Project Summary. Human ALIX (also known as PDCD6IP) functions in endo-lysosomal pathway, apoptosis, enveloped virus budding, and other essential cell signaling and membrane scission processes. These diverse functions are regulated by its posttranslational modifications (PTMs), specifically tyrosine phosphorylation and ubiquitination. We recently uncovered that ALIX, through its proline-rich domain (PRD), forms liquid-like condensates and amyloid fibrils, and that both these assemblies dissolve on phosphorylation and reform on dephosphorylation of its tyrosine residues. Projects in this ESI-MIRA proposal expand upon these exciting discoveries and will uncover the dynamic functional interplay between phase separation, fibrillization, and PTMs of ALIX. Specifically, we will: determine the structural characteristics of ALIX’s assemblies, their regulation by tyrosine de/phosphorylation and membranes, and their formation in mammalian cells (direction 1), elucidate the functional relevance of ALIX polymerization, and the mechanisms of the time-dependent transitions of ALIX condensates to fibrils (direction 2), and characterize the interactions between ALIX and ubiquitin, and determine the cross-talk between ALIX ubiquitination and its phosphorylation-mediated polymerization (direction 3). Structural characterization of ALIX’s higher- order assemblies in direction 1.1 will reveal the interactions hotspots that govern its phase separation and novel atomic- resolution details of how a PRD can form β-sheet rich fibrils. Mechanistic studies in direction 1.2 will elucidate regulation and modulation of ALIX condensates and fibrils by lipid membranes and tyrosine de/phosphorylation, revealing how a kinase accesses its sites within these assemblies, and the identity of tyrosine residues whose dephosphorylation triggers ALIX polymerization. Cellular studies in direction 1.3 will examine ALIX polymerization in mammalian cells. In direction 2.1, we will determine how polymerization affects ALIX’s functions. Mechanistic studies in direction 2.2 will elucidate time-dependent hardening of ALIX condensates into fibrils, yielding new insights into the role of phase separation in fibrillization. Structural and kinetic studies in direction 3.1 will elucidate the interactions between ALIX and ubiquitin. Finally, in direction 3.2, we will determine the impact of ALIX ubiquitination on its phosphorylation-mediated polymerization. The above studies build upon our discoveries of the unique ALIX assemblies, their modulation by PTMs, the slow maturation of ALIX condensates into rigid fibrils, residue-specific details of ALIX – late endosomal membrane interactions, and how ALIX’s phosphorylation inhibits these interactions. Extensive preliminary results, including highly homogenous samples of ALIX assemblies enabling their structural characterization, the discoveries of selective recruitment of ALIX’s signaling partners in its condensates, and of ALIX – ubiquitin interactions in ...