Exploring the Mechanics of Golgi-localized Tul1 Selective Degradation

Abstract

The eukaryotic proteome is in constant flux, as cellular proteins are continuously synthesized, folded, post-translationally modified, trafficked, and degraded to maintain a healthy proteomic balance. Maintaining this balance is critical to organismal health and disrupted cellular protein homeostasis is omnipresent in human disease. Cellular proteostasis is maintained and/or restored by networked protein quality control systems. Endoplasmic reticulum (ER) quality control surveils nascent secretory and membrane proteins synthesized into the ER lumen before trafficking through the secretory pathway. Organelles that receive ER-synthesized proteins contain additional quality control systems that recognize and respond to proteostatic threats by either rerouting substrates and/or facilitating their degradation. In S. cerevisiae, two examples of post-ER degradative quality control systems are the Tul1 (transmembrane ubiquitin ligase 1) sub-complexes, which cycle through the Golgi apparatus/endosomal compartments or localize to the vacuole (the yeast lysosome). Two features of the Golgi-localized Tul1 system distinguish it from all other degradative quality control systems. First, Tul1 is the only known integral membrane ubiquitin ligase that localizes to the Golgi/endosomes in yeast. Second, Golgi-localized Tul1 complexes facilitate protein substrate degradation through two different pathways: the vacuole and the cytosolic proteasome. Our current understanding of Golgi-localized Tul1 substrate degradation is quite limited. However, the pathway by which a substrate is degraded seems fixed and specific to the recognized protein; proteasomal substrates are not re-routed for degradation in the vacuole and vice versa. We sought to elucidate how Tul1 complexes specify a substrate for the proteasome versus the vacuole, beginning with a dissection of the central component in the complex the Tul1 ubiquitin ligase. In this thesis, we established deep mutational scanning tools and biochemical characterization assays to perform a residue-level structure-function analysis of Tul1. From our efforts, we defined lumenal mutations that impaired Tul1 complex formation and inhibited its function, meaning it was unable to degrade proteasomal and vacuolar substrates. Surprisingly, we identified mutations within the Tul1 RING domain that changed substrate specificity. These mutants were nonfunctional for degradation of proteasomal substrate, but hypomorphic for vacuolar substrate degradation. We did not identify Tul1 single-residue mutants that were singularly functional for only proteasomal or only vacuolar substrate degradation, which led us to conclude that Tul1 is important for selecting substrates for either degradation pathway, but there are likely other factors involved. Based on our results, we propose models for how the Golgi-localized Tul1 system can selectively degrade substrates. Of these, we favor a model in which differing interactions with the ubiquitin conjugating enzyme Ubc4 influences Tul1 to selectively conjugate differing lengths of ubiquitin chains on to proteasomal and vacuolar substrates, which ultimately directs selective substrate engagement with degradation machinery. Further exploration of this, and other proposed models, can be easily achieved by applying or adapting tools that we introduce. In summary, the work presented in this thesis lends further insight into how the Golgi-localized Tul1 protein quality control system contributes to maintaining cellular proteostasis by selectively degrading substrates through proteasomal and vacuolar pathways.