Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter.
The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP.
Tate
C G
CG
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom. cgt@mrc-lmb.cam.ac.uk
Whiteley
E
E
Betenbaugh
M J
MJ
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D037281:Calnexin; D037282:Calreticulin; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D017510:Protein Folding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D012261:Ribonucleoproteins; D050486:Serotonin Plasma Membrane Transport Proteins; D037281:Calnexin; D017485:1-Deoxynojirimycin; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D003042:Cocaine