Project Summary/Abstract Maintaining protein homeostasis– the delicate balance between protein synthesis, folding, and degradation– is essential for cellular health. Over a third of the cellular proteome and most membrane proteins are inserted into the endoplasmic reticulum (ER) to become resident in this organelle or to enter into the secretory pathway. Quality control (QC) mechanisms constantly monitor the proteome across secretory compartments– such as the ER and Golgi apparatus– and promptly recognize and degrade misfolded proteins. But within each compartment, the mechanisms by which QC machineries detect misfolded substrates for degradation remain unknown. Whether the mechanistic principles for substrate recognition are shared across compartments also remains an unanswered question. In the ER, when a protein cannot reach its native folded state, it is retro-translocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a pathway termed ER-associated degradation (ERAD). Degradation of membrane-spanning proteins (ERAD-M) is mediated by the ubiquitin ligase Hrd1 but how substrates are recognized by Hrd1 inside the membrane is not well understood. In Aim 1 (K99), I will test the hypothesis that Hrd1 locally thins the membrane to trap and select substrates for degradation. This model would explain how Hrd1 can recognize a broad range of substrates that expose diverse hydrophilic residues to the membrane – a hallmark of ERAD-M. The Hrd1 complex can also handle misfolded proteins in the ER lumen (ERAD- L). During ERAD-L, the substrate completely traverses the ER membrane from lumen to the cytosol, but how Hrd1 mediates this process is unclear. I will address this question in Aim 2 (K99) by “trapping” the substrate on Hrd1 as it crosses the ER membrane and by visualizing the complex using cryo-electron microscopy (cryo-EM). This work will clarify important biological questions as ERAD exemplifies a unique protein translocation mechanism that functions without the use of a signal sequence and a well-defined transmembrane channel. Next, in the independent phase (Aim 3, R00), I will focus on the Tul1 ubiquitin ligase residing in Golgi membranes and I will elucidate how it recognizes and handles its substrates. I will test the hypothesis that Tul1, like Hrd1, thins the membrane to specifically recognize and select membrane-spanning substrates. I will address these questions using expertise gained in the mentored phase, by visualizing the Tul1 complex in vitro by cryo-EM and by employing novel degradation assays in cells. With continued guidance of my mentor Dr Tom Rapoport, the Advisory Committee that will provide support in key aspects of my training, and the exceptional resources provided by the Harvard Medical School, I am well positioned to successfully finish this work. The experience I acquire during this award will fully equip me for career independence, where I will employ my skillsets to elucidate the mechanistic principles ...