UC San Diego

The biological roles and functional utility of disorder within proteins is poorly understood relative to globular domains despite the predicted presence of disorder within over 50% of the human proteome. Disorder is generally characterized to facilitate protein-protein interactions; however many biophysical investigations employ only a small fragment from a disordered region to characterize a given biological interaction, likely overlooking any possible contributions from the remainder of the disordered region. In this respect, this dissertation discusses biophysical characterization and mechanistic insights into the functions of the intact proline-rich domain belonging to ALIX, a central ESCRT component protein critical to the fundamental process of cell division.Chapter 1 provides perspective over the biological roles of disordered proteins and proline-rich domains, and relevant open questions surrounding the molecular biology of ALIX. Chapter 2 begins an exploration of the ALIX proline-rich domain, describing its multifaceted interaction with partner TSG101-UEV domain, and its novel formation of phosphorylation-reversible amyloid. Chapter 3 continues with a characterization of the phosphorylation-dependent intramolecular association between ALIX Bro1 and proline-rich domains to modulate membrane binding and spatial localization of ALIX. Chapter 4 then concludes the present work on ALIX by characterizing the intact protein, focusing on the contributions of the proline-rich domain towards ALIX phosphorylation-reversible phase separation, and regulation of cytokinetic abscission machinery CHMP4B and CHMP4C. Altogether the observations made here display the propensity of the proline-rich domain of ALIX to mediate the formation of phosphorylation-reversible amyloids and condensates, and rationally propose biological functions and molecular mechanisms in which such behavior is utilized.