Direct evidence that two cysteines in the dopamine transporter form a disulfide bond.
We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.
Chen
Rong
R
Department of Pharmacology, The Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, OH 43210, USA.
Wei
Hua
H
Hill
Erik R
ER
Chen
Lucy
L
Jiang
Liying
L
Han
Dawn D
DD
Gu
Howard H
HH
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D013329:Structure-Activity Relationship
D004220:Disulfides; D050483:Dopamine Plasma Membrane Transport Proteins; D050505:Mutant Proteins; D003545:Cysteine; D004229:Dithiothreitol; D004298:Dopamine