Deciphering the ubiquitin code in stress signaling and membrane trafficking

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Project Summary Ubiquitin is a 76 amino acid peptide that can be covalently conjugated to substrates to alter protein fate in diverse ways, regulating protein degradation, trafficking, subcellular localization and protein-protein interactions. Given its versatility, ubiquitin regulates many fundamental cellular processes, and its dysregulation is associated with many human diseases ranging from neurodegeneration to cancer. Ubiquitin networks include conjugating and deconjugating enzymes as well as effector pathways comprised of ubiquitin binding proteins that direct the fate of ubiquitin-modified substrates. All of these elements work together to “write”, “read”, and “edit” the ubiquitin code – which ultimately consists of ubiquitin polymers of different lengths and topologies that determine which effector pathways are engaged. Here, we describe two main research directions that will result in a deeper understanding of the ubiquitin code and how it regulates diverse cellular functions, including stress signaling and membrane trafficking. The first research direction will address how phosphorylation of ubiquitin at the Ser57 position regulates stress responses in yeast and human cells. The proposed studies will build on our recent discovery of a small group of Ser57 ubiquitin kinases conserved from yeast to humans and will include genetic, biochemical, and proteomic approaches. Specifically, we will determine how these kinases and Ser57 phosphorylation of ubiquitin contribute to the cellular stress response, and we will address how ubiquitin phosphorylation alters its interaction profile and engagement with effector pathways. This research will contribute transformative new insights into the biology of ubiquitin and proteostasis. The second research direction will address how human glucose transporters are regulated by ubiquitin modification and endocytic trafficking. Glucose transporters of the GLUT family are key regulators of cellular glucose homeostasis, and yet regulation of their trafficking and quality control remain poorly characterized. Here, we describe lines of investigation based on our recent findings that GLUT1 endocytic trafficking is regulated by specific ubiquitin modifications. These studies have important implications for cellular glucose homeostasis and human diseases including GLUT1 Deficiency Syndrome and many types of cancer. Together, these research directions will result in a deeper understanding of the ubiquitin code, membrane trafficking, and stress responses in eukaryotic cells.