Approximately one-third of all newly synthesized proteins pass through the secretory pathway. These proteins use cues within their primary structure as well as a conserved chaperone system in order to fold into their native, functional conformations. When folding is disrupted, i.e. by an inherited genetic mutation, misfolded proteins are "sensed" by the cellular proteostasis machinery and may then be targeted for ER-associated degradation (ERAD). ERAD substrates have been classified based on the location of the folding lesion within their ER lumenal, membrane, or cytoplasmic domains. Specifically, it appears that an ERAD substrate can be recognized at different points during its synthesis depending on the location of the "folding lesion". Ste6p is an ABC transporter found in the yeast S. cerevisiae, and is required for export of the a-factor mating pheromone. Truncation of Ste6p's large C-terminal cytoplasmic domain (Ste6p*) converts the protein into a substrate for the ERAD-C (cytoplasmic) pathway. Because the mutation that renders Ste6p* into an ERAD substrate resides at the extreme C-terminus, the "decision" for degradation must occur post-translationally. Recent evidence from the Brodsky lab indicates that when the truncated C-terminus of Ste6p* is transferred to other proteins it is sufficient to induce the degradation of the chimera, raising the possibility that the sequence can act as a "degron" to target proteins for degradation. I propose to dissect the ERAD pathway taken by chimeric proteins containing the Ste6p* degron. The first goal of this research is to determine the chaperones, ubiquitination machinery, and requirements for retrotranslocation for an ERAD substrate with a post-translational ERAD-C-type lesion. Second, since little is known about how integral membrane hydrophobicity influences ERAD efficiency, I propose to generate chimeric constructs including the Ste6p* degron with varied transmembrane domains. The goal of these studies is to determine if the difference in transmembrane hydrophobicity of an ERAD substrate correlates with degradation and/or membrane extraction efficiency. These studies will help to extend our knowledge of the complex mechanisms used by cells to degrade misfolded proteins, identify new factors that catalyze ERAD, and identify potential drug targets for disease-associated ERAD substrates.