S-Palmitoylation of the serotonin transporter promotes its cell surface expression and serotonin uptake

The neurotransmitter serotonin (5-HT) is transported back into serotonergic neurons by the serotonin transporter (SERT). SERT is a main target of antidepressants, and much effort has therefore focused on finding relationships between SERT and depression. However, it is not fully understood how SERT is regulated at the cellular level. Here, we report post-translational regulation of SERT by S-palmitoylation, in which palmitate is covalently attached to cysteine residues of proteins. Using AD293 cells (a human embryonic kidney 293-derived cell line with improved cell adherence) transiently transfected with FLAG-tagged human SERT, we observed S-palmitoylation of immature SERT containing high-mannose type N-glycans or no N-glycan, which is presumed to be localized in the early secretory pathway, such as the endoplasmic reticulum. Mutational analysis by alanine substitutions shows that S-palmitoylation of immature SERT occurs at least at Cys-147 and Cys-155, juxtamembrane cysteine residues within the first intracellular loop. Furthermore, mutation of Cys-147 reduced cellular uptake of a fluorescent SERT substrate that mimics 5-HT without decreasing SERT on the cell surface. On the other hand, combined mutation of Cys-147 and Cys-155 inhibited SERT surface expression and reduced the uptake of the 5-HT mimic. Thus, S-palmitoylation of Cys-147 and Cys-155 is important for both the cell surface expression and 5-HT uptake capacity of SERT. Given the importance of S-palmitoylation in brain homeostasis, further investigation of SERT S-palmitoylation could provide new insights into the treatment of depression.