title: Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum.
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.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Miranda
authors.forename: Tatiana
authors.initials: T
authors.lastname: Sorkina
authors.forename: Tom N
authors.initials: TN
authors.lastname: Grammatopoulos
authors.forename: W Michael
authors.initials: WM
authors.lastname: Zawada
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000473:pathology; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D050483:Dopamine Plasma Membrane Transport Proteins; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005998:Glycine / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013552:Swine; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493216:Slc6a3 protein, rat; C054562:yellow fluorescent protein, Bacteria; D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D008239:Lysine; D000409:Alanine; D005998:Glycine