Saturday, May 10, 2025
5/10/2025
Mechanisms of alpha-arrestin-mediated protein trafficking - O’Donnell, A. F., University of Pittsburgh PROJECT SUMMARY Cells must rapidly reorganize the membrane proteome to survive changing environments and restore homeostasis. How does the cell ‘decide’ to relocalize or degrade a membrane protein? Cell signaling events modify protein trafficking adaptors, which then bind selectively to membrane proteins (referred to as cargo) to control relocalization. This cargo reshuffling restores cellular balance, and its importance is highlighted by the cardiovascular, neurodegenerative, and metabolic diseases linked to defects in protein trafficking adaptors. Our research focuses on a little studied class of trafficking adaptor, the a-arrestins, that are conserved across eukaryotes. Foundational studies of a-arrestin function, including those from our lab, have defined their role in regulating selective endocytosis of nutrient transporters in the yeast Saccharomyces cerevisiae. However, critical gaps remain in our understanding of a-arrestins and how they regulate cellular homeostasis: How do a- arrestins recognize cargos? What trafficking pathways are regulated by a-arrestins? How does cell signaling control a-arrestin activity? The proposed research will address each of these knowledge gaps. Our studies will provide a deep, mechanistic understanding of a-arrestin function in protein trafficking. Our preliminary work suggests that a-arrestins are master regulators of protein trafficking as they control a varied set of cargo from diverse cellular membranes, including the Golgi and endosomes. We will use innovative and systems-level approaches to define the cargo repertoire for a-arrestins. Leveraging these new cargos, we will use mutational scanning to define amino acid motifs needed for a-arrestin-cargo interactions. Trafficking adaptors interact with vesicle forming proteins to mediate cargo packaging. We identified a-arrestin interactions with the clathrin-mediated endocytosis (CME) machinery, and we will define the amino acids needed for these associations. Excitingly, we found that a-arrestins also act as cargo selective adaptors in clathrin-independent endocytosis (CIE), often considered a bulk endocytic route. We posit that a-arrestins analogously couple cargo to the CIE machinery, and we will define the cargo and trafficking machinery interfaces needed for a-arrestins to function in CIE. Further adding to a-arrestin function, we have revealed a new role for a-arrestins in intracellular protein sorting that causes increased trafficking of cargo to the cell surface. As we have done for other trafficking pathways, we will determine the molecular determinants needed for a-arrestin function in intracellular sorting. Finally, given their breadth of trafficking activities, a-arrestin functions must be controlled by cell signaling. We have defined many kinases, phosphatases, and ubiquitin modifiers that alter a-arrestin-mediated trafficking and will now elucidate how these synergistically control a- arrestin function and stability. In yeast, we will establish the rules that govern a-arrestin interactions, stability, and function, which we will then apply to our longer-term studies of mammalian a-arrestins, an emerging class of tumor suppressor. Specific Aims Page
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