S-palmitoylation is a post-translational modification of Alix that regulates its interaction with the CD9 tetraspanin

Background: The multifunctional protein Alix is a bona fide extracellular vesicle (EV) regulator. Skeletal muscle (SkM) cells can release Alix-positive nano-sized EVs directly from their plasma membrane, offering a new paradigm for understanding how myofibres communicate within skeletal muscle and other organs. S-palmitoylation is a reversible lipid post-translational modification (PTM) that is involved in different biological processes, such as the trafficking of membrane proteins and stabilization of protein interaction. Methods: Here, we have evaluated the extent to which S-palmitoylation is functionally linked to Alix and EVs by: (i) a comparative analysis of publicly available palmitoyl- and exosome-proteome data sets and (ii) altering protein palmitoylation, using a specific inhibitor (2-Br-Palmitate; 2BP) and evaluating S-palmitoylation of Alix as well as its subcellular distribution and interaction in SkM cells. Results: We found a higher percentage of S-palmitoylated proteins in exosomes, compared to all the other cellular compartments. This finding suggests that this PTM could be a distinctive signature for exosomal proteins. By coupling bioinformatic observation with biochemical analyses, we have also determined that endogenous Alix undergoes S-palmitoylation. In particular, exosomal Alix is palmitoylated to a larger extent than cellular Alix, and the inhibition of palmitoylation altered its subcellular localization. Furthermore, endogenous Alix interacts with CD9, and S-palmitoylation supports this interaction, as it also does for tetraspanin complexes in the tetraspanin enriched microdomains. Summary/Conclusion: Thus, we propose that S-palmitoylation might regulate the proper function of Alix in facilitating interactions among exosome-specific regulators in SkM-derived exosome biogenesis. Essential discoveries related to SkM-derived EVs may help in designing engineered exosomes which can be employed in the tissue regeneration field, e.g. to help in recovery from muscle atrophy and/or injury. Funding: The research leading to these results has been funded by the Italian Ministry for Education, University, and Research in the framework of the Flagship Project NanoMAX.