TheRoleofERp44inMaturationofSerotoninTransporter …This article has been withdrawn by Samuel...

The Role of ERp44 in Maturation of Serotonin TransporterProtein*

Received for publication, January 23, 2012, and in revised form, March 22, 2012 Published, JBC Papers in Press, March 26, 2012, DOI 10.1074/jbc.M112.345058

Samuel Freyaldenhoven, Yicong Li, Arif M. Kocabas, Enrit Ziu, Serra Ucer, Raman Ramanagoudr-Bhojappa,Grover P. Miller, and Fusun Kilic1

From the Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences,Little Rock, Arkansas 72205

Background: ERp44 favors maturation of disulfide-linked oligomeric proteins.Results: ERp44 bound to SERT but preferentially to Cys mutants; in ERp44-silenced cells, 5-HT uptake was down-regulated;MTSEA-biotin labeled SERT with a higher affinity, indicating more free Cys on SERT in silenced cells.Conclusion: A disulfide link between Cys-200 and Cys-209 is a prerequisite for SERT oligomerization.Significance: This is the first study showing the involvement of ERp44 in maturation of SERT.

Inheterologous and endogenous expression systems,we stud-ied the role of ERp44 and its complex partner endoplasmic retic-ulum (ER) oxidase 1-� (Ero1-L�) in mechanisms regulatingdisulfide bond formation for serotonin transporter (SERT), anoligomeric glycoprotein. ERp44 is an ER lumenal chaperoneprotein that favors the maturation of disulfide-linked oligo-meric proteins. ERp44 plays a critical role in the release of pro-teins from the ER via binding to Ero1-L�. Mutation in the thio-redoxin-like domain hampers the association of ERp44C29Swith SERT,which has threeCys residues (Cys-200, Cys-209, andCys-109) on the second external loop. We further explored therole of the protein chaperones through shRNA knockdownexperiments for ERp44 and Ero1-L�. Those efforts resulted inincreased SERT localization to the plasma membrane butdecreased serotonin (5-HT) uptake rates, indicating the impor-tance of theERp44retentionmechanism in thepropermaturationofSERTproteins.Thesedatawerestronglysupportedwiththedatareceived from the N-biotinylaminoethyl methanethiosulfonate(MTSEA-biotin) labelingofSERTonERp44shRNAcells.MTSEA-biotin only interacts with the free Cys residues from the externalphase of the plasmamembrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible tolabelingbyMTSEA-biotin.However, in the control cells, theseCysresidues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERTmutants (C200S, C209S, and C109A) when compared with wildtype. These interactionswith the chaperonemay reflect the inabil-ity ofCys-200 andCys-209 SERTmutants to formadisulfide bondand self-association as evidenced by immunoprecipitation assays.Based on these collective findings, we hypothesize that ERp44together with Ero1-L� plays an important role in disulfide forma-tion of SERT, whichmay be a prerequisite step for the assembly ofSERTmolecules in oligomeric form.

The serotonin transporter (SERT2; SLC64A) is a member ofthe Na�- and Cl�-dependent monoamine transporter family,which includes the dopamine transporter and the norepineph-rine transporter (1). These neurotransmitter transporters shareextensive sequence homology (1–4) with several commonstructural characteristics, including oligomeric properties(5–7), multiple sites forN-linked glycosylation (8–10), and thecysteine residues connected by a disulfide bond on the secondextracellular loop (EL2) (1, 3, 11, 12). Proper post-translationalmodifications are essential regulatory factors in neurotransmit-ter uptake functions of SERT (9–11), norepinephrine trans-porter (8), and dopamine transporter (6, 12) and occur in ahost-dependent fashion (13). Following these modificationstransporters adopt more stable, lower energy conformations(14–17) to initiate for their correct folding and assembly. ForSERT, these alterations of its structure impact the extracellularuptake function of serotonin (5-HT) and hence its biologicalrole in neurons and peripheral tissues (1–3). Consequently,identifying themechanisms regulating post-translational mod-ifications would advance our understanding of the importanceof SERT conformations and oligomerization in biologicalprocesses.Conformational maturation of monoamine transporter fam-

ily members involves different pathways for post-translationalmodifications of the respective proteins (18–21). Formation ofintra- or intermolecular disulfide bonds is one of the majorrate-limiting factors. SERT has three Cys residues on the sec-ond external loop. Disulfide bond formation between two resi-dues, Cys-200 and Cys-209, is required for SERT folding, sur-face expression, and transport activity (11). Disruption of thedisulfide bond by a single mutation of Cys-200 or Cys-209 pro-duces mutants with a terminally exposed thiol that are retainedintracellularly (11, 22). However, mutation of both Cys in thepair allows SERT to reach the plasma membrane but compro-mises transport activity (22). Previous studies highlighting the

* This work was supported, in whole or in part, by National Institutes of HealthGrants HD058697, HD053477, HL091196, and HL091196-01A2W1 (to F. K.).

1 To whom correspondence should be addressed: Dept. of Biochemistry andMolecular Biology, College of Medicine, University of Arkansas for MedicalSciences,4301W.MarkhamSt.,#516,LittleRock,AR72205.E-mail:[email protected].

2 The abbreviations used are: SERT, serotonin transporter; 5-HT, serotonin;MSH, �-mercaptoethanol; ER, endoplasmic reticulum; Ero1-L�, ER oxidase1-�; Ab, antibody; WB, Western blot; IP, immunoprecipitation; QQ, twoglycosylation sites mutated to glutamine; MTSEA-biotin; N-biotinylamino-ethyl methanethiosulfonate; PM, plasma membrane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 21, pp. 17801–17811, May 18, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MAY 18, 2012 • VOLUME 287 • NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 17801

This article has been withdrawn by Samuel Freyaldenhoven, Yicong Li, Arif M. Kocabas, Grover P. Miller, and Fusun Kilic. Despite attempts, Enrit Ziu, Serra Ucer, and Raman Ramanagoudr-Bhojappa could not be reached for

conference on this decision. Dr. Kilic contacted the editorial office to report errors in Fig. 3A and Fig 8 of their article. The Journal requested the

original data for Figs. 1A, 1D, 3A, 6A, 8A, 8B, and 9A. Due to the dated material, she could not provide all of them. The data that were provided

to the Journal was not 300 ppi. The investigation by the Journal determined the following. Lanes 2 and 3 of the total SERT immunoblot in Fig. 1A were duplicated in lanes 5 and 6. The actin immunoblot from Fig. 1A was reused in Figs. 8B and 9A as actin. The first two lanes of the SERT

(PM) immunoblot in Fig. 3A were duplicated. Additionally, the first lane of the Total SERT immunoblot in Fig. 3A was reused in lane 4. In Fig. 6A, lanes

2 and 5 of the actin immunoblot were duplicated.

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importance of a disulfide bond for SERT folding and surfaceexpression suggest a thiol-dependent quality control mecha-nism in SERT maturation (11, 22).Moreover, the modification of exposed thiols with �-mer-

captoethanol (MSH) altered the density of SERT molecules onthe plasmamembrane and the 5-HTuptake rates (10). Pretreat-ment of the cells with MSH led to much lower 5-HT uptakerates when compared with control cells, although MSH treat-ment released more SERTmolecules to the plasma membrane.The association between SERT molecules also was obstructedby MSH treatment (10). Altogether, these findings indicate theinvolvement of SERT in a thiol-mediated retentionmechanism.However, neither themediators aiding in disulfide bond forma-tion nor the role of disulfide bonds in SERT maturation hasbeen vigorously studied yet.To explore factors involved in SERT maturation, we exam-

ined whether the SERTmaturation pathway utilizes ERp44 andER oxidase 1-� (Ero1-L�) because of their roles in the matura-tion and quality control of disulfide-containing oligomeric pro-teins (15, 19, 23–29). An exposed thiol for a protein localized tothe ER favors formation of mixed disulfide bonds with thiore-doxin family members, notably ERp44 (16, 23–27). During thisprocess, ERp44 preferentially associates with unassembled sub-units of disulfide-containing oligomeric proteins (15, 26, 27). Inthis study, we employed biochemical and molecular biologicaltechniques using endogenous and heterologous expression sys-tems to assess an association betweenERp44 and SERT, the roleof Ero1-L� in the maturation process, and the correspondingimpacts on SERT localization and function at the plasmamem-brane. ERp44-silenced cells led to increased levels of SERT atthe cell surface. The functional state of SERT was also compro-mised based on changes in the mechanism andmaximal rate of5-HT uptake by these cells. Moreover, SERT mutants (C200S,C209S, and C109A) with compromised disulfide bond forma-tion and hence structure and function were shown to preferen-tially associate with ERp44. Taken together, these studies sug-gest that (i) disulfide bond formationmay present a critical stepin SERT folding to a fully active form, and (ii) SERT utilizesERp44 as well as its partner Ero1-L�, an oxidoreductase, in thedisulfide bond formation process.

MATERIALS AND METHODS

JAR cells were provided by the American Type Culture Col-lection (Manassas, VA). Protein A-Sepharose beads and non-immune rabbit serum were purchased from Zymed Laborato-ries Inc. (South San Francisco, CA). 3H-Labeled 5-HT waspurchased from PerkinElmer Life Sciences. HA-tagged anduntagged forms of ERp44 and the Ero1-L� construct were agenerous gift fromDr. Sitia Roberto (Salute SanRaffaele,Milan,Italy). Lentiviral small hairpin RNA (shRNA) plasmid, anti-ERp44, and Ero1-L� antibodies were generous gifts from Dr.Scherer at the University of Texas Southwestern and used byWang et al. (30). The second-generation packaging plasmid,psPAX2, andVSV-Gwere purchased fromAddgene Inc. (Cam-bridge, MA). Expression vectors, cell culture materials, Lipo-fectin, and Lipofectamine 2000 were purchased from Invitro-gen. ERp44 and Ero1-L� antibody (Ab) were purchased fromCell Signaling Technology (Beverly, MA). NHS-SS-biotin, the

Micro BCA protein assay reagent kit, and Pico-West Supersig-nal ECL substrate were purchased from Pierce. Scintillationmixture was purchased from Fisher. A monoclonal SERT Abrecognizing amino acid residues 51–66 on the N terminus waspurchased fromMab Technologies (Stone Mountain, GA).Plasmids, Constructs, andCell Line Expression Systems—JAR

cells were cultured inRPMI 1640mediumwith 10% fetal bovineserum, 2 mM L-glutamine, 100 units/ml penicillin, and 100�g/ml streptomycin, referred to as “full RPMI.” Cells (2 � 105cells/assay) were used in biotinylation, Western blot (WB),membrane preparation, transport assay, and immunoprecipi-tation (IP) assays 48 h postseeding.Transporters with both glycosylation sites mutated to gluta-

mine, QQ (N208Q and N17Q), were constructed utilizing aStratagene QuikChange XL site-directed mutagenesis kit asdescribed previously (7, 10).The three Cys residue (C109A, C200S, and C209S) muta-

tionswere introduced by site-directedmutagenesis using oligo-nucleotides 5�-CTT CCC CTA CAT AGC TTA CCA GAATGG AG-3�, 5�-CTG CCC TGG ACC AGC TCC AAG AACTCC TGG AAC AC-3�, and 5�-CCT GGA ACA CTG GCAACT CCA CCA ATT ACT TCT CCG AG-3�, respectively, onSERTand the FLAG- andMyc-tagged forms of SERT.Using thesame primers, the double mutant was generated.We confirmed the subcloning processes by sequencing the

genes at the University of Arkansas for Medical Sciences DNASequencing Facility. In addition, mutants with Cys-200mutated to serine were prepared using the same method, andmutations were confirmed by sequencing.These mutants were expressed in JAR cells by using the vac-

cinia-T7 transient expression system as described (10). Trans-fected cells were incubated for 16–20 h at 37 °C before theywere used for transport or IP experiments. Protein concentra-tion was obtained by means of the Micro BCA protein assayreagent kit (Pierce).5-HTUptake Assay—Before seeding the cells, a 24-well plate

was coated with poly-D-lysine (0.1–0.5 mg/ml in sterile water)for 30 min and washed three times with sterile water. JAR cellswere seeded 36–48 h in a polylysine-coated 24-well plate priorto initiating the transport assay. Uptake assays were performedby incubation of cells (2 � 105 cells/assay) in 20.5 nM [1,2-3H]5-HT (3400 cpm/pmol) in PBS/CM (phosphate-bufferedsaline, 0.1 mM CaCl2, and 1 mM MgCl2). The intact cells werewashed quickly with ice-cold PBS to stop the activity, harvestedin 2% SDS in PBS, and transferred to scintillation vials contain-ing 5 ml of scintillation mixture, and the radioactivity wasdetermined in a Beckman scintillation counter. An equal num-ber of cells per cell line was confirmed by cell counting with ahemocytometer, and a group of cells was treated with a highaffinity cocaine analog, 0.1 �M 2�-carbomethoxy-3-tropane, tomonitor 5-HT influx in the background (2�-carbomethoxy-3-tropane was provided by the National Institute of MentalHealth) (10).The resulting data were fit to equations for two different

models describing the relationship between the uptake rate and5-HT concentrations. The traditional model describes a hyper-bolic kinetic profile in which the uptake rate reflects contribu-tions from a single transporter at a constant concentration and

Maturation of Serotonin Transporter in Endoplasmic Reticulum

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the transporter binds 5-HT in a 1:1 stoichiometry. When theseconditions are not satisfied, the kinetic profilemay deviate froma simple hyperbola, and thuswe also fit data to theHill equationdescribing cooperative effects of 5-HT concentrations on theuptake rate. Equation 1 depicts the Hill equation such that � isthe observed uptake rate,Vmax is themaximal uptake rate,Kh is[5-HT] at themidpoint of the curve, and n is the Hill coefficientormeasure of cooperativity.When theHill coefficient is 1.0, theequation reduces down to the one used for fitting the data to thetraditional transport model. The fits of the data to these kineticmodels were compared, and the most probable one was identi-fied by the Akaike Information Criterion using GraphPadPrism software (San Diego, CA).

� � �Vmax � [5-HT]n��Khn � �5-HT]n� (Eq. 1)

Preparation of shRNA Lentiviral Particles—Lentiviral shRNAexpression constructs were created utilizing a second-genera-tion packaging plasmid, psPAX2, obtained from Addgene.HEK-293FT cells were cultured for 48 h and transiently trans-fected with the respective shRNA construct graciously pro-vided by Dr. Scherer (University of Texas Southwestern),psPAX2, and VSV-G in a 1:2 ratio of Lipofectamine 2000 toOpti-MEMmedium. The medium was replaced 12 h later withfull DMEM. After 48 h post-transfection, the medium was col-lected, filtered with a 0.45-�m PDVF filter, and precipitated inPEG 8000 Na�/Cl� solution for 20 h. The lentiviral particleswere collected with centrifugation at 4 °C and resuspended inPBS. Viral particles were stored at �80 °C.Transfection with Lentiviral Expression Constructs—JAR

cells were grown to 60% confluence for 24 h in full medium.Cells were transfected with lentiviral constructs in full mediumwith 5 �g/ml Polybrene and 1:1000 dilution with respectiveviral particles for 16 h. The mediumwas replaced the followingday, and the cells were allowed to divide. The cells were sortedfor positive GFP expression on a FACSAria at the University ofArkansas for Medical Sciences Core Flow Cytometry Facility72 h post-transfection, and the positive cells were cultured infull medium for further experiments. These cells (2� 105 cells/assay)were used in the assays or transiently transfectedwith thethree constructs, SERT, QQ, and C200S, under a CMV pro-moter as described previously (5, 7).WB Analysis—Cells (2 � 105 cells/assay) were solubilized in

PBS containing 0.44% SDS, 1 mM phenylmethylsulfonyl fluo-ride (PMSF), and protease inhibitormixture, which contained 5mg/ml pepstatin, 5mg/ml leupeptin, and 5mg/ml aprotinin. Inlysis buffer, the alkylating agent N-ethylmaleimide at a finalconcentration of 5 mM was added to prevent oxidation andformation of nonspecific disulfide bonds during lysis and toretain the native monomeric structures in the gel (7). Sampleswere analyzed by 10% SDS-PAGE and transferred to nitrocel-lulosemembrane.WB analysis was performedwith anti-ERp44(diluted 1:1000), anti-SERT (diluted 1:500), or anti-Ero1-L�(diluted 1:1000) Ab and then with horseradish peroxidase-con-jugated anti-rabbit secondary Ab (diluted 1:7500), respectively.The signals were visualized using the ECL WB detection sys-tem. Blots were visualized using the VersaDoc 1000 gel visual-ization and analysis system and three separate readings in order

to calculate the average intensity (Bio-Rad) (10). Statisticalanalysis of densitometric scans was done using a two-sided ttest.Cell Surface Biotinylation—The surface expression of the

SERT and themutant formsweremonitored by biotinylation asdescribed (5). In brief, cells (2 � 105 cells/assay) were treatedwith the membrane-impermeant biotinylating reagent NHS-SS-biotin (Pierce) or 1 mM MTSEA-biotin (Toronto ResearchChemicals, Downsview, Canada). These reagents selectivelymodify only the external lysine residues onmembrane proteins.The unbound reagent was washed away and quenched withglycine (in the case of NHS-SS-biotin), and the cells were lysedin Tris-buffered saline containing 1% SDS, 1% Triton X-100,and protease inhibitor mixture/PMSF. The biotinylated pro-teins were recovered with streptavidin-agarose beads (Pierce).Quenching unreactedMTSEA-biotinwas not required becauseof its rapid hydrolysis in aqueous buffers. The labeled proteinswere resolved by 8% SDS-PAGE, were transferred to nitrocel-lulose, and were detected with anti-SERT, anti-FLAG, or anti-Myc antibodies as described (5, 7, 10). NHS-SS-biotin containsa sulfonic acid moiety with a fixed negative charge and wasshown to selectivelymodify lysine amino groups exposed on thecell surface, and MTSEA-biotin was shown to selectively mod-ify external Cys sulfhydryl groups under the conditions used (5,10). The primary Ab was detected using horseradish peroxi-dase-conjugated secondary Ab and the ECL detection system.Immunoprecipitation—JAR cells were used as an endoge-

nous expression system to assay SERT, ERp44, and Ero1-L�association. Before collection, cells were treated with 10 mM

N-ethylmaleimide for 30 min. Cells were lysed in IP buffer (55mM triethylamine (pH 7.5), 111mMNaCl, 2.2mMEDTA, 0.44%SDS, 1%TritonX-100, 1mMPMSF/protease inhibitormixture)containing 5mMN-ethylmaleimide (1, 2). Cell lysates were firstprecleared by incubation with protein A beads. The preclearedlysate was treated with primary Ab-coated protein A beads(polyclonalHAAb in the heterologous and polyclonal SERTAbin the endogenous expression system) overnight at 4 °C. Thelysate immune complexes were recovered by brief centrifuga-tion in a bench topmicrocentrifuge (Beckman), washed severaltimes with high and low salt IP buffers, and eluted in Laemmlisample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% brom-phenol blue, 10% glycerol, and 1% mercaptoethanol). Sampleswere separated on a 10%SDS-polyacrylamide gel. After electro-phoresis, gels were analyzed by WB with either horseradishperoxidase-conjugatedmonoclonal FLAGAb (diluted 1:750) orpolyclonal anti-ERp44 or -Ero1-L�-Ab (diluted 1:1000). Thesignals were developed with the ECL detection system.Data Analysis—Analysis of variance was performed to assess

group differences for 5-HT end points, and comparisonsbetween groups weremade using two-sided t tests based on theanalysis of variance mean squared error. Data are presented asmean and S.D. of multiple experiments.

RESULTS

In order to evaluate ER thiol-mediated SERT retention atthe plasma membrane and its role in SERT function, humanplacental JAR cells were pretreated with MSH at differentconcentrations, as it was previously studied for IgG (26). The




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surface expression of SERT in MSH-pretreated JAR cellsincreased in parallel with MSH concentrations (Fig. 1A),whereas their 5-HT uptake rates were significantly lower

than the control cells (Fig. 1B). The results indicate theinvolvement of the disulfide bond formation in the matura-tion process of SERT in ER.

FIGURE 1. A, JAR cells expressing SERT were pretreated with MSH at different concentrations (0 –10 mM), and the intact cells were biotinylated (5, 7, 10).Biotinylated PM proteins were resolved on SDS-PAGE followed by WB analysis using a polyclonal SERT and actin Ab. The level of actin in each lane shows thecell population in each group. B, the 5-HT uptake rates of MSH-treated JAR cells were measured as described previously (5, 7, 10). C, WB analysis of endogenousSERT, Ero1-La, and ERp44 expressions in JAR cells. D, the association between SERT and ERp44 or Ero1-L� was determined in JAR cells. JAR cell lysates wereprepared and subjected to IP in the presence (�Ab) or absence (�Ab) of monoclonal anti-SERT Ab, as described under “Materials and Methods.” The IP andcorresponding supernatants (SERT-Ab-depleted cell lysate) were subjected to WB with polyclonal anti-ERp44, -Ero1-L�, -SERT, and -actin Abs as indicated. Arepresentative of three separate experiments is shown. All lanes contain protein recovered from the same number of cells equivalent to 30% of one well froma confluent 24-well dish. Three wells of each condition were pooled, and an aliquot of this mixture was run on the gel. E, the densitometric scanning ofimmunoblots in D shows the level of association between SERT-ERp44 and SERT-Ero1-L�. Data with error bars are represented as mean � S.D. for triplicatesamples.




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ERp44 and Ero1-L� Interact with Endogenous SERT—JARcells endogenously express SERT and have proven to be veryuseful in studies relating to the regulatory aspects of the placen-tal transporter.We took advantage of this system inmonitoringthe processing of SERT. The JAR cell line in the transient trans-fection systemoffers an in situ environment in processing of thenewly synthesized SERT. As reported previously (10, 31), tran-sient transfection produced an at least 50-fold higher level ofprotein than the endogenous system. Therefore, using JAR cellsonly mimics the endogenous condition for the transientlytransfected plasmids. The endogenous expression of SERT,ERp44, and Ero1-L� were confirmed in JAR cells through WBassays with the corresponding Abs (Fig. 1C).Associations between SERT and these chaperones were then

demonstrated by IP assays. Cellular proteins pulled down bymonoclonal SERT-Ab were analyzed with WB assays usinganti-ERp44 or Ero1-L� polyclonal Abs (Fig. 1D). Densitometricquantification of the levels of ERp44 and Ero1-L� proteins onSERT-Ab showed that a significant level of chaperone proteinswere bound to SERT-Ab compared with the bands when theprimary Ab was omitted (Fig. 1E, gray filled bars). Althoughthese results suggest the presence of an association betweenthese ER proteins and SERT in the endogenous expressionsystem, we further examined the specificity of our IP assay con-ditions by analyzing the levels of ERp44, Ero1-L�, and SERT inSERT-Ab-depleted cell lysate (Fig. 1D). In SERT-Ab-depletedcell lysate, the levels of these three proteins appeared signifi-cantly less than their expression levels in the whole cell lysate(SERT-Ab-excluded IP). The intensities of the bands werequantified as representative of their expression level (Fig. 1E,black filled bars). These data show that 72.8% of ERp44 and45.8% of EroL-1� of the whole cell were bound to SERT-Ab(gray bars versus black blocks in the graph). Actin was used as aloading control for protein lysate and was similar betweenlysate-loaded lanes (Fig. 1D).Characterization of SERT in ERp44 shRNA or Ero1-L�

shRNA Cells—The functional role of the ERp44 and Ero1-L�interaction with SERT was analyzed in JAR cells stably infectedwith shRNA for ERp44 or Ero1-L� as described under “Mate-rials and Methods.” We confirmed 60 and 90% reduction ofERp44 (lane 2) or Ero1-L� (lane 3) in cell lysates from theirrespective shRNA-expressing cells comparedwith their expres-sions in control JAR cells (Fig. 2A, lane 1). ERp44 is the primaryER localization mechanism of Ero1-L�; in ERp44 shRNA-ex-pressing cells, we observed a 30% reduction in Ero1-L� level(Fig. 2).SERT Expression and Transport Activity in shRNA-express-

ing Cells—In order to evaluate the role of interactions betweenSERT and two ER chaperones, ERp44 and Ero1-L� we mea-sured the plasma membrane and the whole cell expression ofSERT in ERp44 or Ero1-L� shRNA-expressing JAR cells firstand then compared the 5-HT uptake abilities of these cells.WBanalysis of ERp44- and Ero1-L�-silenced JAR cell lysatesshowed no difference in the total cellular expression of SERTcompared with the control (scrambled plasmid) or JAR cells(Fig. 3A).The density of SERT molecules on the surface of ERp44 or

Ero1-L� shRNA-transfected JAR cells was measured by a cell

surface biotinylation assay as described under “Materials andMethods.” We observed a 50 and 100% increase in SERTexpression on the plasma membrane in ERp44 and Ero1-L�shRNA-expressing cells, respectively (Fig. 3A). The quantifica-tion of the levels of SERT on the plasma membrane of thesecells determined a 75 and 100% increase in SERT expression.Despite the increase in the cell surface of SERT expression inERp44- or Ero1-L�-silenced JAR cells, therewas an unexpectedgreater than 50% reduction in 5-HT uptake rates (Fig. 3B).SERT-ERp44 Protein Associations in Ero1-L� shRNA-ex-

pressing JAR Cells—SERT-ERp44 and SERT-Ero1-L� associa-tions were tested with IP assays in Ero1-L�- and ERp44-shRNAexpressing JAR cells, respectively. Detergent-soluble shRNA-infected JAR cell lysate was incubated with monoclonal SERTAb-coated protein A beads. SERT Ab-bound cellular proteinswere eluted and resolved on SDS-PAGE followed by WB anal-ysis with polyclonal Abs recognizing ERp44 or Ero1-L� (Fig.4A). Although the association between ERp44 and SERT waselevated by 100% in Ero1-L�-silenced JAR cells compared withthe control cell line (lane 2 versus lane 1 in the top blot), therewas a 50% reduction in the level of association betweenEro1-L�and SERT in ERp44-silenced JAR cells (Fig. 4A, lane 3 versuslane 1 of the middle blot). The expression levels of SERT pro-teins in whole JAR cells did not show a difference from that inshRNA-transfected JAR cells (Fig. 4A, bottom blot).Loss of ERp44 Compromises 5-HTTransport—We compared

5-HT transport by SERT expressed in JAR cells whether ERp44was present or silenced (Fig. 5). When ERp44 was present, thekinetic profile for 5-HT uptake was qualitatively more sigmoi-dal (or cooperative) than hyperbolic (Fig. 5). A comparative

FIGURE 2. ERp44 and Ero1-L� expressions in cell lines expressing ERp44or Ero1-L� shRNA. JAR cells infected with the shRNA for ERp44 or Ero1-L�were sorted for positive GFP expression on a FACS Aria at the University ofArkansas for Medical Sciences Core Flow Cytometry Facility 72 h post-trans-fection, equal numbers of positive cells (2 � 105 cells/assay) were lysed, deter-gent-soluble cell lysates were resolved on SDS-PAGE, and then WB analysiswas performed with a polyclonal ERp44, Ero1, SERT, or actin Ab, as indicated.Densitometric scanning of the immunoblots in A was performed using a Versa-Doc 1000 gel documentation system (Bio-Rad). Data show that in ERp44-silenced JAR cells, the expression of Ero1-L� is not altered, whereas in Ero1-L�-silenced JAR cells, the expression of ERp44 is significant lowered. Theasterisks indicate samples that significantly differ from their expressions inJAR cells (two-sided t test, p 0.05). Results from three independent experi-ments are shown (mean � S.D. (error bars)).




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analysis of transportmodels demonstrated that the cooperativeHill equation was significantly favored (99.9% probability)over the traditional uptake equation based on the Akaike infor-mation criterion. The data demonstrated strong positive coop-erativity, as reflected in a Hill coefficient (n) of 2.4 � 0.2. Inother words, the uptake rate rose faster as a function of 5-HTconcentration than expected for the traditional transportermodel. Based on the fit of the data to the Hill equation, Vmaxwas 1.20 � 0.02 (pmol/min/mg protein), and Kh was 1.83 �0.06 �M.Loss of ERp44 significantly altered the kinetic profile for

5-HT transport from a sigmoidal curve to an almost hyperbolicone. The fit of the data to the Hill equation was still favored butnot as strong as observed previously (60% versus 99.9%). Thesource of this shift was the almost complete abolishment ofcooperativity, as reflected in a Hill coefficient of 1.25 � 0.18.This value is close to 1.0, which corresponds to the traditionalmodel. Despite an increase in SERT localization to the plasmamembrane (Fig. 3A), the absence of ERp44 decreased Vmax to0.52 � 0.04 (pmol/min/mg protein) and had no effect on theaffinity of 5-HT for SERT (Kh of 2.1� 0.3�M). Thus, analysis ofthe data demonstrated that the loss of ERp44 activity impacted(i) the mechanism of transport and (ii) the maximal uptake ratefor 5-HT.ERp44 Preferentially Associates with SERT Mutants—SERT

has three Cys residues, Cys-109, Cys-200, and Cys-209, onextracellular loops 1 and 2 (11). Here the Cys residues werechanged one at a time, and a FLAG epitope was introduced into

each mutant protein. We next investigated the associationbetween ERp44 and FLAG epitope-tagged SERT mutants.Additionally, the form of the transporter with bothN-glycosyl-ation sites mutated, SERT-QQ, which is unable to be glycosy-lated and oligomerized (10), was included in these studies. JARcells were transfected for 24 h with HA-ERp44 cDNA in a 1:1ratio with FLAG-SERT, FLAG-QQ, FLAG-C200S, or FLAG-C109A, as described under “Materials andMethods” (2). The IPanalysis showed a 50 and 100% increase in association betweenERp44 and SERT mutant forms, -QQ (which is unable to beglycosylated and oligomerized) (lane 1) and -C200S (whichcannot form a disulfide bridge) (lane 2), respectively, comparedwith the association between SERT and ERp44 (lane 4) (Fig. 6).Next, the interaction between ERp44-C29S and SERT was

tested in the same heterologous expression system. As reportedpreviously, ERp44-C29S cannot interact with Ero1-L� (27,32–34), The absence of a complex between ERp44 and Ero1-L�compromises the ability of ERp44 to interact with SERT asshown by the lack of SERT pulled down with ERp44-C29S (Fig.6A, lane 3). The association between FLAG-SERT and HA-ERp44 was observed with less affinity than the SERT mutantinteractions with ERp44 (lane 4).Functional Role for Three Cys Residues on Second Extracellu-

lar Loop of SERT—Previous studies using a Cys-specific mem-brane-impermeable form of methanethiosulfonate (MTS)reagents showed the involvement of Cys-200 and Cys-209 res-idues in the 5-HT uptake process (1, 2). In this study, cellsexpressing these mutants and the wild-type SERT constructwere assayed for 5-HT transport activity. Fig. 7 shows that5-HT uptake rates of ERp44-silenced JAR cells were 3-foldlower than in physiological JAR cells. C109A is resistant to theMTS reagents as described previously (5). The 5-HT uptakerate of C109A is slightly lower than that observed for wild-typeSERT. Expressing C109A in ERp44-silenced JAR cells reducedthe 5-HT uptake rates to 3-fold the rate in JAR cells. Comparedwith the wild-type transporter, the relative 5-HT uptake ratesfor C200S, C209S, and the double mutant (C200S/C209S) were18.76, 3.38, and 9.66%, respectively.Glycosylation is an important prerequisite for the self-asso-

ciation of transporter protein (10), and the SERT monomerscannot be fully functional because they are in the oligomericform (5). In relating these findings together with the kineticcharacteristics of SERT in ERp44-silenced cells, next the role ofCys residues in the self-association ability of transporter pro-tein was studied.Self-association Abilities of C200S, C209S, and C109A and

Role of ERp44 in This Process—We tested the self-associationability of SERTmutants as a measure of the possible role of thedisulfide-bridge in SERT oligomerization.JAR cells transfected with a 1:1 mixture of Myc- or FLAG-

tagged mutant plasmids and either FLAG- or Myc-tagged pro-teins were precipitated from the mixture by using protein Abeads coated with polyclonal FLAG or monoclonal Myc Ab.FLAG-SERT or Myc-SERT and any associated proteins elutedfrom the beads were analyzed inWB assays. The FLAG-SERT-associated proteins were probed with monoclonal Myc Ab;Myc-SERT and associated proteins were blotted with poly-clonal FLAG Ab.

FIGURE 3. SERT expression and transport activity of JAR cells expressingERp44 or Ero1-L� shRNA. A, JAR cells were infected by either control shRNAor shRNA for ERp44 or Ero1-La plasmids. The cells were sorted for GFP expres-sion on a FACS Aria at the University of Arkansas for Medical Sciences CoreFlow Cytometry Facility 72 h post-transfection, and equal numbers of GFP-positive cells (2 � 105 cells/assay) were analyzed for the whole cell and sur-face expressions of SERT via biotinylation of intact cells followed by WB anal-ysis as described previously (5, 7, 10). The whole cell lysate or biotinylated PMproteins were resolved on SDS-PAGE followed by WB analysis using a poly-clonal SERT-Ab. Densitometric scanning of the immunoblots in B was per-formed, and asterisks indicate samples that significantly differ from JAR cells.There was no difference in the levels of SERT on PM of control shRNA-express-ing and JAR cells. Results from three independent experiments are shown(mean � S.D. (error bars)). The whole cell expression of SERT in these cell lineswas also analyzed with WB for SERT and actin Ab as indicated B, 5-HT uptakerates of these four cells were measured as described previously (5, 7, 10). Theasterisks indicate that 5-HT uptake rates of ERp44- or Ero1-L�-silenced JARcells are significantly different from the uptake rates of JAR cells (two-sided ttest, p 0.05). Results from three independent experiments are shown(mean � S.D.).




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The self-association abilities of SERTmolecules were shownunder both IP assay settings, with either FLAG or Myc Ab-coated protein A beads (Fig. 8,A or B, respectively); Myc-SERTwas found in association with FLAG-SERT, indicating thatalthough the two forms ofMyc-SERT/FLAG-SERT and FLAG-C109A/Myc-SERT remained associated after detergent disrup-tion of the cells, the two other Cys mutants, C200S and C209S,associated neither by themselves nor with wild-type SERT.Surface Labeling of Cells with MTSEA-Biotin—When SERT

is present at the cell surface, there are three exposed Cys resi-dues that are susceptible to modification and thus can be inter-rogated to assess the presence of disulfide bonds. MTSEA-bio-

tin in the external medium reacts with free Cys residues onSERT and the mutants containing exposed Cys residues (1, 2,15). JAR and ERp44-silenced JAR cells were transfected withSERT or one of the Cys mutants (C109A, C200S, and C209S).The next day, cells were treated with MTSEA-biotin asdescribed previously (5); after removing unreacted reagent, thecells were lysed in detergent, and Streptavidin beads were usedto precipitate biotinylated surface proteins. Fig. 9A shows thatJAR-C109A cells were not labeled with MTSEA-biotin at all,indicating the absence of free Cys residue on the external loop ofthe C109A form; however, JAR-SERT cells were labeled at a level50% less than the labeling of JAR-C200S or JAR-C209S cells.However, MTSEA-biotin labeling of ERp44-silenced JAR

cells expressing SERT or one of the Cys mutant forms of SERTshowed a different pattern. The level ofMTSEA-biotin labelingof the JAR cells expressing C109A, C200S, and C209S wasalmost 50% of the labeling level of SERT in JAR cells.These data show that in JAR cells, SERT must have only one

Cys that can interact with MTSEA-biotin, but in ERp44-si-lenced cells, it has more free Cys residues to be labeled byMTSEA-biotin. In a similar way, in JAR cells, C109A should nothave free Cys residues, but in ERp44-silenced JAR cells, C109Amust have two free Cys residues.Next, we wanted to verify these findings in an endogenous

expression system. JAR, ERp44 or Ero1-L� silenced JAR cellswere treated with MTSEA-biotin. Biotinylated proteins werepulled down and analyzed using anti-SERT-Ab. The level ofbiotin labeling of JAR cells wasmuch less than that in ERp44- orEro1-L�-silenced JAR cells (Fig. 9B).

DISCUSSION

Despite a wealth of knowledge on key amino acid residuesneeded for SERT activity, there are limited data on the protein

FIGURE 4. SERT-chaperone associations in shRNA-infected JAR cells. A, ERp44- or Ero1-L�-silenced JAR cells were first sorted for positive GFP expression,and equal (2 � 105 cells/assay) numbers of positive cells were lysed and prepared for IP (5, 7, 10). Monoclonal SERT-Ab-bound proteins were recovered andresolved on SDS-PAGE followed by WB analysis either with polyclonal SERT-Ab or with ERp44 Ab or Ero1 Ab. B, densitometric scanning of the immunoblots inA shows an enhanced association between SERT and ERp44 in Ero1-L�-silenced JAR cells and between SERT and Ero1-L� in ERp44-silenced JAR cells. Theasterisks indicate samples that are significantly different from their expression with SERT in JAR cells (two-sided t test, p 0.05). Results from two differentexperiments are shown (mean � S.D. (error bars)).

FIGURE 5. Substrate dependence of transport in JAR and chaperone-si-lenced JAR cells. Initial rates of 5-HT influx were measured over the indicatedrange of 5-HT concentrations using 20 nM [3H]5-HT with added unlabeled5-HT to the final concentration. A comparison of the fits of the data to the Hillequation and the traditional equation for transport kinetics (Equation 1) dem-onstrated that the Hill equation was statistically favored. Consequently, the fitof the data to the Hill equation is shown in the figure. This analysis yieldedvalues for Kh, Vmax, and n (the Hill coefficient). Results from two different setsof experiments done in triplicate are shown (mean � S.D. (error bars)).




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mediators and quality control checkpoints in SERTmaturation.Studies have shown that an exposed thiol at Cys-200 orCys-209on EL2 is sufficient for the intracellular retention of SERT, butSERTmutantswithoutCys residues on the second extracellularloop are able to reach the PMdespite the lack of a disulfide bond(11). These studies suggest a quality control mechanisminvolved in SERTmaturation, which recognizes exposed Cys inSERTmolecules and retains them intracellularly. The ability ofCys mutants of SERT to reach the PM further implies that thequality control mechanism does not recognize non-nativestructure, such as hydrophobic patches or immature glycans,but rather, the retention of Cys mutants of SERT is entirelythiol-dependent.The retention of proteins through reversible disulfide bonds

has become the primary feature of thiol-mediated retentioninvolving two ER resident proteins, ERp44 and Ero1-L� (15, 26,27, 30). ERp44 and Ero1-L� have been described as a qualitycontrol mechanism in the maturation of disulfide-containingoligomeric proteins (16, 26, 27, 30). Given the importance of

disulfide bond formation in the maturation of SERT and thenature of thiol-mediated retention in ER, herein, we tested anovel hypothesis of ERp44-mediated disulfide bond formationand the role of disulfide in oligomerization of SERTmonomers.In initial experiments, we were able to demonstrate that

endogenous SERT interacts with ERp44 and Ero1-L� (Fig. 1C).ERp44 mediates retention of cargo proteins at the ER though athiol-dependent process (15, 26, 27, 30, 32); thus, we examinedthe effects of a terminally reactive thiol on the associationbetween SERT and ERp44 as a step in SERT maturation at theER. A single mutation of Cys-200 or Cys-209 disrupts disulfidebond formation and produces an exposed Cys in SERT (11). Inaddition, C200S or C209S mutants of SERT are not able toreach the PM and are retained intracellularly (11). A 50%increase in association between a single Cys mutant of SERTand ERp44 demonstrates that an exposed Cys at Cys-209 is

FIGURE 6. Increased association of ERp44 with SERT mutants. A, JAR cellswere transiently transfected with HA-ERp44 or HA-ERp44-C29S cDNA in a 1:1ratio with FLAG-SERT, FLAG-C200S, FLAG-C109A, or FLAG-QQ, as indicated.Cell lysates were mixed with HA Ab-treated protein A-Sepharose beads. HAAb-bound proteins were eluted and resolved, and SDS-PAGE and WB analysiswere performed with FLAG and ERp44 Ab, as indicated. B, quantification ofthe results from A was performed by densitometric scanning as describedunder “Materials and Methods” and presented as the percentage of SERT-ERp44 association. The associations between ERp44 and QQ (lane 1) or C200S(lane 2) are enhanced but not the one that is not involved in the disulfidebridge, C109A (lane 4), compared with the association with SERT (lane 5). Themutation in the thioreductase domain of ERp44C29S does not recognizeSERT (lane 3). These results are significantly different from the level of SERT-ERp44 association (two-sided t test, p 0.05). Results from three indepen-dent experiments are shown (mean � S.D. (error bars)).

FIGURE 7. 5-HT uptake rates of mutant transports in JAR and in ERp44-silenced JAR cells. JAR cells were infected with shRNA for ERp44 or Ero1-L�.Seventy-two hours postinfection, JAR cells were sorted for GFP expression atthe FACS Flow Cytometry Facility, and equal numbers of GFP-positive cellsand JAR cells (2 � 105 cells/assay) were seeded. The next day, they weretransfected with SERT, C109A, C200S, C209S, and double mutant C200S/C209S plasmids. 5-HT uptake rates of these cell lines were measured asdescribed previously (5, 7, 10). The rate of uptake is expressed as the meanand S.D. values (error bars) of triplicate determinations from three indepen-dent experiments. * and **, results of Student’s t test with both p 0.001(compared with mutant versus wild-type SERT and SERT in JAR versus inERp44-silenced JAR uptake rates, respectively).

FIGURE 8. Association between FLAG- and Myc-tagged SERT and mutantproteins. JAR cells co-expressing FLAG- and Myc-tagged transporters or withFLAG-SERT (A) or Myc-SERT (B) alone were solubilized and treated with pro-tein A beads and the Abs against FLAG or Myc. The IPs were separated bySDS-PAGE. WB analyses of the proteins pulled by polyclonal FLAG Ab or MycAb were blotted with monoclonal Myc Ab or polyclonal FLAG Ab, respec-tively. Both blots were stripped and reprobed with actin Ab.




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sufficient for the retention of SERT (Fig. 6A), and furthermore,ERp44 may be involved in the quality control of disulfide bondformation.ERp44 also preferentially interacts with unassembled sub-

units of oligomeric proteins (27). This association enablesERp44 to selectively retain cargo proteins, like SERT, in an envi-ronment suitable for their maturation and concentrate sub-units in a local environment to promote their polymerization(15, 27, 30). SERT mutants (e.g. QQ) that cannot undergo gly-cosylation demonstrate compromised oligomerizations and areunable to properly transport 5-HT (10); here, this mutant formof SERT was included in our studies to investigate if SERTundergoes disulfide bond-mediated oligomerization process.Data showed that if SERT molecules cannot associate in anoligomeric form, then this facilitates the association betweenERp44 and SERT (Fig. 6).Because ERp44 associates with cargo proteins through

exposed thiols, our findings suggest that QQ mutants may notbe able to form a disulfide bond, and further, glycosylation ofSERT may contribute to its disulfide modification. Althoughnot previously reported for SERT, glycosylation has beenshown to facilitate disulfide bond formation in other proteins,such as epidermal growth factors and human insulin receptor(33, 34). This result implies the requirement of disulfide bondformation in functional oligomerization of SERT monomers.Sequential modification would allow the stepwise maturationof SERT, and quality control checkpoints intimately associatedwith the folding process ensure that unmodified or misfoldedproteins do not proceed to the next stage in maturation (15). In

this manner, the ER folding machinery is able to couple thematuration and quality control of proteins within the secretorypathway (16, 23). However, additional studies are needed inorder to confirm with certainty the interdependence amongpost-translational modifications of SERT.Once associated with ERp44, previous data have shown that

cargo molecules are displaced by Ero1-� through binding withCys-29 of ERp44 (36). Interactions with Cys-29 have also beenshown to facilitate the association between ERp44 and othercargo proteins, such as adiponectin and IgM subunits (27, 30).However, using amutant form of ERp44, C29S, we showed thatSERT does not interact with ERp44 through Cys-29, but ratherdisruption of interactions through Cys-29 through mutationsincreases its association with the transporter. Considering therole of Ero1-L� in displacement of other cargo proteins fromERp44, these findings suggest that Ero1-L� is required to inter-act with ERp44 for efficient release of SERT, and the relativeratio of the two chaperones, ERp44 and Ero1-L�, to each otheras well as SERT is a crucial determinant for proper SERTmaturation.Our co-IP assays suggested a possible association between

ERp44 and SERT; however, studying protein-protein interac-tions in co-IP experiments with detergent-disturbed cell lysatesuffers from drawbacks, such as possible nonspecific interac-tions and variations in the efficiency of the IP. Therefore, weperformed an analysis of these associations on 5-HT uptakefunction of SERT proteins. The kinetic studies along with theMTSE-biotinylation assays strongly indicate that ERp44 andEro1-L� contribute to the maturation of SERT. Consequently,

FIGURE 9. MTSEA-biotin labeling of SERT and Cys mutants. A, JAR cells were infected with shRNA for ERp44 or Ero1-L�. Seventy-two hours postinfection, JARcells were sorted for GFP expression at the FACS Flow Cytometry Facility, and equal numbers of GFP-positive cells and JAR cells (2 � 105 cells/assay) wereseeded in 24-well plates. The next day, cells were transfected with SERT, C109A, C200S, and C209S. Twenty-four hours post-transfection, intact cells weretreated with MTSEA-biotin (see “Materials and Methods” and Refs. 7 and 10). After removing unreacted reagent, the cells were lysed in detergent, andStreptavidin beads were used to precipitate biotinylated surface proteins. The biotinylated proteins were eluted and separated by SDS-PAGE, and WB analyseswere performed with SERT Ab. Blots were stripped and reprobed with actin Ab. Densitometric scanning of immunoblots was calculated as the percentage oflabeling of SERT in JAR or in ERp44-silenced JAR. Results from three different experiments are shown (mean � S.D. (error bars)). B, JAR cells expressing shRNAfor ERp44 and Ero1-L� were prepared as described under “Materials and Methods.” As described above, intact cells were labeled with MTSEA-biotin, and WBanalysis was performed with SERT-Ab. The blot was stripped and reprobed with actin Ab.




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we examined the functional contribution of the interaction.Wewere able to create stable knockout cell lines of ERp44 andEro1-L� through the delivery of RNA interference (RNAi). Ini-tial attempts at protein reduction with transient transfection ofprotein-specific shRNA to degrade targeted mRNA constructswere unsuccessful due to the long half-life of ERp44 and Ero1-L�. As a solution, lentiviral particles containing shRNA tar-geted to ERp44 or Ero1-L� were produced and transfected intotarget cell lines for long term RNAi. The expression levels ofERp44 and Ero1-L�were successfully reduced to 60%of endog-enous levels in JAR cells. Furthermore, as a primary ER local-izationmechanism of Ero1-L�, we observed a 30% reduction inEro1-L� when ERp44 protein levels were significantly reduced.Our data confirm the previously published studies (26, 35) onthe saturability of the ERp44-dependent retention mechanism.According to these studies, if the Ero1-L�/ERp44 ratio exceedsits threshold for retention, a portion Ero1-L� escapes from theER (26, 35).In our knockout expression system, SERT is endogenously

expressed in JAR cells; thus, we are able to measure the func-tional consequence of reduced ERp44 or Ero1-L� proteinexpression on SERT activity. However, these could be an indi-rect effect of the gene silencing on other proteins which arerequired for the SERT folding and maturation. Therefore, thekinetic and biochemical studies together with the data fromMTSEA-biotinylation assays are more specific in demonstrat-ing the direct action of ERp44 on SERT.For untreated cells, the kinetic profile for 5-HT uptake was

strongly sigmoidal, indicating that increasing 5-HT concentra-tions led to positive cooperativity. In other words, 5-HT levelsimproved the rate of transport not simply by saturating thetransporter but by altering its functional efficiency and/or con-centration. Based on our previous studies (31, 36, 37), we favorthe biphasic relationship between the extracellular 5-HT levelswith the number of SERTmolecules on the plasma membrane.Specifically, the number of SERT molecules on the plasmamembrane and the 5-HT uptake rates of cells initially rise asextracellular 5-HT levels are increased but then fall below nor-mal as the 5-HT level continues to rise. Indeed, our in vivo andin vitro studies confirm a dynamic relationship between extra-cellular 5-HT elevation, loss of surface SERT, and depletion ofplatelet 5-HT (31, 36, 37). The rise in concentration of oligo-merized, active SERT improves 5-HT activity more thanexpected, as reflected in the sigmoidal kinetic profile.Thismechanism depends on ERp44 function tomaintain the

appropriate response of SERT to changing 5-HT levels and theresulting transport activity.Without ERp44 chaperone activity,relative levels of SERT at the cell surface were high prior to theaddition of 5-HT. As 5-HT levels increased, the sigmoidicity ofthe kinetic profile was essentially abolished, which is probablydue to the absence of any change in cellular SERT localization.Nevertheless, once SERT reached the cell surface, its activitywas compromised based on a decreased maximal uptake rate.SERT maturation into an active transporter then requiresERp44 activity as well. Ero1-L� is another contributor to theseprocesses through its interactions with ERp44. Taken together,these data suggest that ERp44 plays a critical role in the matu-ration and membrane trafficking of SERT within cells.

To identify the cause of reduced SERT activity, we measuredthe SERT surface expression by biotinylation in shRNA-ex-pressing cells. Despite decreased transport activity, we discov-ered a greater than 1- and 2-fold increase in SERT expressionon the PM in cells expressing ERp44 or Ero1-L� shRNA,respectively. A reduced transport activity can be attributed toan increase in nonfunctional SERT and/or a decrease in fullyactive SERT molecules at the cell surface, which was demon-strated by our biochemical studies in ERp44-silenced cells (Fig.5). Therefore, significantly lowered ERp44 or Ero1-L� is suffi-cient to allow improperly folded SERT molecules to reach thePM, and ERp44 and Ero1-L� probably act as a quality controlmechanism ensuring the fidelity of SERT maturation.Previous data implicate ERp44 and Ero1-L� in the quality

control of cargo proteins, and a dynamic relationship existsbetween ERp44 and Ero1-L� for the efficient retention andrelease cargo (27, 30, 32). It was previously shown that an inter-action betweenCys-29 of ERp44 andEro1-L� aids the release ofthe cargo protein from ERp44 (15, 26–28). We hypothesizedthat this mechanism is also required for SERT release fromERp44. However, the increased SERT surface expression incells with 30% of endogenous Ero1-L� expression seemed con-tradictory; thus, we measured the association between ERp44and SERT in JAR cells expressing Ero1-L� shRNA.We discov-ered an almost 100% association between SERT and ERp44when Ero1-L� is not present at endogenous levels, and thisresult further implies the saturability of ERp44-mediated reten-tion (Fig. 4). When Ero1-L� levels are not able to adequatelyrelease cargo proteins from ER retention as in our Ero1-L�shRNA-expressing cell line, ERp44 becomes saturated. Thisoutcome causes the cargo/chaperone ratio to increase, and as aresult, misfolded SERT molecules are unable to be properlyretained and bypass quality control.SERT is a complex oligomeric glycoprotein, which needs sig-

nificant post-translational modification to fold into its nativestructure (5, 9–11). The formation of such a large, multimericcomplex is inherently error-prone and requires more time tofold. Whereas ERp44-mediated quality control recognizesimproperly folded SERT molecules and prevents them fromreaching the PM, ERp44-dependent ER retention may serve toincrease the time SERT is in the secretory pathway and thus theopportunity for SERT to fold into its native structure. Further-more, ERp44 is able to couple quality control and oxidativefolding by directing non-native SERT to the ER machineryneeded for disulfide bond formation.In addition to a quality control mechanism, the regulation of

ERp44 and Ero1-L�may offer an additional layer of post-trans-lational control of SERT activity. Increased expression of bothchaperones, ERp44 and Ero1-L�, has been shown to stimulatethe functional oligomerization and expression of other cargoproteins involved in thiol-mediated retention (30). However,up-regulation of only ERp44 has been shown to favor the reten-tion of cargo molecules, and Ero1-L� overexpression stimu-lates their release (30). This is especially relevant due to thetissue- and sex-specific expression of ERp44 and Ero1-L�. Forexample, there is a 50% reduction in ERp44 and Ero1-L�expression in male mice as compared with female mice and agreater than 80% reduction in ERp44 and Ero1-L� in ob/ob




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mice (30). Thus, our in vitro shRNAexperiments are physiolog-ically relevant and provide evidence that regulation of ERp44and Ero1-L� expression and activity could affect SERT in vivo.

Acknowledgments—We thank Dr. Roberto Sitia and Dr. PhilipScherer for kindly supplying ERp44 and Ero1-L� plasmids and anti-bodies. We thank Dr. Zhao Wang and Dr. Anelli Tiziana for criticalreview of the manuscript.

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Ramanagoudr-Bhojappa, Grover P. Miller and Fusun KilicSamuel Freyaldenhoven, Yicong Li, Arif M. Kocabas, Enrit Ziu, Serra Ucer, Raman

The Role of ERp44 in Maturation of Serotonin Transporter Protein

doi: 10.1074/jbc.M112.345058 originally published online March 26, 20122012, 287:17801-17811.J. Biol. Chem.

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VOLUME 287 (2012) PAGES 17801–17811DOI 10.1074/jbc.W118.007073

Withdrawal: The role of ERp44 in maturation ofserotonin transporter protein.Samuel Freyaldenhoven, Yicong Li, Arif M. Kocabas, Enrit Ziu, Serra Ucer,Raman Ramanagoudr-Bhojappa, Grover P. Miller, and Fusun Kilic

This article has been withdrawn by Samuel Freyaldenhoven, YicongLi, Arif M. Kocabas, Grover P. Miller, and Fusun Kilic. Despite attempts,Enrit Ziu, Serra Ucer, and Raman Ramanagoudr-Bhojappa could not bereached for conference on this decision. Dr. Kilic contacted the editorialoffice to report errors in Fig. 3A and Fig 8 of their article. The Journalrequested the original data for Figs. 1A, 1D, 3A, 6A, 8A, 8B, and 9A. Dueto the dated material, she could not provide all of them. The data thatwere provided to the Journal was not 300 ppi. The investigation by theJournal determined the following. Lanes 2 and 3 of the total SERTimmunoblot in Fig. 1A were duplicated in lanes 5 and 6. The actinimmunoblot from Fig. 1A was reused in Figs. 8B and 9A as actin. Thefirst two lanes of the SERT (PM) immunoblot in Fig. 3A were duplicated.Additionally, the first lane of the Total SERT immunoblot in Fig. 3A wasreused in lane 4. In Fig. 6A, lanes 2 and 5 of the actin immunoblot wereduplicated.

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70 J. Biol. Chem. (2019) 294(1) 70 –70

© 2019 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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