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

Sucic S, El-Kasaby A, Kudlacek O, Sarker S, Sitte HH, Marin P, Freissmuth M - J. Biol. Chem. (2011)

Bottom Line: The combination of all siRNAs was not more effective than that directed against SEC24C.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

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Recruitment of COPII complex proteins by SERT C terminus. A, protein extracts from mouse brains (10 mg of protein/experiment) were incubated with 10 μg of the synthetic peptide corresponding to the C terminus of mouse SERT (SERT C terminus) immobilized on Sepharose beads or with Sepharose beads only (control). The areas of two-dimensional gels representative of four experiments performed independently, which contain spots identified as COPII complex proteins (indicated by arrows), are illustrated. B, annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE) retained by affinity in pulldowns using SERT C terminus as bait.
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Figure 1: Recruitment of COPII complex proteins by SERT C terminus. A, protein extracts from mouse brains (10 mg of protein/experiment) were incubated with 10 μg of the synthetic peptide corresponding to the C terminus of mouse SERT (SERT C terminus) immobilized on Sepharose beads or with Sepharose beads only (control). The areas of two-dimensional gels representative of four experiments performed independently, which contain spots identified as COPII complex proteins (indicated by arrows), are illustrated. B, annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE) retained by affinity in pulldowns using SERT C terminus as bait.

Mentions: Mouse brain extracts were incubated with the synthetic peptide corresponding SERT C terminus immobilized on Sepharose beads. Control experiments were carried out in the absence of peptide. Proteins retained by affinity were separated on two-dimensional gels and stained with silver. Representative areas of two-dimensional gels (from four individual experiments) containing the spots identified as COPII complex proteins by MALDI-TOF mass spectrometry (SEC23A and SEC24C, respectively, indicated by arrows) are depicted in Fig. 1A. Fig. 1B shows the annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE). supplemental Tables S1 and S2 contain a comprehensive list of peptides matching with mouse Sec24C and Sec23A sequences, respectively. It is noteworthy that other SEC23 or SEC24 isoforms were not identified in pulldown assays where the SERT C terminus was used as bait.


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

Sucic S, El-Kasaby A, Kudlacek O, Sarker S, Sitte HH, Marin P, Freissmuth M - J. Biol. Chem. (2011)

Recruitment of COPII complex proteins by SERT C terminus. A, protein extracts from mouse brains (10 mg of protein/experiment) were incubated with 10 μg of the synthetic peptide corresponding to the C terminus of mouse SERT (SERT C terminus) immobilized on Sepharose beads or with Sepharose beads only (control). The areas of two-dimensional gels representative of four experiments performed independently, which contain spots identified as COPII complex proteins (indicated by arrows), are illustrated. B, annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE) retained by affinity in pulldowns using SERT C terminus as bait.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091253&req=5

Figure 1: Recruitment of COPII complex proteins by SERT C terminus. A, protein extracts from mouse brains (10 mg of protein/experiment) were incubated with 10 μg of the synthetic peptide corresponding to the C terminus of mouse SERT (SERT C terminus) immobilized on Sepharose beads or with Sepharose beads only (control). The areas of two-dimensional gels representative of four experiments performed independently, which contain spots identified as COPII complex proteins (indicated by arrows), are illustrated. B, annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE) retained by affinity in pulldowns using SERT C terminus as bait.
Mentions: Mouse brain extracts were incubated with the synthetic peptide corresponding SERT C terminus immobilized on Sepharose beads. Control experiments were carried out in the absence of peptide. Proteins retained by affinity were separated on two-dimensional gels and stained with silver. Representative areas of two-dimensional gels (from four individual experiments) containing the spots identified as COPII complex proteins by MALDI-TOF mass spectrometry (SEC23A and SEC24C, respectively, indicated by arrows) are depicted in Fig. 1A. Fig. 1B shows the annotated MALDI-TOF MS spectrum of SEC24C (Q8CGF4_MOUSE). supplemental Tables S1 and S2 contain a comprehensive list of peptides matching with mouse Sec24C and Sec23A sequences, respectively. It is noteworthy that other SEC23 or SEC24 isoforms were not identified in pulldown assays where the SERT C terminus was used as bait.

Bottom Line: The combination of all siRNAs was not more effective than that directed against SEC24C.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus