Distinct functions of dimeric and monomeric Alix in regulating F-actin assembly and loading of exosomal cargo

ABSTRACT: Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes, relating to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are questions still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that Alix dimer is enriched in exosome and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix−/− primary cells underlined Alix indispensable role in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of Alix deletion mutants, we could define the function of the Bro1 domain, which is solely required for Alix’ exosomal localization, and that of the V domain, which is needed for recruiting syntenin into exosomes. We reveal an essential role for Cys814 within the disordered proline rich domain (PRD) for Alix dimerization. By mutating this residue, Alix remains exclusively monomeric and, in this configuration, is more effective in loading syntenin into exosomes. In contrast, loss of dimerization affects Alix ability to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that by 3 shifting the ratio between its dimeric and monomeric forms, Alix selectively executes two of its main functions, exosomal cargo loading or cytoskeleton remodeling.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Jeroen Demmers  

LAB HEAD: Jeroen Demmers

PROVIDER: PXD031450 | Pride | 2022-10-14

REPOSITORIES: Pride