What question did this study set out to answer?

This research aims to characterize the S-acylation process of ARL15 and identify the S-acyltransferases involved.

April 19, 2026Open Access

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

TCTakeshi ChinoMeiji Pharmaceutical University MAMakoto ArakiMeiji Pharmaceutical University YAYuki AshiMeiji Pharmaceutical University

Key Points

This research aims to characterize the S-acylation process of ARL15 and identify the S-acyltransferases involved.
Characterized S-acylation using acyl-PEGyl exchange gel-shift assays in HEK293T cells.
Performed single cysteine-to-serine mutations to assess the impact on S-acylation.
Conducted confocal imaging and subcellular fractionation to evaluate membrane association.
Utilized siRNA knockdown and CRISPR/Cas9-mediated gene disruption to screen for candidate enzymes.
ARL15 was found to be triply S-acylated at three N-terminal cysteine residues (Cys17, Cys22, and Cys23).
Single mutations significantly reduced S-acylation, while triple mutations completely abolished it.
Loss of S-acylation disrupted ARL15's association with membranes.
ZDHHC7 and ZDHHC3 were identified as the primary S-acyltransferases for ARL15, with dual inhibition affecting its localization.

Abstract

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 (APEGS) 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.

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