Switching the Clientele: A LYSINE RESIDING IN THE C TERMINUS OF THE SEROTONIN TRANSPORTER SPECIFIES ITS PREFERENCE FOR THE COAT PROTEIN COMPLEX II COMPONENT SEC24C*

The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif (⁶⁰⁷RI⁶⁰⁸ in SERT). Substituting Lys⁶¹⁰ with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters.     

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.     

A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.     

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.     

Glycogen phosphorylase in Dictyostelium discoideum: demonstration of two developmentally regulated forms, purification to homogeneity, immunochemical analysis, cAMP induction, in vitro translation, and molecular cloning

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.     

The serotonin transporter is an exclusive client of the coat protein complex II (COPII) component SEC24C

The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif ((607)RI(608)) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.