Most cases of early-onset torsion dystonia (EOTD) are caused by a deletion of one glutamic acid in the carboxyl terminus of a protein named torsinA. The mutation causes the protein to aggregate in perinuclear inclusions as opposed to the endoplasmic reticulum localization of the wild-type protein. Although there is increasing evidence that dysfunction of the dopamine system is implicated in the development of EOTD, the biological function of torsinA and its relation to dopaminergic neurotransmission has remained unexplored. Here, we show that torsinA can regulate the cellular trafficking of the dopamine transporter, as well as other polytopic membrane-bound proteins, including G protein-coupled receptors, transporters, and ion channels. This effect was prevented by mutating the ATP-binding site in torsinA. The dystonia-associated torsinA deletion mutant (⌬E-torsinA) did not have any effect on the cell surface distribution of polytopic membrane-associated proteins, suggesting that the mutation linked with EOTD results in a loss of function. However, a mutation in the ATP-binding site in ⌬E-torsinA reversed the aggregate phenotype associated with the mutant. Moreover, the deletion mutant acts as a dominant-negative of wild-type torsinA through a mechanism presumably involving association of wild-type and mutant torsinA. Taken together, our results provide evidence for a functional role for torsinA and a loss of function and a dominant-negative phenotype of the ⌬E-torsinA mutation. These properties may contribute to the autosomal dominant nature of the condition. deletion mutant ͉ cell surface ͉ endoplasmic reticulum ͉ transmembrane ͉ polytopic

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 wildtype 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. The dopamine transporter (DAT)1 belongs to a large family of Na ϩ /Cl Ϫ -dependent plasma membrane transporters that also includes the closely related norepinephrine and serotonin transporters (NET and SERT, respectively), and carriers for GABA, glycine, proline, taurine, and betaine. In the central nervous system, DAT mediates the re-uptake of released dopamine (DA) from the synaptic cleft back into the nerve terminal for subsequent storage and release. Pharmacological and genetic studies highlight the DAT-mediated re-uptake process as the main mechanism for the termination of dopamine neurotransmission (1). In addition, DAT represents the main target site for commonly abused drugs such as cocaine and amphetamine as well as some therapeutic agents used in the management of affective disorders (2). Hydrophobicity analysis of their deduced amino acid sequence reveals that Na ϩ /Cl Ϫ -dependent plasma membrane neurotransmitter transporters are proteins containing twelve transmembrane domains (TMs) with both the amino and the carboxyl termini located on the intracellular side of the membrane. This topological arrangement has been confirmed for several members of the family, including DAT (3). Since the molecular cloning of this transporter gene family, a great deal of information has been accumulated concerning the relationship between the structure and function of this class of proteins (4). Studies...

The Na+/Cl--dependent dopamine transporter (DAT) is critical in terminating dopaminergic transmission by removing the transmitter away from the synapse. Several lines of evidence suggest that transporter-interacting proteins may play a role in DAT function and regulation. In this report, using the yeast two-hybrid system, we have identified a novel interaction between DAT and the multiple Lin-11, Isl-1, and Mec-3 (LIM) domain-containing adaptor protein Hic-5. This association involves the N-terminal portion of the intracellular tail of DAT and the LIM region of Hic-5. In human embryonic kidney 293 cells, Hic-5 colocalizes with DAT at polarized sites and reduces DAT uptake activity through a mechanism involving a decrease in the cell-surface levels of the transporter. A fragment of Hic-5 containing the LIM domains is sufficient to bind DAT but lacks the ability to inhibit transporter activity. In addition, the LIM fragment prevents the effect of the full-length Hic-5 on DAT localization and function. In the brain, Hic-5 protein is expressed in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and striatum, suggesting a role for this protein in the nervous system. The association of the endogenous Hic-5 and DAT proteins was confirmed biochemically by coimmunoprecipitation from brain striatal extracts. Moreover, immunostaining of rat midbrain neurons in culture revealed a presynaptic colocalization of Hic-5 and DAT. Because Hic-5 has been shown to interact with several signaling molecules, including the nonreceptor protein tyrosine kinases focal adhesion kinase and Fyn, this raises the possibility that this adaptor protein may link DAT to intracellular signaling pathways.