Structure-function analysis of Ubp3, Bre5 and the Ubp3/Bre5 complex

Ubiquitination is a post-translational protein modification in eukaryotes, with broad signaling functions. In this process, the small molecule ubiquitin is covalently attached to target proteins through a complex three-step enzymatic cascade, which can result in the formation of polyubiquitin chains. Ubiquitin-mediated signaling is involved in a multitude of cellular functions and quality control mechanisms, requiring precise regulation. These quality control pathways include different types of selective autophagy such as ribophagy or mitophagy. To maintain a balance between various quality control mechanisms, deubiquitinating enzymes can remove or modify ubiquitin signals by cleaving ubiquitin or polyubiquitin chains. In Saccharomyces cerevisiae, one such deubiquitinating enzyme is Ubp3, which relies on its cofactor Bre5 for specific activity. The Ubp3-Bre5 complex plays a vital role in various cellular processes, particularly in stress response. Interestingly, it exhibits a dual role in cellular functions. For instance, it protects mitochondria from mitophagy while simultaneously promoting other forms of general autophagy. This investigation aims to elucidate how these distinct functions are coordinated and to explore the nature of the interaction between Ubp3 and Bre5. We demonstrate the establishment of a heterologous expression and purification strategy, to obtain protein for detailed structure-function analyses. With this in vitro approach, isolation of individual full-size proteins and their complex is targeted. This is achieved through a series of chromatography steps utilizing different affinity tags. Isolated proteins are investigated regarding their activity, structure and interactions. With this approach, it should be possible to elucidate the molecular mechanisms that govern their natural function and the subsequent hypotheses can then be verified in vivo. This study should therefore help to entangle the complex regulatory network in which Ubp3 is involved and advance our understanding of deubiquitinases.