S-acylation and membrane localization of the small GTPase ARL15 are mediated by the Golgi S-acyltransferases ZDHHC7 and ZDHHC3.

ARL15 is a member of the ARF-like (ARL) family of small GTPases, implicated in the regulation of ion homeostasis and metabolic signaling pathways. Although ARL15 has been suggested to undergo S-acylation-a reversible lipid modification that governs membrane association and trafficking-the stoichiometry of this modification and the responsible S-acyltransferases have remained unclear. Here, we systematically characterized the S-acylation of ARL15 and identified the enzymes mediating this modification. Using acyl-PEGyl exchange gel-shift assays, we show that ARL15 is triply S-acylated at three conserved N-terminal cysteine residues (Cys17, Cys22, and Cys23) in HEK293T cells. Single cysteine-to-serine mutations substantially reduced S-acylation, whereas substitution of all three cysteines abolished it entirely. Loss of S-acylation disrupted membrane association of ARL15, as shown by confocal imaging and subcellular fractionation. A candidate screen using siRNA knockdown and CRISPR/Cas9-mediated gene disruption revealed that the Golgi-localized S-acyltransferases ZDHHC7 and ZDHHC3 mediate ARL15 S-acylation in a partially redundant or parallel manner. Dual inhibition of both enzymes led to a marked reduction in S-acylation and redistributed ARL15 from membranes to the cytosol. These findings elucidate the stoichiometry and enzymatic regulation of ARL15 S-acylation and provide mechanistic insight into its subcellular localization.