HUMAN MITOCHONDRIAL FOLATE/ANTIFOLATE TRANSPORT

描述（由申请人提供）：线粒体的内膜有几个嵌入的转运蛋白，促进代谢物和辅助因子转运到线粒体。这些蛋白质与外膜中的机制协同工作，将阳离子和阴离子代谢中间体从细胞质带到哺乳动物细胞的基质中。酵母基因组中有35种可识别的线粒体壁内转运蛋白；只有13个函数是已知的。我们最近克隆了一个cDNA，该cDNA编码线粒体内蛋白，促进叶酸辅助因子转运到线粒体。有数据表明，在人体中发现的四氢叶酸抗代谢物的毒性的一个组成部分可能是由于这种转运体的抑制。缺乏这种转运体的哺乳动物细胞不能在缺乏甘氨酸的情况下存活。

DESCRIPTION (provided by applicant): The inner membrane of the mitochondria has several embedded transport proteins that facilitate the translocation of metabolites and cofactors into the mitochondria. These proteins work in concert with mechanisms in the outer membrane to bring cationic and anionic metabolic intermediates from the cytosol to the matrix of mammalian cells. There are 35 inner mitochondrial wall transporters recognizable in the yeast genome; the function of only 13 are known. We have recently cloned a cDNA that encodes an inner mitochondrial protein that facilitates the transport of folate cofactors into the mitochondria. There are data suggesting that a component of the toxicity of the tetrahydrofolate antimetabolites seen in man may be due to inhibition of this transporter. Mammalian cells deficient in this transporter cannot survive in the absence of glycine. In this application, we propose to study the mechanism of this transporter, defining the substrate specificity for the various forms of the folates found in cells and also for the various antifolates that have been used to treat human cancers. Transport process will be studied in isolated mitochondria and in recombinant protein reconstituted into proteolipid bilayers. The proteins in the outer and inner mitochondrial membrane that bind folates will be defined, as will any binding partners involved in the transport through inner or outer membrane of the mitochondria. The phenotype of mice genetically engineered to lack this transporter will be studied as a model for human genetic deficiencies of mitochondrial folate transport. The binding site for folates in this transporter and the orientation of the transmembrane domains will be determined by site-directed mutagenesis, random mutagenesis of peptides, and epitope mapping. These studies will lead to an understanding of the basic biochemistry of this transport process and will lead to an understanding of the role of the inner membrane folate transporter in antifolate toxicity and in human genetic disorders of folate metabolism.