Membrane proteins comprise ~30% of the proteome. Due to their complex topology, these proteins present a unique challenge for protein folding systems, which must integrate folding information within the cytoplasm, membrane, and ER lumen to determine whether substrates are appropriately folded or are misfolded and warrant degradation. These systems play critical roles in many disease states, including Cystic Fibrosis (CF) where a genetically encoded mutation (DF508) causes the 12 transmembrane CFTR chloride channel to misfold. Misfolded CFTR is degraded through a process termed endoplasmic reticulum associated degradation (ERAD). Manipulations that improve CFTR folding or inhibit ERAD stabilize mutant CFTR and partially restore chloride secretion to epithelial cells, the underlying defect in CF. A better understanding of ERAD mechanisms could provide therapeutic insights into CF and other related diseases. Yeast genetics and mammalian cell biochemistry have demonstrated that core components of ERAD are evolutionarily conserved. While these studies have provided a cellular perspective to the study of ERAD, genetic approaches for investigating ERAD in a live animal setting have not yet been developed. C. elegans has proven to be an outstanding in vivo system for the study of misfolded cytoplasmic substrates (e.g. polyQ, SOD-1, TDP-43). To explore mechanisms regulating misfolded membrane proteins in this system, we introduced the human DF508 mutation into C. elegans PGP-3, a 12 transmembrane ABC transporter closely related to human CFTR. When expressed in intestine and muscle, PGP-3wt and PGP-3DF508 show identical levels of mRNA but exhibit striking differences at the protein level. In both tissues PGP-3wt is stable and efficiently trafficked to the membrane, however membrane localization of PGP-3DF508 is strongly reduced. In intestinal cells PGP-3DF508 exhibits dramatically reduced protein levels, while in muscle PGP-3DF508 is stable but accumulates in both intracellular puncta and reticular patterns. Both physiological (adaptation to 200 mM NaCl) and genetic (Osr mutants
osm-7 and
osm-11) activation of the osmotic stress response pathway post-transcriptionally stabilize PGP-3DF508 but not PGP-3wt. To search for additional stabilizer mutants, we performed a forward genetic screen using the COPAS Biosort. We identified several mutants that post-transcriptionally stabilize the misfolded PGP-3DF508 protein and are currently working on cloning the affected loci. Our studies introduce C. elegans as a new and powerful in vivo model for the study of membrane protein misfolding, reveal tissue-specific differences in ERAD, and suggest that these pathways can be influenced by cell-non-autonomous mechanisms.