Comprehensive S-acylation profiling of the porcine epididymis and exosomes reveals a role in cargo sorting and long-distance trafficking.

The epididymis orchestrates sperm maturation through microenvironmental regulation and epididymosome-mediated cargo delivery. Despite emerging evidence implicating protein S-acylation in vesicular trafficking, its compartment-specific dynamics and functional implications in epididymal physiology remain poorly characterized. Here, we employed acyl-biotin exchange-based 4D proteomics to decode the S-acylation proteomic of porcine caput/cauda epididymidis and their exosomes. Comparative analysis identified 2780 and 2084 S-acylated proteins in caput and cauda tissues, respectively, with 317 upregulated and 579 downregulated S-acylated proteins in cauda versus caput. Functional enrichment revealed S-acylation-dependent regulation of signal transduction, vesicle trafficking, and immune pathways, particularly through lysosomal activity, AMPK signaling, and glutathione metabolism. Exosomal profiling demonstrated conserved S-acylated protein signatures between caput and cauda derived exosomes, with 114 S-acylated proteins shared among caput tissue and both exosomal populations, implicating long-distance transport of caput-specific cargoes. Validation identified 5 caput-enriched S-acylated proteins, including evolutionarily conserved OCLN, CDH1, PDZK1, BAG5, and SCRN1, which were detected in S-acylated forms within caput-derived exosomes and cauda exosomes, but absent in cauda tissue. This study reveals a potential role of S-acylation in mediating exosomal cargo trafficking during porcine epididymis. Our findings advance understanding of post-testicular sperm functionalization and highlight S-acylation as a potential therapeutic target for male infertility. SIGNIFICANCE: This study provides the comprehensive S-acylation proteomic atlas of the porcine epididymis and its exosomes, revealing how this reversible lipid modification spatiotemporally regulates exosome-mediated protein trafficking to support sperm maturation. We demonstrate that S-acylation governs key pathways including vesicle transport, immune regulation, and metabolic signaling in the epididymal microenvironment. Crucially, we identify a cohort of caput-enriched S-acylated proteins that are packaged into exosomes and transported distally to the cauda region, suggesting a previously unrecognized mechanism for long-distance intercellular communication. These findings establish S-acylation as a central regulator of epididymal function and offer molecular insights into post-testicular sperm maturation. The identified S-acylated proteins and associated pathways may serve as diagnostic biomarkers or therapeutic targets for male infertility, particularly in cases of defective sperm functionalization.