A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S -acyltransferases catalyze long-chain S -acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC ‘hole’ mutants and ‘bumped’ chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical–genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S -acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S -acylation biology for a wide range of models and organisms.