Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX

• Published in: The Journal of biological chemistry 2021-11, Vol.297 (5), p.101328-101328, Article 101328
• Main Authors: Elias, Ruben D., Ramaraju, Bhargavi, Deshmukh, Lalit
• Format: Article
• Language: English
• Subjects: Amyloid - chemistry; Amyloid - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - metabolism; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - metabolism; endosomal sorting complexes required for transport; Endosomal Sorting Complexes Required for Transport - chemistry; Endosomal Sorting Complexes Required for Transport - metabolism; Endosomes - chemistry; Endosomes - metabolism; Humans; Intracellular Membranes - chemistry; Intracellular Membranes - metabolism; NMR; Nuclear Magnetic Resonance, Biomolecular; paramagnetic relaxation enhancement; Phosphorylation; post-translational modifications; programmed cell death 6 interacting protein; Protein Domains; Structure-Activity Relationship; Tyrosine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2021.101328

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like β-sheet–rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B–mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.