A critical aspect of cellular physiology is the selective transport of ions, nutrients, proteins, and signaling molecules across cellular and organellar membranes, mediated by membrane transporter proteins. The Saccharomyces cerevisiae STE6 protein is a member of the ATP binding cassette (ABC) superfamily which includes a large number of membrane proteins that mediate transport and channel functions in eukaryotes and prokaryotes. Clinically important members of the ABC family include MDR, the mammalian multidrug resistance protein, and CFTR, the protein defective in patients with cystic fibrosis. The STE6 transporter in yeast mediates export of the lipopeptide mating pheromone a-factor, .and thus is required for mating by yeast. We will use biochemical, genetic, and molecular approaches to dissect the structure and function of STE6 and to identify cellular components involved in its folding and transit to the membrane. One major focus of the work proposed here is to define in molecular terms how transport of a-factor across the membrane is achieved and to determine how STE6 recognizes its a-factor substrate.
Specific aims related to this goal include: 1) development of an in vitro system for STE6-mediated translocation of a-factor that will allow examination of nucleotide hydrolysis and substrate specificity of STE6, 2) identification of residues within STE6 that are critical for substrate recognition by isolation of ste6 suppressor mutants that recognize an altered a-factor, 3) genetic conversion of MDR from a drug transporter into an optimized a- factor transporter, and 4) identification of functional domains of STE6 by isolation of """"""""dominant negative"""""""" mutants. These mutant proteins will be powerful reagents for biochemical dissection of STE6 transport in the in vitro system developed in the first aim. The function of a membrane protein depends upon its proper folding and membrane insertion. Little is known about factors that assist these processes for multispanning membrane proteins such as the ABC transporters. A second major focus of this proposal is to identify cellular components involved in the folding and assembly of STE6 by: 5) isolation of ste6 mutants whose loss of function is due to rapid degradation, and 6) subsequent isolation of suppressors which restore stability to an unstable STE6 mutant protein; these suppressors are expected to identify molecular chaperones and foldases which execute folding, assembly, and intracellular transport of STE6. These experiments will provide a high resolution view of the function, folding, and trafficking of STE6, that will also be relevant to other ABC proteins. This information can provide insight into preventing multi-drug resistance to chemotherapeutic agents by MDR, and into treating cystic fibrosis, which results from misfolded or misfunctional CFTR.
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