Discovering Palmitoylation Sites on the Dopamine Transporter
Authors: Stanislowski Daniel J. , Vaughan Roxanne A. , Foster James D. 
Published: November 2023

The dopamine transporter (DAT) controls synaptic dopamine (DA) levels by reuptake after presynaptic neuronal vesicular release. Parkinson disease, addiction, and ADHD are neurological disorders associated with abnormal DA homeostasis causing DAT dysregulation to be hypothesized as a contributing factor in these disorders. Several binding partner interactions and posttranslational modifications combine to regulate DAT function. One such modification is S‐palmitoylation, the reversible, catalytic addition of palmitate to cysteine via a thioester linkage. S‐palmitoylation can regulate protein‐protein interactions, protein trafficking, transmembrane domain tilting, and membrane stability, and DAT palmitoylation increases DA transport Vmax without altering DAT surface expression and opposes DAT degradation. Because S‐palmitoylation is thought to be an intracellular process, we generated five cytosolic accessible cysteine to alanine mutants at residues Cys6, Cys135, Cys341, Cys522, and Cys580 and analyzed them for palmitoylation by [3H]palmitic acid metabolic labeling. A significant decrease in labeling was seen in only the C580A mutant versus WT control; however, approximately fifty percent of the signal remained, implying the presence of additional S‐palmitoylation site(s). To this end the more sensitive acyl‐biotinyl exchange (ABE) method was used to reanalyze these same mutants, and similar results were obtained confirming Cys580 as a major palmitoylation site with similar palmitoylation reduction in the C580A DAT and approximately 50% remaining residual palmitoylation. To further assess palmitoylation at individual cysteines, all but one of the intracellular cysteines were mutated to alanine, leaving a single cysteine available for palmitoylation. ABE analysis of these mutants showed significantly elevated palmitoylation of DATs containing Cys6 or Cys580 relative to those containing Cys135, Cys341, or Cys522 and mutants containing both Cys6 and Cys580 displayed near WT levels of palmitoylation. To assess if endogenous N‐terminal palmitoylation is present, we performed peptide mapping of rat striatal DAT using endoproteinase Asp‐N digestion prior to ABE and epitope specific immunoblotting. Asp‐N DAT cleavage occurs at Asp174, producing a characteristic 19 kDa peptide fragment containing Cys6 and Cys135 when blotted with a DAT N‐terminal antibody. This analysis revealed palmitoylation of this fragment, indicating one or both of these residues is palmitoylated suggesting further analysis is necessary to distinguish between these sites. Together these results provide evidence supporting DAT palmitoylation at Cys6, implying the potential for tethering of the distal N‐terminus to the plasma membrane. Additionally, Cys6 is adjacent to a known PKC‐mediated phosphorylation site, Ser7, suggesting the potential for interplay between phosphorylation and palmitoylation at these sites and a role for these modifications in DAT regulation.Support or Funding InformationSupported by National Institute on Drug Abuse grants R15 DA031991 (J.D.F.) and R01 DA13147 (R.A.V.) and NIH grants P20GM103442/P20RR016471 (to UND).