Shiga toxin follows a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface glycolipids, the toxin is internalized and trafficked in retrograde fashion to the Golgi and endoplasmic reticulum (ER). From the ER lumen, the toxin must gain access to the cytoplasm, where it enzymatically inactivates the 28S ribosomal RNA, inhibiting protein synthesis. This recently discovered pathway from the endoplasmic reticulum lumen to the cytoplasm represents a cellular quality control mechanism responsible for degrading host proteins that misfold in the ER lumen (termed ER-associated degradation, or ERAD). Shiga toxin apparently "pretends" to be a misfolded host protein, and thereby utilizes the quality control pathway to gain access to ribosomes in the cytoplasm. In yeast, ERAD is known to require a pore called Sec61, as well as chaperones within the ER lumen. The host molecules involved in this pathway in higher eucaryotes are largely unknown. Recently, however, cholera toxin was found to be transported from the ER through Sec61. We devised a genetic screen for molecules involved in shiga toxin trafficking and isolated a cDNA encoding a novel chaperone. This chaperone, which we named HEDJ, was found to be localized to the ER lumen and to interact with Bip, a molecule known to be involved in protein translocation out of the ER. We demonstrate here that HEDJ interacts with shiga toxin in the ER lumen prior to toxin transport. We propose that STx, cholera and other toxins co-opt HEDJ and other chaperones for transport across the ER membrane. However, unlike host molecules transported in this manner, we propose that STx bypasses proteosomal degradation within the cytosol. Using in vitro assays developed in our laboratory, the experiments proposed here will address the role of HEDJ and other chaperones in toxin translocation to the cytoplasm. Sequential co-immunoprecipitation experiments will allow a detailed dissection of the ER-localized chaperones involved in toxin transport. Turning our attention to the toxin, structure-function analyses will determine which portion(s) of STx enables the molecule to interact with chaperones and exit the ER. Additionally, we will determine whether STx avoids proteosomal degradation and will begin to examine the mechanism of proteosomal avoidance.