(Taken directly from the application) CFTR mediates the conductance of chloride ions across the apical membrane of epithelial cells. When it is dysfunctional or absent from this membrane due to mutations in the CFTR gene, epithelial surfaces are improperly hydrated so that viscous mucous secretions accumulate and the resultant disease pathology ensues. Many of the more than 500 disease causing mutations, including the most common, delta F508, present in more than 90% of patients, prevent the protein from reaching the apical membrane. The delta F508 protein is synthesized and can function as a regulated Cl-channel but does not proceed beyond the endoplasmic reticulum (ER). Both the wild-type and the delta F508 protein are rapidly degraded (T 1/2 about 1/2 h) at the ER but about 25% of the wild type molecules achieve a mature conformation enabling them to escape degradation and be transported to the Golgi. The objective of this proposal is to gain a better understanding of the factors which are responsible for the maintenance of a positive balance between maturation and degradation and to identify the rate limiting step in transport from ER to Golgi. This will be done through five specific aims. First, the degradation pathways will be characterized in more detail. We have recently shown that the ubiquitin-proteasome pathway plays an important role but there are others to be identified. Proteasome inhibition does not augment maturation of delta F508 but inhibition of the other proteolytic mechanisms might. Second, the influence of modifications that occur at the ER during synthesis will be elucidated. The efficiency of maturation of the wild-type protein can be improved by altering the number and location of N-glycosylation sites which are used and by dephosphorylation. The influence of these modifications on delta F508 synthesis will now be tested. Third, we shall identify chaperones (in addition to calnexin and hsc70) which interact with CFTR at the ER and attempt to manipulate these interactions as we have done with calnexin. Fourth, we shall further test the hypothesis that a complete native tertiary structure of the cytoplasmic aspect of CFTR is required for it to be recognized as competent for exit from the ER. Fifth, we shall explore an alternative model to the active retention of delta F508 CFTR at the ER. Instead, the mutant molecule would be defective in recognition by the ER to Golgi vesicular transport machinery. We shall identify components of this mechanism which control CFTR export, employing a new in vitro assay which reconstitutes vesicle budding from ER microsomes. The potential role of a specific family of chaperones (p24/25) in """"""""escorting"""""""" CFTR during export will also be examined.
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