The long-term objective of this research proposal is to dissect the molecular events that allow the catalytic A1 polypeptide of cholera toxin (CT) to enter the cytosol of intoxicated eukaryotic cells. CTA1 ADP-ribosylates and irreversibly activates the stimulatory alpha subunit of the heterotrimeric G protein at the cytoplasmic face of the eukaryotic plasma membrane. The resulting downstream signaling events induce the life-threatening watery diarrhea seen in over one million cases of cholera since 1995. After it is secreted into the extracellular milieu by Vibrio cholerae, CT binds to the plasma membrane of eukaryotic cells and is transported in retrograde fashion to the endoplasmic reticulum (ER). CTA1 dissociates from holotoxin in the ER and then crosses the ER membrane to enter the cytosol. It is hypothesized that the ER-associated degradation (ERAD) pathway facilitates CTA1 translocation from the ER to the cytosol. ERAD is a quality control mechanism that recognizes misfolded proteins in the ER and exports them to the cytosol for ubiquitination and proteosomal degradation. A hydrophobic region in CTA1 is thought to trigger ERAD activity and stimulate CTA1 translocation to the cytosol; degradation in the cytosol is presumably avoided because CTA1 has a paucity of the lysine residues that serve as ubiquitin attachment sites. This project will test and elaborate upon the ERAD/CTA1 translocation model. Previous methods to measure CTA1 translocation were based on the downstream cytosolic effects of CTA1 activity, but this work will instead utilize a recently developed biochemical assay that directly monitors the CTA1 translocation event. The assay will be used to (i) identify the structural features of CTA1 that are required for translocation to, and persistence in, the cytosol; (ii) identify the putative ERAD factors that interact with CTA1 during the translocation process; (iii) delineate the physiological parameters required for the ER-to-cytosol transfer of CTA1, and (iv) establish a yeast-based system to study CTA1 translocation. These research activities will meet the short-term goal of establishing a productive research lab with the capacity to train undergraduate, graduate, and post-doctoral personnel. The proposed work will contribute significantly to the understanding of cholera pathogenesis, will generate important observations relating to ERAD function, and will produce information relevant to other toxins that follow the CT trafficking and translocation itinerary.
