Endoplasmic Reticulum (ER) Associated Degradation of Membrane Proteins in Yeast

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DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common, inherited lethal disease in Caucasians in North America, and arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The majority of disease-causing mutations block the maturation of this secreted protein, such that CFTR becomes trapped in the endoplasmic reticulum (ER) and is degraded by the proteasome. This process is referred to as ER associated degradation (ERAD), and >30 ERAD substrates from yeast to man have been identified, many of which are linked to specific diseases. ERAD substrate selection and targeting are catalyzed by molecular chaperones, but to date it has been difficult to define how and specifically at which step the chaperones impact CFTR degradation. Moreover, it has been challenging to define how a membrane protein, like CFTR, is delivered to the proteasome, and to identify uncharacterized genes required for maximal ERAD efficiency. To surmount existing technical barriers, the PI's laboratory established a yeast CFTR expression system and showed that unique chaperones play distinct roles during ERAD. To identify novel factors that catalyze ERAD, a micro-array "screen" was performed and a chaperone class with no previous connection to ERAD was found to facilitate CFTR degradation in yeast. In parallel with these studies, an in vitro system was established that recapitulates the polyubiquitination of CFTR and a CFTR homologue in yeast membranes. Based on these new data and tools, the goals of this grant application are to determine at which step in the CFTR degradation pathway known and newly identified chaperones function. And, for the first time, the requirements for substrate de-ubiquitination and proteasome targeting during ERAD will be investigated in a defined system. Importantly, data obtained from the in vitro assay will be complemented through in vivo studies in wild type and mutant yeast strains. This project reflects the PI's long-term interest in defining the molecular machines responsible for protein biogenesis in the ER, and this grant application constitutes the primary focus of ongoing research in the PI's laboratory. Finally, the results obtained from the experiments described in this application will direct future efforts to delineate the CFTR maturation pathway in mammalian cells, an effort that is vital as ongoing chaperone-based therapies to treat CF and other protein conformational diseases are entering clinical trials.