Abstract
The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.
Highlights
Dopaminergic neurotransmission plays a critical role in modulation of a variety of central nervous system functions, including locomotor activity, cognition, and reward [1]
These results suggested that the PDZ-binding motif is not sufficient for endoplasmic reticulum (ER) export of the transporter and that sequences within the juxtamembrane portion of the carboxyl terminus are critical for efficient delivery of dopamine transporter (DAT) to the plasma membrane
A large number of mutants were used to examine the mechanisms by which DAT trafficking from the ER is regulated by its carboxyl-terminal sequences
Summary
Plasmids and Mutations—YFP-DAT, CFP-DAT, YFP-⌬N-DAT (deletion of the first 65 amino acid residues), and YFP-DAT-CЈ615 (truncation of the last 5 amino acid residues) were described previously [14]. To generate DAT CЈ618, CЈ615, CЈ610, and CЈ576 truncation mutants, a stop codon was created at the positions corresponding to amino acid residues 619, 616, 611, and 577, respectively, in the DAT-pcDNA3.1 using a QuickChange site-directed mutagenesis. To generate the EGF receptor (EGFR)-DAT chimera, the XhoI restriction site was introduced into wtEGFR-pEGFP-N1 [17] at the position corresponding to amino acid residues 644 – 645 of the EGFR by using site-directed mutagenesis. Immunofluorescence Staining—The cells grown on glass coverslips were washed with Ca2ϩ, Mg2ϩ-free PBS (CMF-PBS), fixed with freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 15 min at room temperature, and mildly permeabilized using a 3-min incubation in CMF-PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin at room temperature. To visualize CFP and YFP, consecutive images were acquired through corresponding filter chan-
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