Genetic Analysis of Complex Membrane Protein Insertion

这项研究的目的是：a)更好地了解膜蛋白跨跨段之间的相互作用在膜插入和折叠中所起的作用，以及b)帮助表征突变膜蛋白的细胞代谢。这项研究将使用大肠杆菌乳胶渗透酶作为模型来分析跨越片段的相互作用是否有助于蛋白质的两个周质结构域的膜转位。这项分析将包括：a)开发一种用于未融合的紫胶渗透酶的单个结构域的周质定位的分析方法。B)相互作用在拓扑结构折叠中的作用(识别改变折叠的蛋白质部分(tm9-tm12)的错义变化)。这项研究还将调查涉及突变膜蛋白的两个现象。首先，改变的膜蛋白的产生通常对细胞有毒，可能是由于与膜本身或膜插入装置的异常相互作用。分离出降低这种毒性的突变体将有助于确定其来源。其次，改变的膜蛋白通常会被细胞内的蛋白酶迅速降解；突变筛选可以识别导致这种不稳定性的一种或多种蛋白酶。细胞被膜包围，这些膜保护它们免受外部环境的影响，同时允许营养物质向内传递。膜主要由脂类和蛋白质分子组成。这项研究通过检测蛋白质如何插入到合适的功能结构中，来研究细胞组装膜的机制。这项研究将分析一种特定的膜蛋白(大肠杆菌的乳糖渗透酶)的突变形式，它应该显示异常插入。改变的插入模式应该为正常的膜组装过程提供洞察力。

The objectives of the research proposed are, a) to provide a better understanding of the role that interactions between spanning segments of a membrane protein play in membrane insertion and folding, and b) to help characterize the cellular metabolism of mutant membrane proteins. The studies will use Escherichia coli lac permease as a model to analzye whether spanning segment interactions contribute to the membrane translocation of two of the protein's periplasmic domains. This analysis will include: a) developing an assay for the periplasmic localization of individual domains of unfused lac permease. b) the role of interactions in the folding of the topological structure (identifying missense changes in part of the protein (tm9-tm12) that alter folding). This research will also investigate two phenomena involving mutant membrane proteins. First, the production of altered membrane proteins is commonly toxic to cells, perhaps due to abnormal interaction with the membrane itself or the membrane insertion apparatus. Isolating mutants reducing this toxicity will help identify its source. Second, altered membrane proteins are often rapidly degraded by cellular proteases; Mutant screens can identify the protease or proteases responsible for this instability. %%% Cells are surrounded by membranes which protect them from the outside environment while allowing inward passage of nutrients. Membranes are composed mainly of a combination of lipid and protein molecules. The research proposed investigates the mechanisms used by cells to assemble membranes by examining how proteins are inserted into them in the appropriate functional structures. The research will analyze mutant forms of one particular membrane protein (lactose permease of the bacterium Escherichia coli) which should show aberrant insertion. The patterns of altered insertion should provide insights into the normal membrane assembly process.