title: Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization.
abstract: The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585and591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The584KW585motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the591PYRKR595sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Robinson
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Hardaway
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Hardie
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Jane
authors.initials: J
authors.lastname: Wright
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Glynn
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Bermingham
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Han
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Sturgeon
authors.affiliation: Department of Biology, Fisk University, Nashville, TN 37208, United States.
authors.forename: Phyllis
authors.initials: P
authors.lastname: Freeman
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States; Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN 37232-8548, United States; Department of Biomedical Sciences, Charles E. Schmidt College of Medicine, Jupiter, FL 33458, United States. Electronic address: rblakely@fau.edu.
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000818:Animals; D001370:Axonal Transport; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D013569:Synapses / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; C532156:DAT-1 protein, C elegans; D050483:Dopamine Plasma Membrane Transport Proteins; D020690:GTPase-Activating Proteins; D021382:Protein Sorting Signals; C000607941:SEC-24 protein, C elegans
keywords: Dopamine; Nematode; Sec proteins; Synaptic trafficking; Transporter