The majority of wild type and nearly all of the disease-causing AF508 variant of CFTR fold inefficiently in the ER, and are ubiquitinated and destroyed by the cytoplasmic proteasome; however, the molecular mechanism by which CFTR is targeted for degradation is not completely clear. To begin to define which factors are specifically required for CFTR proteolysis, the protein was expressed in the yeast S. cerevisiae and found to be degraded by the ubiquitin-proteasome pathway, but was stabilized in yeast mutated for the cytoplasmic Hsc70, a molecular chaperone that associates with immature CFTR in the mammalian ER. In yeast, CFTR localizes to a unique site in the ER in the proteasome mutant, whereas it is dispersed throughout the ER in wild type or Hsc70 mutant cells. These combined results indicate that Hsc70 facilitates CFTR degradation and acts up-stream of the proteasome. To more precisely define the degradation pathway, CFTR-associated proteins will be identified by co-opting established yeast biochemical and proteomic methods. Analyzing the stability of CFTR in cells mutated or depleted for the corresponding genes will indicate which MR-interacting factors are required for proteolysis. Because CFTR turn-over can be abrogated at two distinct steps (catalyzed by Hsc70 and the proteasome, respectively), components acting at each point in the degradation pathway will be identified. As a complementary approach, it is hypothesized that genes required for the removal of CFTR will be induced in the CFTR-expressing Hsc70 and proteasome mutant strains. Candidate genes will be identified by screening microarrays using cDNA probes prepared from the appropriate strains. As above, CFTR degradation will be assayed in yeast mutated or depleted for the candidate genes. In sum, these studies serve as a prelude for examining whether mammalian homologues of the yeast factors play a similar role during CFTR degradation in human cells, long-term studies that will be catalyzed by continued collaborations with the local CF research community.
| Zhang, Hui; Schmidt, Bela Z; Sun, Fei et al. (2006) Cysteine string protein monitors late steps in cystic fibrosis transmembrane conductance regulator biogenesis. J Biol Chem 281:11312-21 |
| Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L et al. (2004) The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family. J Biol Chem 279:21992-2001 |
| Youker, Robert T; Walsh, Peter; Beilharz, Traude et al. (2004) Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast. Mol Biol Cell 15:4787-97 |
| Huyer, Gregory; Piluek, Wachirapon F; Fansler, Zoya et al. (2004) Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J Biol Chem 279:38369-78 |
