MITOCHONDRIAL LOCALIZATION OF PROTEINS CONTAINING COILED COIL-HELIX DOMAINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 具体目标： 将ChChd3、ChChd6和Mia40克隆到带有GFP标签或Flag标签的哺乳动物表达载体中，用于荧光显微镜下的可视化。 将重组ChChd3、ChChd6和Mia40 DNA导入哺乳动物组织培养细胞，并确定野生型蛋白的定位。 利用生物信息学技术确定蛋白质上潜在的重要突变位点。 引入ChChd3、ChChd6和Mia40突变，并监测其对表达蛋白亚细胞定位的影响。 以确定蛋白质中的哪些氨基酸是将蛋白质靶向线粒体所必需的。 通过突变探索其他保守的序列基序及其对蛋白质的影响。 假设： 含有类似螺旋螺旋结构域(CHCH)的蛋白质有一个预测的线粒体定位。CHCH结构域中关键氨基酸的突变将破坏含有ChChd3和ChChd6蛋白的新CHCH结构域的靶向。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aims: To clone ChChd3, ChChd6 and Mia40 into mammalian expression vectors with either GFP tags or Flag tags for visualization in an epifluorescent microscope. To introduce recombinant ChChd3, ChChd6, and Mia40 DNA into mammalian tissue culture cells and determine localization of the wild type proteins. To use bioinformatics techniques to determine potentially important mutation sites on the proteins. To introduce mutations in ChChd3, ChChd6, and Mia40 and monitor the effect on subcellular localization of the expressed proteins. To determine which amino acids in the proteins are necessary for targeting the proteins to the mitochondria. To explore other conserved sequence motifs through mutation and the effect on the proteins. Hypothesis: Proteins containing similar coiled coil-helix domains (CHCH) have a predicted mitochondrial localization. Mutation of key amino acids in the CHCH domain will disrupt targeting of the novel CHCH domain containing proteins ChChd3 and ChChd6.