Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter.
The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wild-type transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter.
Torres
Gonzalo E
GE
Howard Hughes Medical Institute, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Carneiro
Ana
A
Seamans
Katie
K
Fiorentini
Chiara
C
Sweeney
Ava
A
Yao
Wei-Dong
WD
Caron
Marc G
MG
D015153:Blotting, Western; D002460:Cell Line; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D004305:Dose-Response Relationship, Drug; D005799:Genes, Dominant; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D007930:Leucine / Q000737:chemistry; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014162:Transfection
D018076:DNA, Complementary; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D007930:Leucine