CFTR, the protein whose defect results in cystic fibrosis (CF), is a member of a superfamily of structurally related membrane proteins designated the ATP binding cassette (ABC) proteins. ABC proteins are comprised of two homologous halves, each half containing six predicted membrane spanning segments and an ATP nucleotide binding fold (NBF) domain. Another membrane of the ABC superfamily is the STE6 protein of Saccharomyces cerevisiae, a transporter which mediates export of the mating pheromone a-factor. In this project, we will use STE6 as a model for investigating the biogenesis and structure of CFTR; the similar overall design of the two proteins suggests they are likely to require similar cellular components to ensure their proper biogenesis, i.e. membrane insertion, folding, and trafficking to the cell surface. The most prevalent CF allele, deltaF508, appears to cause misfolding of CFTR, resulting in aberrant intracellular trafficking and degradation of the mutant protein. Little is known about factors that aid the biogenesis of complex multispanning membrane proteins such as CFTR. A major goal of this project is to utilize the power of yeast genetics to identify genes encoding cellular components that ensure the proper folding and progression of ABC proteins, in particular STE6 and CFTR, from their site of synthesis to the cell surface. A second goal is to probe intramolecular interactions within STE6 (and STE6-CFTR chimeras) as a means of determining which regions within these proteins interact to promote the proper conformation of the molecule. At third goal is to produce high levels of properly folded CFTR in yeast. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate mutations within STE6 that result in its mislocalization or rapid degradation, 2) identify suppressors of ste6 mutants obtained in aim 1; such suppressors will genetically pinpoint components of the cellular machinery involved in membrane insertion, folding, quality control, and trafficking of STE6, 3) Use STE6-CFTR chimeras to identify additional components of the cellular folding and quality control machinery, 4) Express properly folded CFTR at high levels in S. cerevisiae, and 5) Dissect the intramolecular interactions that govern assembly of STE6, particularly focusing on the role of charged residues in the transmembrane spans. These studies will allow us to identify candidate cellular components that assist CFTR in its biogenesis, and will provide a more detailed view of the structure and folding of CFTR. This information could serve as a basis for devising strategies to revitalize the defective gene product in certain CF patients.
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