E COLI MIN PROTEINS

该项目的长期目标是确定细菌在细胞中选择合适的分裂位点的机制，而不是位于细胞内其他地方的其他潜在分裂位点。由于minC、minD和minE这三个min基因的产物负责这个位点选择过程，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。先前的研究表明，在缺乏其他Min蛋白的情况下，MinD定位于极性和中细胞的潜在分裂位点，并且MinE或MinE结构域的共表达导致膜相关Gfp-MinD重新分布到结构中，其位置取决于MinE拓扑特异性结构域的存在。该提案的主要直接目标将是：i.确定其定位到极点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；2。利用荧光显微镜、分子遗传学和膜生物化学的结合，确定Gfp-MinD与MinE和MinE的n端区共表达诱导的不同膜相关MinD结构的分子基础；3。确定MinC的定位模式及MinC对MinD和MinE定位的影响；iv.完成MinE拓扑特异性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.描述在minCDE位点之外的新的微型细胞突变体，并确定它们中是否有任何编码MinD和MinE定位的拓扑目标。

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.