Date of Award
Doctor of Philosophy (PhD)
Cancer and Developmental Biology
Linda M. Hendershot, Ph.D.
John Cox, Ph.D.; Mondira Kundu, M.D., Ph.D.; Junmin Peng, Ph.D.; Michael A. Whitt, Ph.D.
ER-Associated Degradation, ER-Quality Control, Protein Degradation, Protein Folding
Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Proteins that fail quality control must be recognized and eliminated to maintain ER proteostasis. The ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. While multiple ER-Associated Degradation (ERAD) components have been identified and their roles elucidated, it remains less clear how folded domains on ERAD clients complicate their extraction from the ER and degradation. To address this, we used several luminal ERAD substrates with well-defined structural properties. Deglycosylation and digitonin permeabilization assays were used to monitor client extraction from the ER. Our fully unfolded clients were released into the cytosol when the proteasome was inhibited. Conversely, ERAD clients possessing a single folded domain were completely retrotranslocated but were not released from the ER membrane without proteasome function. These clients were fully reduced, but still retained structure in their folded domain. Overall, our data argue that ubiquitinated clients with well-folded domains can be dislocated from the ER in a p97-dependent manner, but proteasome activity is required to fully release them from the cytosolic side of the ER membrane and the ERAD extraction machinery.To decipher key steps in the extraction of non-glycosylated ER luminal proteins, and to determine how Hrd1, p97, and the proteasome contribute to the retrotranslocation of clients, we developed a novel in-cell biotin-based reporter system. The bacterial BirA biotin ligase was tethered to FAM8A1, a component of the retrotranslocon that interacts with Hrd1, thus positioning the biotin ligase near the cytosolic exit site. The non-secreted immunoglobulin (Ig) κ light chain (NS1 κ LC), which is composed of a poorly folded N-terminal domain and a well-folded C-terminal domain was used in the biotinylation assays. NS1 was modified at either the N- (BAP-NS1) or C- (NS1-BAP) terminus with a biotin acceptor peptide (BAP) allowing us to detect cytosolic exposure of both ends of this ERAD client. We established that BAP-tagged NS1 constructs were still ERAD substrates and that BirA-tagged FAM8A1 still assembled with the retrotranslocon. We found that both termini of NS1 were readily biotinylated when the proteasome was inhibited by MG132 treatment, or when dominant negative constructs of Hrd1 and p97 were expressed. To differentiate between full extraction of NS1 and partial exposure of the termini to the cytosolic side of the ER, we permeabilize the plasma membrane with digitonin. Cytosolic proteins were released, but both biotinylated and non-biotinylated NS1 remained cell-associated. This argues that the biotinylated light chain was not fully extracted from the ER when NS1 degradation was inhibited with either MG132 or with the Hrd1 and p97 mutants. The digitonin permeabilized cells expressing BAP-NS1 were treated with proteinase K to assess how much of this client was exposed to the cytosolic side of the ER. We found that the NS1 κ light chain was only partially extracted when cytosolic ERAD components (proteasome and p97) were impaired. These data indicate that the termini of this ERAD client can be inserted into the retrotranslocon and “sample” the cytosolic side but require the action of an E3 ligase, the p97 complex and the proteasome to be fully extracted.
Oikonomou, Christina (https://orcid.org/0000-0002-5969-1323), "Delineating the Mechanisms of Misfolded Endoplasmic Reticulum (ER) Luminal Protein Retrotranslocation for ER-Associated Degradation" (2019). Theses and Dissertations (ETD). Paper 504. http://dx.doi.org/10.21007/etd.cghs.2019.0489.