E COLI MIN PROTEINS

该项目的长期目标是确定细菌在中细胞选择适当分裂位点的机制，而不是位于细胞内其他地方的其他潜在分裂位点。 由于三个min基因minC、minD和minE的产物负责该位点选择过程，因此我们将尝试定义每个基因产物在位点选择过程中的作用机制。 以前的工作表明，MinD定位于潜在的分裂位点在两极和中间细胞在没有其他Min蛋白，MinE或MinE域的共表达导致重新分配的膜相关的GFP-MinD到结构，其位置取决于MinE拓扑特异性域的存在。 该建议的主要近期目标是：定义MinD中负责其定位于两极和中间细胞的决定因素，其在MinE环形成中的作用，以及其激活MinC分裂抑制剂的能力; ii.使用荧光显微镜、分子遗传学和膜生物化学的组合，确定由Gfp-MinD与MinE和MinE的N-末端区域的共表达诱导的不同膜相关MinD结构的分子基础; iii.确定MinC的定位模式以及MinC对MinD和MinE定位的影响; iv.完成MinE拓扑特异性结构域的高分辨率三维结构，并将MinE的拓扑特异性突变映射到三维结构; V.表征映射到minCDE基因座外部的新的小细胞突变体，并确定它们中的任何一个是否编码MinD和MinE定位的拓扑靶标。

The long-range goal of this project is to determine the mechanism used by bacteria to select the proper division site at midcell in preference to other potential division sites that are located elsewhere within the cell. Since the products of the three min genes, minC, minD and minE, are responsible for this site-selection process, we will attempt to define the mechanism of action of each of the gene products in the site-selection process. Previous work has shown that MinD localizes to potential division sites at poles and midcell in the absence of the other Min proteins, and that coexpression of MinE or domains of MinE causes redistribution of membrane-associated Gfp-MinD into structures whose position depends on the presence of the MinE topological specificity domain. The major immediate aims of the proposal will be: i. To define the determinants in MinD that are responsible for its localization to poles and midcell, its role in formation of the MinE ring, and its ability to activate the MinC division inhibitor; ii. To determine the molecular basis of the different membrane-associated MinD structures that are induced by coexpression of Gfp-MinD with MinE and with the N-terminal region of MinE, using a combination of fluorescence microscopy, molecular genetics, and membrane-biochemistry; iii. To determine the localization pattern of MinC and the effects of MinC on localization of MinD and MinE; iv. To complete the high-resolution three-dimensional structure of the MinE topological specificity domain and to map topological specificity mutations of MinE to the three-dimensional structure; v. To characterize new minicell mutants that map outside of the minCDE locus and determine whether any of them code for the topological targets for MinD and MinE localization.