A minimal functional ZDHHC5 defines distinct requirements for NOD2 binding and S-acylation

Protein S-acylation by ZDHHC5 promotes membrane recruitment of the innate immune receptors NOD1 and NOD2. ZDHHC5 contains an extended cytosolic tail implicated in substrate recruitment, accessory-protein interactions, and enzyme trafficking, but its contribution to NOD1 and NOD2 modification remains unclear. Here, we used mutagenesis and complementation of ZDHHC5 knockout cells to define the regions required for NOD association, S-acylation, and plasma membrane localization. Substitution of three S-acylated cysteines in the tail preserved ZDHHC5 localization and its ability to restore NOD2 membrane recruitment. Progressive truncation revealed that most of the cytosolic tail was dispensable, with a construct comprising residues 1-230 retaining activity towards both NOD1 and NOD2. Further truncation to amino acids 1-218 impaired NOD S-acylation and membrane recruitment without eliminating NOD2 association. Thus, substrate association and productive modification have distinct requirements. C-terminal tail removal also differentially affected accessory-protein association: full-length ZDHHC5 co-immunoprecipitated with both GOLGA7A and GOLGA7B, whereas only GOLGA7B was detectably associated with the 1-230 construct. Together, these findings define a minimal functional ZDHHC5 that supports NOD S-acylation and distinguish the requirements for substrate association, catalytic activity, and accessory-protein interactions. They establish that the extended tail is dispensable for NOD modification under the conditions examined, while identifying differential requirements for GOLGA7A and GOLGA7B association with ZDHHC5.