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Background: The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments., Methodology/principal Findings: We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter., Conclusions/significance: Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.

The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT](ex)) is elevated. It is well reported that stimulation of cells with high [5HT](ex) induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT](ex), Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616-624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT](ex)"paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation.