Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter

Abstract

The serotonin transporter (SERT) is anN-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 theN-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. The serotonin transporter (SERT) is anN-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 theN-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. The serotonin transporter (SERT) 1The abbreviations used are: SERT, serotonin transporter; SERT-QQ, unglycosylated serotonin transporter mutant; dNJM, 1-deoxynojirimycin; CXN, calnexin; CRT, calreticulin; BiP, immunoglobulin heavy chain binding protein; RTI55 (β-CIT), 2β-carbomethoxy-3β-(4-iodophenyl)tropane; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline-Tween 20; p.i., post-infection. 1The abbreviations used are: SERT, serotonin transporter; SERT-QQ, unglycosylated serotonin transporter mutant; dNJM, 1-deoxynojirimycin; CXN, calnexin; CRT, calreticulin; BiP, immunoglobulin heavy chain binding protein; RTI55 (β-CIT), 2β-carbomethoxy-3β-(4-iodophenyl)tropane; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline-Tween 20; p.i., post-infection. 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 Na+ and Cl− ions down their concentration gradients (reviewed in Refs 1Borowsky B. Hoffman B.J. Int. Rev. Neurobiol. 1995; 38: 139-199Crossref PubMed Scopus (98) Google Scholar, 2Rudnick G. Clark J. Bichim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (367) Google Scholar, 3Worrall D.M. Williams D.C. Biochem. J. 1994; 297: 425-436Crossref PubMed Scopus (71) Google Scholar). SERT is of particular pharmacological interest, because it is the primary binding site in the brain for antidepressant drugs, and it also interacts with cocaine and amphetamines (4Ritz M.C. Cone E.J. Kuhar M.J. Life Sci. 1990; 46: 635-645Crossref PubMed Scopus (438) Google Scholar, 5Rudnick G. Wall S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1817-1821Crossref PubMed Scopus (404) Google Scholar). 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. This particular loop is glycosylated in all members of the family, although the number of potential N-glycans varies between 1 and 4, and the sites of glycosylation are not absolutely conserved. SERT has two N-glycans in this region, and their importance for its activity has been tested by altering the consensusN-glycosylation sequence from Asn-X-Ser/Thr to Gln-X-Ser/Thr; deletion of both N-glycosylation sites in SERT neither the for serotonin uptake the for binding of the inhibitor RTI55 J. 1994; PubMed Google Scholar). the number of functional of to that of the levels of fully glycosylated SERT, suggesting an in the folding of SERT for its in the membrane J. 1994; PubMed Google Scholar). members of the neurotransmitter transporter family were shown to share this with SERT J. 1995; PubMed Scopus Google Scholar, S. Google Scholar, J. PubMed Scopus Google to the large extracellular loop between transmembrane domains 3 and also contain a between two of expression of functional SERT the transporter, of both led to a in activity A. J. 1995; Scholar, S. G. PubMed Scopus Google Scholar). The two to the are absolutely this neurotransmitter transporter family, suggesting that the be structural for chaperones are a of proteins that interact with and the protein correctly (reviewed in Rev. Biochem. PubMed Google and Biochem. Sci. 1994; PubMed Scopus Google Scholar). the protein its the molecular is the protein the interaction between the molecular and the protein be Rev. Biochem. PubMed Google Scholar, Biochem. Sci. 1994; PubMed Scopus Google Scholar). A membrane J. PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google D.M. J. 1994; PubMed Google Scholar, S. Williams J. 1994; PubMed Google Scholar, S. J. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google and A. J. 1994; PubMed Google Scholar, J. 1994; Scopus Google Scholar, PubMed Scopus Google been shown to interact in vivo with various molecular chaperones. molecular immunoglobulin heavy chain binding protein (BiP), interact with the chain of the protein G. S. J. J. 1995; PubMed Scopus (98) Google two molecular chaperones and been shown to interact with a N-glycan to the proteins A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A. Williams J. 1995; PubMed Scopus Google Scholar, A. A. J. 1995; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). a transmembrane domain that it to the ER, it is to a in of proteins A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A. 1994; PubMed Scopus Google Scholar). the sequence calreticulin and calnexin binding to an protein A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The protein ERp57 also on proteins S. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google this is because of its by calnexin calreticulin A. D.C. J. PubMed Scopus Google Scholar). The of the neurotransmitter for N-glycosylation for functional expression that molecular chaperones and be in their this this by the co-expression of calnexin, calreticulin, and ERp57 with SERT in cells using the baculovirus expression baculovirus expression system is used to glycosylated integral membrane proteins in quantities for and structural Rev. Biophys. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of SERT in cells using in of functional SERT by a binding using the cocaine and amounts of inactive transporter J. 1994; PubMed Google Scholar). The of transporter is increase in the of functional SERT for this is that the folding of SERT, PubMed Scopus Google Scholar, J. PubMed Scopus Google has from membrane and protein folding has the rate-limiting the of SERT, the amounts of molecular chaperones that SERT to increase the of functional SERT a of folding the N-glycan binding molecular chaperones an family member (BiP), and the ERp57 were co-expressed with SERT in cells to their in the of active The folding by SERT in vivo are not molecular chaperones that the of functional SERT in cells be as potential for the folding of SERT in serotonin transporter is a integral membrane protein of 12 transmembrane that for folding and J. 1994; PubMed Google and the of a putative for activity S. G. PubMed Scopus Google Scholar). shown that the of Myc-SERT is enhanced by molecular chaperones. The between Myc-SERT and the molecular chaperones was investigated in cells using the baculovirus expression system. The of large quantities of inactive Myc-SERT in cells that folding be rate-limiting for the of functional Myc-SERT using the baculovirus expression system. this the of molecular chaperones in active Myc-SERT was and that co-expression of molecular chaperones increased the of functional SERT The of chaperones be to Myc-SERT in a and prevent was the of the co-expressed molecular functional Myc-SERT expression by nearly in functional Myc-SERT expression were also on co-expression of calreticulin and BiP. between Myc-SERT and calnexin, and Myc-SERT and calreticulin, were in was not to an interaction between and Myc-SERT in the because of in the The protein did not increase functional Myc-SERT expression in the of molecular expression of functional Myc-SERT was a of molecular activity and not because of in This was from of from cells co-expressing Myc-SERT and the molecular chaperones calnexin and The levels of unglycosylated and glycosylated Myc-SERT did not increase in with the in functional Myc-SERT determined from inhibitor suggesting that the were because of folding of Myc-SERT in the of molecular chaperones. co-expression of calnexin the of inactive Myc-SERT This was on co-expression of Myc-SERT, calnexin, and BiP, on 1 p.i., glycosylated and unglycosylated Myc-SERT and of the levels on a amounts of functional A number of this in Myc-SERT increased of Myc-SERT of because of coupling between folding and for the large increase in functional Myc-SERT expression on co-expression of calnexin are that is calnexin in cells that calnexin not interact with Myc-SERT. This was from Myc-SERT in the of The of the is by is to prevent calnexin from with Myc-SERT in as in the of co-expressed calnexin, has on functional Myc-SERT suggesting that the folding of Myc-SERT in cells is not on an this it be determined is calnexin in the cells is calnexin and that it not interact with Myc-SERT of using to calnexin it is not calnexin cells not be the cells to calnexin because this has also been for A. Williams J. PubMed Scopus Google large increase in levels of functional Myc-SERT by co-expressing calnexin a of the interaction between Therefore, an unglycosylated SERT mutant, SERT-QQ, was co-expressed with calnexin was of the functional expression of that was interaction between calnexin and this not that was between and calnexin, because interaction been by This be calnexin with SERT its transmembrane region, as has been for the between calnexin D.M. J. 1995; PubMed Scopus Google Scholar). The interaction between and calnexin also to be inhibited by The is that interact with calnexin and prevent its as a molecular this not be that is inhibited by dNJM, the N-glycan as is that a small of this as are N-glycosylation sites predicted to be in SERT the the used for J. 1990; PubMed Google Scholar). These also be by an of calnexin on calnexin the folding of molecular in cells that to in all a tagged version of SERT with the the and predicted to be in the The molecular chaperones and BiP, CRT, and ERp57 are all in the and, therefore, interact with The functional domain of calnexin is in the it also has a transmembrane domain and a domain that is in the the on SERT interact with the of although the of calnexin is for the of calnexin in the ER, it is not to be for of calnexin A. D.C. J. PubMed Scopus Google Scholar). the of the molecular it was that the of the on SERT the ability of molecular chaperones to the expression of the transporter the of functional Myc-SERT not from the of functional SERT are co-expression of calnexin with Myc-SERT led to a 3-fold in functional activity as with a in expression of is that the on SERT are a on the folding of a for the co-expressed molecular chaperones to and an in the of SERT be folded using the molecular chaperones in and molecular chaperones shown that the folding of Myc-SERT in vivo be assisted by the molecular chaperones calnexin, calreticulin, and BiP. it has been shown that the a increase the expression of the transporter Biochem. Biophys. PubMed Scopus Google a of the of molecular chaperones and in the folding of SERT not to an of membrane proteins it also to proteins for and structural membrane proteins are in expression Rev. Biophys. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google and this be a of the of molecular chaperones in the be insufficient amounts of molecular chaperones to with the high of protein in expression from cells is enhanced by co-expressing protein M.J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). co-expressing molecular chaperones has not been suggesting that their levels of expression The of chaperones and on and the of as the protein to this The serotonin transporter (SERT) 1The abbreviations used are: SERT, serotonin transporter; SERT-QQ, unglycosylated serotonin transporter mutant; dNJM, 1-deoxynojirimycin; CXN, calnexin; CRT, calreticulin; BiP, immunoglobulin heavy chain binding protein; RTI55 (β-CIT), 2β-carbomethoxy-3β-(4-iodophenyl)tropane; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline-Tween 20; p.i., post-infection. 1The abbreviations used are: SERT, serotonin transporter; SERT-QQ, unglycosylated serotonin transporter mutant; dNJM, 1-deoxynojirimycin; CXN, calnexin; CRT, calreticulin; BiP, immunoglobulin heavy chain binding protein; RTI55 (β-CIT), 2β-carbomethoxy-3β-(4-iodophenyl)tropane; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline-Tween 20; p.i., post-infection. 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 Na+ and Cl− ions down their concentration gradients (reviewed in Refs 1Borowsky B. Hoffman B.J. Int. Rev. Neurobiol. 1995; 38: 139-199Crossref PubMed Scopus (98) Google Scholar, 2Rudnick G. Clark J. Bichim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (367) Google Scholar, 3Worrall D.M. Williams D.C. Biochem. J. 1994; 297: 425-436Crossref PubMed Scopus (71) Google Scholar). SERT is of particular pharmacological interest, because it is the primary binding site in the brain for antidepressant drugs, and it also interacts with cocaine and amphetamines (4Ritz M.C. Cone E.J. Kuhar M.J. Life Sci. 1990; 46: 635-645Crossref PubMed Scopus (438) Google Scholar, 5Rudnick G. Wall S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1817-1821Crossref PubMed Scopus (404) Google Scholar). 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. This particular loop is glycosylated in all members of the family, although the number of potential N-glycans varies between 1 and 4, and the sites of glycosylation are not absolutely conserved. SERT has two N-glycans in this region, and their importance for its activity has been tested by altering the consensusN-glycosylation sequence from Asn-X-Ser/Thr to Gln-X-Ser/Thr; deletion of both N-glycosylation sites in SERT neither the for serotonin uptake the for binding of the inhibitor RTI55 J. 1994; PubMed Google Scholar). the number of functional of to that of the levels of fully glycosylated SERT, suggesting an in the folding of SERT for its in the membrane J. 1994; PubMed Google Scholar). members of the neurotransmitter transporter family were shown to share this with SERT J. 1995; PubMed Scopus Google Scholar, S. Google Scholar, J. PubMed Scopus Google Scholar). to the large extracellular loop between transmembrane domains 3 and also contain a between two of expression of functional SERT the transporter, of both led to a in activity A. J. 1995; Scholar, S. G. PubMed Scopus Google Scholar). The two to the are absolutely this neurotransmitter transporter family, suggesting that the be structural for chaperones are a of proteins that interact with and the protein correctly (reviewed in Rev. Biochem. PubMed Google and Biochem. Sci. 1994; PubMed Scopus Google Scholar). the protein its the molecular is the protein the interaction between the molecular and the protein be Rev. Biochem. PubMed Google Scholar, Biochem. Sci. 1994; PubMed Scopus Google Scholar). A membrane J. PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google D.M. J. 1994; PubMed Google Scholar, S. Williams J. 1994; PubMed Google Scholar, S. J. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google and A. J. 1994; PubMed Google Scholar, J. 1994; Scopus Google Scholar, PubMed Scopus Google been shown to interact in vivo with various molecular chaperones. molecular immunoglobulin heavy chain binding protein (BiP), interact with the chain of the protein G. S. J. J. 1995; PubMed Scopus (98) Google two molecular chaperones and been shown to interact with a N-glycan to the proteins A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A. Williams J. 1995; PubMed Scopus Google Scholar, A. A. J. 1995; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). a transmembrane domain that it to the ER, it is to a in of proteins A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A. 1994; PubMed Scopus Google Scholar). the sequence calreticulin and calnexin binding to an protein A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The protein ERp57 also on proteins S. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google this is because of its by calnexin calreticulin A. D.C. J. PubMed Scopus Google Scholar). The of the neurotransmitter for N-glycosylation for functional expression that molecular chaperones and be in their this this by the co-expression of calnexin, calreticulin, and ERp57 with SERT in cells using the baculovirus expression system. The baculovirus expression system is used to glycosylated integral membrane proteins in quantities for and structural Rev. Biophys. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of SERT in cells using in of functional SERT by a binding using the cocaine and amounts of inactive transporter J. 1994; PubMed Google Scholar). The of transporter is increase in the of functional SERT for this is that the folding of SERT, PubMed Scopus Google Scholar, J. PubMed Scopus Google has from membrane and protein folding has the rate-limiting the of SERT, the amounts of molecular chaperones that SERT to increase the of functional SERT a of folding the N-glycan binding molecular chaperones an family member (BiP), and the ERp57 were co-expressed with SERT in cells to their in the of active The folding by SERT in vivo are not molecular chaperones that the of functional SERT in cells be as potential for the folding of SERT in serotonin transporter is a integral membrane protein of 12 transmembrane that for folding and J. 1994; PubMed Google and the of a putative for activity S. G. PubMed Scopus Google Scholar). shown that the of Myc-SERT is enhanced by molecular chaperones. The between Myc-SERT and the molecular chaperones was investigated in cells using the baculovirus expression system. The of large quantities of inactive Myc-SERT in cells that folding be rate-limiting for the of functional Myc-SERT using the baculovirus expression system. this the of molecular chaperones in active Myc-SERT was and that co-expression of molecular chaperones increased the of functional SERT The of chaperones be to Myc-SERT in a and prevent was the of the co-expressed molecular functional Myc-SERT expression by nearly in functional Myc-SERT expression were also on co-expression of calreticulin and BiP. between Myc-SERT and calnexin, and Myc-SERT and calreticulin, were in was not to an interaction between and Myc-SERT in the because of in the The protein did not increase functional Myc-SERT expression in the of molecular expression of functional Myc-SERT was a of molecular activity and not because of in This was from of from cells co-expressing Myc-SERT and the molecular chaperones calnexin and The levels of unglycosylated and glycosylated Myc-SERT did not increase in with the in functional Myc-SERT determined from inhibitor suggesting that the were because of folding of Myc-SERT in the of molecular chaperones. co-expression of calnexin the of inactive Myc-SERT This was on co-expression of Myc-SERT, calnexin, and BiP, on 1 p.i., glycosylated and unglycosylated Myc-SERT and of the levels on a amounts of functional A number of this in Myc-SERT increased of Myc-SERT of because of coupling between folding and for the large increase in functional Myc-SERT expression on co-expression of calnexin are that is calnexin in cells that calnexin not interact with Myc-SERT. This was from Myc-SERT in the of The of the is by is to prevent calnexin from with Myc-SERT in as in the of co-expressed calnexin, has on functional Myc-SERT suggesting that the folding of Myc-SERT in cells is not on an this it be determined is calnexin in the cells is calnexin and that it not interact with Myc-SERT of using to calnexin it is not calnexin cells not be the cells to calnexin because this has also been for A. Williams J. PubMed Scopus Google large increase in levels of functional Myc-SERT by co-expressing calnexin a of the interaction between Therefore, an unglycosylated SERT mutant, SERT-QQ, was co-expressed with calnexin was of the functional expression of that was interaction between calnexin and this not that was between and calnexin, because interaction been by This be calnexin with SERT its transmembrane region, as has been for the between calnexin D.M. J. 1995; PubMed Scopus Google Scholar). The interaction between and calnexin also to be inhibited by The is that interact with calnexin and prevent its as a molecular this not be that is inhibited by dNJM, the N-glycan as is that a small of this as are N-glycosylation sites predicted to be in SERT the the used for J. 1990; PubMed Google Scholar). These also be by an of calnexin on calnexin the folding of molecular in cells that to in all a tagged version of SERT with the the and predicted to be in the The molecular chaperones and BiP, CRT, and ERp57 are all in the and, therefore, interact with The functional domain of calnexin is in the it also has a transmembrane domain and a domain that is in the the on SERT interact with the of although the of calnexin is for the of calnexin in the ER, it is not to be for of calnexin A. D.C. J. PubMed Scopus Google Scholar). the of the molecular it was that the of the on SERT the ability of molecular chaperones to the expression of the transporter the of functional Myc-SERT not from the of functional SERT are co-expression of calnexin with Myc-SERT led to a 3-fold in functional activity as with a in expression of is that the on SERT are a on the folding of a for the co-expressed molecular chaperones to and an in the of SERT be folded using the molecular chaperones in and molecular chaperones shown that the folding of Myc-SERT in vivo be assisted by the molecular chaperones calnexin, calreticulin, and BiP. it has been shown that the a increase the expression of the transporter Biochem. Biophys. PubMed Scopus Google a of the of molecular chaperones and in the folding of SERT not to an of membrane proteins it also to proteins for and structural membrane proteins are in expression Rev. Biophys. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google and this be a of the of molecular chaperones in the be insufficient amounts of molecular chaperones to with the high of protein in expression from cells is enhanced by co-expressing protein M.J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). co-expressing molecular chaperones has not been suggesting that their levels of expression The of chaperones and on and the of as the protein to this The serotonin transporter is a integral membrane protein of 12 transmembrane that for folding and J. 1994; PubMed Google and the of a putative for activity S. G. PubMed Scopus Google Scholar). shown that the of Myc-SERT is enhanced by molecular chaperones. The between Myc-SERT and the molecular chaperones was investigated in cells using the baculovirus expression system. The of large quantities of inactive Myc-SERT in cells that folding be rate-limiting for the of functional Myc-SERT using the baculovirus expression system. this the of molecular chaperones in active Myc-SERT was and that co-expression of molecular chaperones increased the of functional SERT The of chaperones be to Myc-SERT in a and prevent was the of the co-expressed molecular functional Myc-SERT expression by nearly in functional Myc-SERT expression were also on co-expression of calreticulin and BiP. between Myc-SERT and calnexin, and Myc-SERT and calreticulin, were in was not to an interaction between and Myc-SERT in the because of in the The protein did not increase functional Myc-SERT expression in the of molecular chaperones. expression of functional Myc-SERT was a of molecular activity and not because of in This was from of from cells co-expressing Myc-SERT and the molecular chaperones calnexin and The levels of unglycosylated and glycosylated Myc-SERT did not increase in with the in functional Myc-SERT determined from inhibitor suggesting that the were because of folding of Myc-SERT in the of molecular chaperones. co-expression of calnexin the of inactive Myc-SERT This was on co-expression of Myc-SERT, calnexin, and BiP, on 1 p.i., glycosylated and unglycosylated Myc-SERT and of the levels on a amounts of functional A number of this in Myc-SERT increased of Myc-SERT of because of coupling between folding and The for the large increase in functional Myc-SERT expression on co-expression of calnexin are that is calnexin in cells that calnexin not interact with Myc-SERT. This was from Myc-SERT in the of The of the is by is to prevent calnexin from with Myc-SERT in as in the of co-expressed calnexin, has on functional Myc-SERT suggesting that the folding of Myc-SERT in cells is not on an this it be determined is calnexin in the cells is calnexin and that it not interact with Myc-SERT of using to calnexin it is not calnexin cells not be the cells to calnexin because this has also been for A. Williams J. PubMed Scopus Google Scholar). The large increase in levels of functional Myc-SERT by co-expressing calnexin a of the interaction between Therefore, an unglycosylated SERT mutant, SERT-QQ, was co-expressed with calnexin was of the functional expression of that was interaction between calnexin and this not that was between and calnexin, because interaction been by This be calnexin with SERT its transmembrane region, as has been for the between calnexin D.M. J. 1995; PubMed Scopus Google Scholar). The interaction between and calnexin also to be inhibited by The is that interact with calnexin and prevent its as a molecular this not be that is inhibited by dNJM, the N-glycan as is that a small of this as are N-glycosylation sites predicted to be in SERT the the used for J. 1990; PubMed Google Scholar). These also be by an of calnexin on calnexin the folding of molecular in cells that to The in all a tagged version of SERT with the the and predicted to be in the The molecular chaperones and BiP, CRT, and ERp57 are all in the and, therefore, interact with The functional domain of calnexin is in the it also has a transmembrane domain and a domain that is in the the on SERT interact with the of although the of calnexin is for the of calnexin in the ER, it is not to be for of calnexin A. D.C. J. PubMed Scopus Google Scholar). the of the molecular it was that the of the on SERT the ability of molecular chaperones to the expression of the transporter the of functional Myc-SERT not from the of functional SERT are co-expression of calnexin with Myc-SERT led to a 3-fold in functional activity as with a in expression of is that the on SERT are a on the folding of a for the co-expressed molecular chaperones to and an in the of SERT be folded using the molecular chaperones in and molecular chaperones shown that the folding of Myc-SERT in vivo be assisted by the molecular chaperones calnexin, calreticulin, and BiP. it has been shown that the a increase the expression of the transporter Biochem. Biophys. PubMed Scopus Google a of the of molecular chaperones and in the folding of SERT not to an of membrane proteins it also to proteins for and structural membrane proteins are in expression Rev. Biophys. 1995; PubMed Scopus Google Scholar, PubMed Scopus Google and this be a of the of molecular chaperones in the be insufficient amounts of molecular chaperones to with the high of protein in expression from cells is enhanced by co-expressing protein M.J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). co-expressing molecular chaperones has not been suggesting that their levels of expression The of chaperones and on and the of as the protein to this are to for the Williams for the calnexin for the ERp57 and for the calreticulin