Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes related 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 still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that the Alix dimer is enriched in exosomes and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix−/− primary cells underlined the indispensable role of Alix in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of deletion mutants, we define the function of Alix Bro1 domain, which is solely required for its 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 for Alix dimerization. By mutating this residue, we show that Alix remains exclusively monomeric and, in this configuration, is effective in loading syntenin into exosomes. In contrast, loss of dimerization affects the ability of Alix to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that dimeric and monomeric forms of Alix selectively execute two of the protein's main functions: exosomal cargo loading and cytoskeleton remodeling.
Bibliographical noteFunding Information:
We are grateful to Dr Noelia Escobedo for assistance with mouse embryonic fibroblast isolation. We thank the staff of the Cellular Imaging Shared Resource of St Jude for assistance for immunofluorescence imaging. We want to thank Dr Khaled Khairy and Maria Panlilio for assistance with the development of the ImageJ macro. A.d’A. holds an endowed chair in Genetics and Gene Therapy from the Jewelry Charity Fund. This study was funded in part by National Institute of Health grants AR049867 , GM60905 , GM104981 and DK52025 , the Assisi Foundation of Memphis , and the American Lebanese Syrian Associated Charities (ALSAC).
© 2022 The Authors