Novel Genetic Approaches to Structure and Function of M*

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DESCRIPTION (provided by applicant): The long term goals of the proposed research are to develop and evaluate the utility of new methods for identifying and isolating functional regions of membrane transport proteins. It is anticipated that these methods will aid in assigning specific functions to particular regions of membrane proteins and will aid in determining the structures of membrane transporters. The main basis for the work is the technique of "genetic footprinting". In this technique, very large libraries of random, small, in-frame insertion mutations are generated in the target gene for a transporter. Following selection for transport function in vivo, the patterns of mutant alleles that either do or do not survive selection are displayed on gels. Regions of the gene that are missing from the selected population correspond to a "footprint" and indicate a region that is required for the selected function. In contrast, regions that are visible on the gel indicate the regions that are permissive, that is, can tolerate the insertion without loss of the selected function. The second and third parts of the proposed work are aimed at identifying and isolating stable regions of transport proteins that retain the ability to participate in subunit interactions. These regions may be useful for biochemical or structural studies. Truncated versions of transporter molecules that interfere with the function of the wild-type transporter (i.e. are dominant-negative) will be identified. The work will be carried out using the maltose transport system of E. coli as a model. This system is a well-characterized bacterial ATP binding cassette, (ABC) transporter. The study of bacterial transport systems offers the opportunity to use both genetic and biochemical approaches to investigate the structure of membrane transport proteins and the mechanism of active transport. Significant sequence similarities among bacterial and eukaryotic transport proteins of the ATP binding cassette (ABC) family underscore the concept that knowledge obtained from the study of prokaryotic systems is generalizable to similar systems in higher organisms. Defects in many ABC transporters are associated with severe human diseases. These include cystic fibrosis (CFTR); hyperinsulinemia hypoglycemia (SUR1), macular degeneration (ABCR); adrenoleukodystrophy (ALDP); and multiple drug resistance in tumors (P-glycoprotein, MDR1).