Mechanism and Function of DHHC Palmitoyltransferases

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Members of a family of integral membrane proteins characterized by a DHHC (Asp‐His‐His‐Cys) motif catalyze the addition of palmitate or other long chain fatty acids onto cysteine residues in intracellular substrate proteins. DHHC proteins are a highly diverse family with 23 members in humans. Conserved features include at least 4 transmembrane spans and a cysteine‐rich domain (CRD) that is exposed on the cytoplasmic face of membranes. The biomedical importance of members of this family is underscored by their association with intellectual disability, Huntington's Disease, and cancer in humans and raises the possibility of the enzymes as targets for therapeutic intervention. Our prior work showed that DHHC proteins form an acyl‐enzyme intermediate prior to transferring the acyl group onto substrate proteins. The DHHC cysteine is necessary for both autoacylation and transfer. In cells, we showed that both wild type DHHC3 and the catalytically inactive DHHS3 (C157S) mutant enzyme are palmitoylated, suggesting that cysteine residues other than C157 are palmitoylated. DHHC proteins oligomerize, suggesting a mechanism for intermolecular palmitoylation, and potentially accounting for palmitoylation of the DHHS3(C157S) mutant in cells. Using mass spectrometry, we directly identified palmitate modification of C146 in DHHC3, a conserved residue in the CRD. Mutation of C146 or other conserved cysteines in the CRD compromised autoacylation and transfer of palmitate. These mutations resulted in structural perturbation, as identified by limited proteolysis experiments. These findings suggest that palmitoylation of residues other than the catalytic cysteine may play an important role in structuring the CRD to enable enzyme activity.