(taken from the application) Mammalian CFTR and the Saccharomyces cerevisiae a mating pheromone transporter Ste6p are both members of the ATP binding cassettes (ABC) superfamily. In this project, we will use Ste6p as a model for investigation the biogenesis structure, and activity of CFTR. Because these two proteins share a common overall design, they are likely to require similar cellular machinery to ensure their proper membrane insertion, folding, and activity. The most prevalent CF allele, delta F508, causes misfolding of the mutant CFTR protein, resulting in its recognition by the ER quality control machinery, ER retention, and subsequent degradation by the ubiquitin- proteasome system. Little is known in the folding and biogenesis of secretory and membrane proteins or that recognize when they are not properly folded. One long-term goal of this project is to utilize the power of yeast genetics to identify cellular components that assist in and monitor the proper folding of ABC proteins. A second long-term goal is to purify and reconstitute Ste6p in active form to determine its biochemical properties. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate suppressors that stabilize an ER-retained, rapidly degraded mutant form of Ste6p identified in the previous project period; such suppressors will genetically pinpoint components of the cellular machinery involved in the membrane insertion, folding, and ER quality control of an ABC protein. 2) Determine how the ER quality control machinery distinguishes folded and unfolded cytosolic subdomains of a membrane protein, suing a C-terminal. Step6p truncation series and a """"""""designer chimera"""""""". 3) Purify and functionally reconstitute His-tagged Ste6p into phospholipid vesicles and examine ATPase activity, substrate specificity, and transport activity. The biochemical properties of purified Ste6p, CFTR, and MDR, will be compared in collaboration with P. Maloney, and colleagues. 4) To establish a second yeast CFTR model (ER-retained Ycf1p) that can be used in addition to Ste6p to dissect the ER quality control pathway. We are optimistic that much of the basic knowledge we acquired about yeast Ste6p will apply to human CFTR, and in the long-term will provide a firm foundation for developing directed chemical and physiological strategies to revitalize the defective gene product in CF patients.
