Abstract
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.
Highlights
The serotonin transporter (SERT)1 is a member of a small family of integral membrane proteins that transport neurotransmitters and osmolytes into cells by coupling uptake to the influx of Naϩ and ClϪ ions down their concentration gradients
Co-expression of Molecular Chaperones Affects the Amount of Functional SERT Expressed—The serotonin transporter and the molecular chaperones calnexin, calreticulin and binding protein (BiP), and the foldase ERp57 were incorporated into separate baculovirus vectors for expression in insect cells
It was assumed that if 125I-RTI55 bound to Myc-SERT in a serotonin-protectable manner, Myc-SERT was functional i.e. if it was present at the plasma membrane, it
Summary
The serotonin transporter (SERT) is a member of a small family of integral membrane proteins that transport neurotransmitters and osmolytes into cells by coupling uptake to the influx of Naϩ and ClϪ ions down their concentration gradients (reviewed in Refs 1–3). SERT is of particular pharmacological interest, because it is the primary binding site in the brain for antidepressant drugs, and it interacts with cocaine and amphetamines [4, 5] All members of this family share two structural characteristics, namely 12 putative transmembrane domains and a large extracellular loop between transmembrane domain 3 and transmembrane domain 4. The amount of inactive, unglycosylated, transporter increases dramatically after day 2 post-infection when there is no further increase in the amount of functional SERT expressed One explanation for this is that the folding of SERT, presumably co-translational [37, 38], has become uncoupled from membrane insertion and protein translation. A selection of different folding assistance factors, including the N-glycan binding molecular chaperones (calnexin, calreticulin), an Hsp family member (BiP), and the foldase ERp57 were co-expressed with SERT in insect cells to examine their roles in the generation of active SERT. The specific folding factors required by SERT in vivo are not yet known, so any molecular chaperones or foldases that improve the overexpression of functional SERT in insect cells can be regarded as potential candidates for assisting the folding of SERT in native tissues
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