Identification and Characterization of Factors Involved in ENaC Biogenesis

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DESCRIPTION (provided by applicant): Proteins entering the secretory pathway are monitored by the ER quality control system and subject to Endoplasmic Reticulum Associated Degradation (ERAD) in the event they fail to properly fold. Defects in the ERAD pathway are associated with diseases such as cystic fibrosis, Huntington's disease and nephrogenic diabetes insipidus. Because ERAD plays an important role in a large number and several types of diseases, understanding this process will undoubtedly be critical for the development of therapeutics. However, recent progress in the field has brought to light the tremendous complexity and specificity of this process, suggesting that it will be critical to fully elucidate the ERAD pathway for each disease related substrate. The major focus of the study proposed here is to characterize the ERAD requirements for the disposal of the Epithelial Sodium Channel (ENaC). ENaC is responsible for salt reabsorption across epithelia including lung and kidney, and defects in ENaC degradation are associated with Liddle's syndrome and pseudohypoaldosteronism type 1. In addition, ENaC function has also been connected to the pathogenesis of cystic fibrosis. While a significant literature exists regarding post-ER ENaC degradation and trafficking, the early, ER associated "decisions" that target a large proportion of the protein for ERAD have gone largely unstudied. In this study I propose to: (1) Define the ENaC ERAD pathway by assaying degradation in yeast strains with mutations in known ERAD effectors; (2) Identify novel factors involved in early ENaC biogenesis and degradation by transcriptional profiling. The experiments proposed here will be carried out in a newly developed yeast expression system in which efficient ENaC ERAD has been reconstituted, and from which information on ENaC quality control in vertebrate cells is transferable.