Biochemistry, Department of


Date of this Version



A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Jaekwon Lee. Lincoln, Nebraska: November, 2014

Copyright (c) 2014 Nathan James Smith


Protein synthesis and proper folding is an essential process for all organisms. In eukaryotes proteins of the secretory pathway are synthesized and inserted into the lumen or membrane of the endoplasmic reticulum. Eukaryotic cells maintain a mechanism for removal of proteins unable to fold properly. This process is known as ER-associated degradation (ERAD). A poorly functioning ERAD can lead to a build-up of misfolded proteins which has been implicated in several degenerative diseases such as Alzheimer’s, Amyotrophic lateral sclerosis, and Parkinson’s. Thus, the study of how proteins are recognized, extracted from the ER, and degraded is essential for determining methods for maintaining protein solubility and stability, and prevention of toxic accumulation of protein aggregates. Our lab has previously identified Pca1, a cadmium exporting P1B-type ATPase in Saccharomyces cerevisiae. A genetic knockout screen led to the discovery that Pca1 expression is controlled post-translationally through the ERAD pathway. Specifically, the ERAD-Cytoplasm (ERAD-C, indicating the location of the misfolding) pathway utilizes the E3 ubiquitin ligase Doa10 to ubiquitinylate substrates. We further tested the mechanism by which Pca1 an eight transmembrane domain containing protein was extracted from the ER membrane for degradation in the cytoplasm. Surprisingly, we determined that the proteasome itself is essential for this process. Finally, we sought to determine the requirements of cadmium sensing and rescue from ERAD as well as the molecular factors involved in recognition of the degron of Pca1. Biophysical characterization revealed cadmium specific binding. A random-mutagenesis screen identified residues required for degradation of Pca1. Bioinformatical study of the Pca1 degron structure identified a hydrophobic patch that when broken with amino acid substitution stabilized the protein. It was also determined that interaction with a known recognition factor of ERAD, Ssa1, was much weaker in the presence of a hydrophilic substitution or cadmium supplementation. Collectively, our results revealed a mechanism in which Pca1 is regulated post-translationally through the ubiquitin proteasome system. We were also able to apply our findings of Pca1 to another ERAD-C substrate. Pca1 is an excellent model for the study of the ERAD-C pathway as it is short-lived and rapidly stabilized by the supplementation of cadmium.

Adviser: Jaekwon Lee