. Movement of molecules into and out of mitochondria plays an integral part in the cell's energy metabolism. The mitochondrion has a very complex membrane structure which has not been amendable to biochemical analysis. New techniques of molecular biology are being used to understand the membranes in which energy is generated and molecules are transported.