The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG601,602, RII607–609) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI607,608 is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG601,602 and RI607,608 was impaired folding, because mutated transporters failed to bind the inhibitor [3H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [3H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI607,608AA and SERT-RII607–609AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.

Neuronal nicotinic acetylcholine (ACh) receptors are ligandgated, cation-selective ion channels. Nicotinic receptors containing ␣4, ␣6, ␤2, and ␤3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing ␣6 and ␤3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged ␤3 subunits were fully functional compared with receptors with untagged ␤3 subunits. We find that ␤3-and ␣6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent ␣4 and ␤2 subunits in neuronal soma and dendrites. Fö rster resonance energy transfer (FRET) reveals efficient, specific assembly of ␤3 and ␣6 into nicotinic receptor pentamers of various subunit compositions.Using FRET, we demonstrate directly that only a single ␤3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for ␣4-and ␣6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of ␣6-and ␤3-containing receptors using TIRF.

Na؉ /Cl ؊ -dependent neurotransmitter transporters form constitutive oligomers. The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer. To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus. Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET). Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity. In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini. Hence, a separate interaction domain (i.e. distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT. This was confirmed by expressing the aminoand carboxyl-terminal halves of hSERT. These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET. Hence, both the carboxyl and amino termini contain elements that drive oligomerization. By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and ␤-lactamase protein fragment complementation assay. TM1/2 was also found to selfassociate. Thus, oligomerization of hSERT involves at least two discontinuous interfaces. The currently identified interaction sites drive homophilic interactions. This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers.Under physiological conditions, the serotonin transporter (SERT) 1 mediates the selective re-uptake of 5-hydroxytryptamine (5-HT; serotonin) that has been released into the synaptic cleft by neurons. SERT is of clinical relevance because it is the target of antidepressants. It is also the site of action for drugs of abuse, viz. ecstasy (methylenedioxymetamphetamine) and its congeners; these compounds release serotonin because they induce reverse transport (1). The hydrophobic core of SERT consists of 12 transmembrane domains (TMs); this structural feature is shared by all neurotransmitter transporters and by many other transporters (e.g. P-glycoprotein-like efflux pumps) and membrane proteins (e.g. mammalian isoforms of membrane-bound adenylyl cyclase). Several Na ϩ /Cl Ϫ -dependent neurotransmitter transporters have been shown to form constitutive oligomers in the plasma membrane (2)(3)(4)(5)(6)(7)(8). Although the precise biological role of oligomerization remains enigmatic, there are many arguments that support the conjecture that oligomer formation is a prerequisite for export from the endoplasmic reticulum (ER) (reviewed in Ref. 9). The topological arrangements of the 12 TM helices is not known; in the recently solved structure of two distant...

Lamina-associated polypeptide 2α (LAP2α) is a non-membrane-bound isoform of the LAP2 family implicated in nuclear structure organization. We show that during postmitotic nuclear assembly LAP2α associates with chromosomes prior to accumulation of the membrane-bound isoform LAP2β, although both proteins contain the same putative chromatin interaction domains located in their common N-terminal regions. By transient and stable expression of various N-and C-terminal LAP2α deletion mutants in HeLa cells, we identified an~350-amino-acid-long region in the C-terminal α-specific domain of the protein that is required for retention of LAP2α in interphase nuclei and for association with mitotic chromosomes, while the N-terminal domain seemed to be dispensable for these interactions. In vitro chromosome binding studies using recombinant LAP2α mutants revealed that this LAP2α-specific 'nuclear targeting domain' was essential and sufficient for association with chromosomes. These data suggested a functional diversity of chromosome binding properties of LAP2 isoforms.