Investigating Palmitoylation Sites on the Dopamine Transporter
Authors: Stanislowski Daniel J. , Vaughan Roxanne A. , Foster James D. 
Published: June 2021

A major regulator of synaptic dopaminergic tone is the dopamine transporter (DAT), a presynaptic integral membrane protein whose function is reuptake of dopamine (DA) into the neuron after vesicular neurotransmitter release. DAT is the target of therapeutic and abused psychoactive drugs, and DAT dysfunction is hypothesized to contribute to neurological disorders like attention deficit hyperactive disorder, addiction, and Parkinson disease. DAT function is tightly modulated through binding partners and posttranslational modifications including phosphorylation, glycosylation, and, of importance here, S‐palmitoylation – the labile, enzymatic modification of cysteine sulfhydryl side groups with the fatty acid palmitate, generating a thioester linkage. S‐palmitoylation conveys protein‐specific functions, which can include protein oligomerization membrane microdomain localization, protein maturation, and alleviating transmembrane domain hydrophobic mismatch. DAT palmitoylation acutely increases DA uptake Vmax without altering DAT surface expression and opposes long‐term DAT degradation. As the enzymatic processes of S‐palmitoylation occurs on intracellularly accessible cysteines, we generated cysteine to alanine mutants at each of the intracellular rat DAT cysteine residues (6, 135, 341, 522, and 580). Palmitoylation of each mutant DAT was probed by [3H]palmitic acid metabolic labeling and the acyl‐biotinyl exchange (ABE) method which revealed a 50% loss of labeling only in the C580A mutant. The remaining palmitoylation signal implied the presence of one or more palmitoylation sites. Subsequently, palmitoylation of mutant rDATs possessing a single intracellular Cys residue was assessed. Cys580 and Cys6 displayed similar and significantly increased palmitoylation levels relative to Cys135, Cys341, and Cys522, and mutant rDATs containing both Cys6 and Cys580 had near WT palmitoylation levels indicating Cys6 is a second palmitoylation site. To determine if endogenous palmitoylation is occurring on Cys6, DAT palmitoylation in rat striatal tissue was assessed by ABE after proteolytic or chemical digestion using Asp‐N or CNBr respectively to generate immunospecific N‐terminal DAT fragments. Asp‐N proteolysis releases a peptide containing Cys6 and Cys135 while CNBr digestion releases a peptide that contains Cys6 but not Cys135, and both fragments are detected by an N‐terminal monoclonal DAT antibody. In these experiments, N‐terminal palmitoylated DAT fragments were produced using both digestion methods, strongly suggesting endogenous palmitoylation of rDAT at Cys6 consistent with our mutagenesis results. Additionally, we are assessing transporter surface expression and functional changes incurred by mutation of this novel rDAT palmitoylation site, Cys6. Palmitoylation of this residue may invoke tethering of the cytosolic N‐terminus of DAT to the plasma membrane affecting DAT function and regulation. We are therefore probing for Cys6 palmitoylation mediated tethering of the N‐terminus to the plasma membrane. Interestingly, Cys6 is adjacent to Ser7, a known PKC‐mediated phosphorylation site, and palmitoylation of Cys6 may sterically inhibit phosphorylation of Ser7 and play a significant role in DAT regulation.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research ResourcesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.