Mejuto, L.; Pipito, L.; Ng, C. P.; Tomkinson, N. C. O.; Reynolds, C. A.; Deganutti, G.; Greaves, J.

Reversible S-acylation controls protein trafficking, localisation and activity within cells, yet the limited understanding of the regulatory mechanisms of S-acylation/deacylation cycles represents one of the biggest obstacles in exploiting this prevalent post-translational modification for therapeutic purposes. Here, we identified ABHD13 and ABHD16A as novel deacylases regulating the SNAP25 family of SNARE proteins responsible for vesicular membrane fusion. We determined the mechanism and structural determinants for ABHD13-mediated deacylation and substrate selectivity towards SNAP25b, and identified a novel mechanism of acyl chain extraction for integral membrane deacylases. This is the first report of deacylase selectivity mapped to a single amino acid position in full-length proteins.