Genetic Analysis of Complex Membrane Protein Insertion

点击翻译按钮获取中文摘要

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.