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

Abstract 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.