Dissecting the Mechanics of Hrd1 Mediated ERAD
Creators: name: Peterson, Brian
Publisher: name: My University
Year: 2023
Subjects: subject: endoplasmic reticulum associated degradation (ERAD)
subject: ubiquitin proteasome system (UPS)
subject: deep mutational scanning
subject: retrotranslocation
subject: multiplexed high throughput screening
subject: protein disorder
subject: Biological Chemistry
subject: Health Sciences

description: Approximately one-third of proteins fold within the endoplasmic reticulum (ER), and the ER employs several protein quality control systems to either refold or degrade misfolded proteins. ER-associated degradation (ERAD) selectively degrades misfolded proteins within the ER lumen, membrane, or cytosolic face using the ubiquitin-proteasome system. ERAD requires a membrane-embedded ubiquitin E3 ligase complex that recognizes, ubiquitinates, and facilitates the removal of the substrate from the ER membrane into the cytosol, where the substrate is delivered to the proteasome for degradation. Beyond its role in misfolded protein quality control, ERAD regulates biosynthetic pathways including the pathways involved in cholesterol synthesis. ERAD malfunction, including exploitation of this system by pathogens, contributes to many disease states. A mechanistic understanding of the ERAD system is critical to understanding how cells maintain homeostasis and the basis of numerous human diseases. The work presented in this dissertation is centered around the Hrd1 ubiquitin ligase complex. The Hrd1 complex is the only ERAD system that is capable of retrotranslocation: movement of misfolded lumenal proteins across the ER membrane to the cytosol for degradation. Hrd1 autoubiquitination regulates retrotranslocation, raising the question of how active, ubiquitinated, Hrd1 is spared from degradation. Using a targeted yeast genetic screen, we identified Ubp1, a membrane-anchored deubiquitinating enzyme, as a protein that returns Hrd1 into a non-ubiquitinated, stable, state. We also found that Ubp1’s deubiquitination activity is modulated by a ubiquitin-like domain in a component of the Hrd1 complex called Usa1. Based on this work, we proposed a model that Hrd1 cycles between autoubiquitinated and deubiquitinated states during the ERAD process. In ERAD, Hrd1 degrades two different types of proteins, both lumenal and membrane proteins. We developed a deep mutational scanning platform to test how Hrd1 accomplishes the degradation of these two diverse substrate classes. First, we focused on the Hrd1 mutations that prevented Hrd1-mediated degradation of only lumenal ERAD substrates. These screening results discovered two predicted disordered regions in Hrd1 that were required for lumenal degradation. This was surprising because both regions are on the cytosolic side of Hrd1 but were required for lumenal substrate degradation. Using reconstitution biochemistry, we found that one of these disordered regions promotes Hrd1 ubiquitination activity while the other is required for retrotranslocation. This second region interacts with lumenal substrate, suggesting it is needed to bind and bias the movement of lumenal ERAD substrate across the ER membrane addressing an outstanding question of how directionality is established during substrate retrotranslocation. Finally, the screening results also helped us identify Hrd1 mutations defective in membrane protein degradation. These mutants clustered in the proposed retrotranslocon cavity of Hrd1 implying this cavity has a dual function for degrading both lumenal and membrane protein substrates. The mechanism of how these ERAD-M mutations disrupt substrate degradation is the subject of ongoing work, but these findings provide indispensable genetic information and update models of Hrd1 function. In summary, in this dissertation we dissected mechanistic aspects of the Hrd1 ERAD system, beginning with a new regulatory factor, Ubp1, that allows Hrd1 to cycle through active and inactive ubiquitination states. Next, we provided an exhaustive map of Hrd1 residues and their functional consequences. We interpreted these results and determined how disordered cytosolic regions promote lumenal substrate retrotranslocation. Together, these studies advanced our mechanistic understanding of the ERAD process and provide avenues for future work.