The human apoptosis-linked gene-2 interacting protein X (ALIX) functions within the endosomal sorting complexes required for transport (ESCRT) pathway. The ESCRT pathway mediates membrane scission to facilitate cytokinetic abscission, biogenesis of multivesicular bodies, plasma membrane repair, neuronal pruning, and budding of enveloped viruses such as HIV-1 and Ebola. In addition to ESCRT-mediated membrane remodeling, ALIX is involved in apoptosis, cell adhesion, and endocytosis. Our primary objective is to elucidate the mechanistic, structural, and functional characteristics of ALIX. We recently made the surprising discovery that the C-terminal proline-rich domain (PRD) of ALIX forms amyloid fibrils, which dissolve upon Src-kinase mediated tyrosine phosphorylation. This reversible polymerization is strikingly different from those exhibited by pathological amyloids, including those associated with Alzheimer’s and Parkinson’s diseases that resist disassembly. We also uncovered that the N-terminal portion of ALIX-PRD comprises three proline-rich motifs that compete for binding to its partner, TSG101-UEV. Global fitting of NMR relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In addition to these results which were recently published (1), we will present our latest unpublished data that describe the structural and functional consequences of the phase transition between insoluble PRD fibrils and soluble (phosphorylated) monomers, and the mechanistic underpinnings of ALIX - membrane association. The mechanistic details of PRD-mediated autoinhibition of ALIX will also be presented. To accomplish these, we have used a synergistic combination of biochemical and biophysical methods, including heteronuclear NMR spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and mass spectrometry. These studies define the molecular mechanisms that dictate the broad functional repertoire of ALIX and significantly expand our understanding of its role in the ESCRT pathway.
1. Elias, R. D., Ma, W., Ghirlando, R., Schwieters, C. D., Reddy, V. S., and Deshmukh, L. (2020) Proline-rich domain of human ALIX contains multiple TSG101-UEV interaction sites and forms phosphorylation-mediated reversible amyloids. Proc Natl Acad Sci U S A 117, 24274-24284