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Originally published In Press as doi:10.1074/jbc.M404850200 on September 21, 2004 Originally published In Press as doi:10.1074/jbc.M404850200 on August 19, 2004

J. Biol. Chem., Vol. 279, Issue 44, 46104-46112, October 29, 2004
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TSAP6 Facilitates the Secretion of Translationally Controlled Tumor Protein/Histamine-releasing Factor via a Nonclassical Pathway*{boxs}

Nathalie Amzallag{ddagger}§, Brent J. Passer{ddagger}§, David Allanic{ddagger}, Elodie Segura||, Clotilde Théry||, Bruno Goud**, Robert Amson{ddagger}, and Adam Telerman{ddagger}

From the {ddagger}Molecular Engines Laboratories, 20 Rue Bouvier, 75011 Paris and Institut Curie, ||INSERM U365, **CNRS UMR144, 12 Rue Lhomond, 75005 Paris, France

Received for publication, April 30, 2004 , and in revised form, August 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5–6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TSAP61 was originally identified as a differentially expressed transcript in the LTR6 myeloid leukemia cell line that carries the Val-135 temperature-sensitive p53 mutant (1). These cells shifted to 32 °C acquire wild-type p53 function and subsequently undergo apoptosis (2). TSAP6 transcripts were shown to be up-regulated as early as 4 h after p53 activation (1). More recently, we identified a functional p53-responsive element within the promoter region of TSAP6 (3).

TSAP6, the human homolog of the rat pHyde (4, 5), appears to belong to a family of related proteins that includes STEAP, TIARP, and STAMP1 (6–8). Amino acid sequence analyses suggest that these proteins have 5–6 putative transmembrane regions and, with the exception of STEAP, also possess an NH2-terminal GXGXX(G/A) motif, a signature sequence for the core Rossman fold. Blast search analyses using the NH2-terminal of TSAP6 identified significant homologies with NADP/NADPH-containing oxidoreductases, which were previously reported for TIARP (7). Immunofluorescence and immunohistochemical studies demonstrated that STEAP, STAMP1, and TIARP1 are expressed at the plasma membrane (6–8), the latter two appearing more intense at the cell-cell junctions (7, 8). STAMP1 and TIARP are localized to vesicular-like structures that appear to be associated with invaginations near the cell surface (7, 8). Furthermore, time-lapse imaging of GFP-STAMPl in COS cells demonstrated its anterograde-retrograde trafficking to and from the Golgi complex in the form of vesicular tubular structures (8). Taken together, these data suggest that TSAP6-related proteins could be involved in vesicular trafficking and secretory processes (7, 8). Previously, we reported that TSAP6 associated with Nix, a pro-apoptotic protein, and Myt1 kinase, a regulator of the G2/M transition (3). Based on these studies, we proposed that TSAP6 could amplify an apoptotic signal by cooperating with Nix and, furthermore, that TSAP6 could delay the G2/M transition of the cell cycle by maintaining Myt1 kinase in its active form. It seems that TSAP6 can promote different types of activities that are related to apoptosis, cell cycle regulation, or vesicular trafficking.

In the present study, TSAP6 was used as a "bait" in a yeast two-hybrid hunt and identified TCTP as an interactor. TCTP was initially described in sarcoma ascites tumor cells as having a serum-inducible mRNA whose expression is regulated at both the transcriptional and translational levels (9–12). Biochemical and immunofluorescence studies demonstrated that TCTP is a tubulin-binding protein that associates with microtubules in a cell cycle-dependent fashion (13). In accordance, polo-like kinase was shown to interact directly with and phosphorylate TCTP, further implicating TCTP in cell cycle-related activities (14). More recently, NMR analysis revealed that TCTP has a structural homology with the mammalian suppressor of Sec4 (MSS4/DSS4), a protein that functions as a guanine nucleotide-free chaperone (15). In line with these findings, we recently identified a guanine nucleotide dissociation inhibitor activity for TCTP (16). Furthermore, TCTP was identified in a high throughput screening, while searching for differentially expressed genes between tumor cells and their revertant counterparts (17). This analysis revealed that TCTP was significantly down-regulated in tumor revertant cells, which have a suppressed malignant phenotype. In agreement, knockdown of TCTP by small interfering RNA in breast cancer cells resulted in the striking reappearance of ductal-like structures reminiscent of normal ones. Moreover, TCTP protein levels were clearly augmented in a variety of human tumor tissues as compared with their normal tissue controls (17). Overall, these studies suggest that perturbations of TCTP expression levels could have different consequences on cell growth related activities.

Extracellularly, TCTP was initially isolated as a secreted factor from U937-derived cultured supernatants based on its ability to promote histamine release (18). Subsequent studies confirmed that indeed TCTP is a secreted protein implicated in immune responses. TCTP has been shown to trigger the release of histamine from eosinophils, basophils, and mast cells and was also found to be a potent stimulus for interleukin-4 and interleukin-8 production (19, 20). In addition, TCTP was shown to have a B-cell stimulating activity (21). Under allergic conditions, TCTP was detected in nasal and skin blister fluids from patients with late phase allergic inflammation (22) and in bronchoalveolar lavages from patients with idiopathic eosinophilic pneumonia (23). Moreover, homologs of TCTP from the parasites Plasmodium falciparum and Schistosoma mansoni have been detected in the plasma of infected hosts (24, 25). The presence of both malarial and schistosome TCTP in infected hosts has been suggested to contribute to inflammatory responses and possibly to parasitic dissemination (25). Although the role of secreted TCTP is well documented, how TCTP is eventually exported out of the cell is unknown. Here we attempt to elucidate the secretory pathway of TCTP and the potential role of TSAP6 in this process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Anti-TCTP and anti-eEF1B{beta} antibodies were raised in rabbits after injection of the full-length proteins as reported previously (16). Anti-TSAP6 was made in the chicken (3). Anti-{beta}-tubulin (H-235), anti-TSG101 (M-19), anti-actin (I-19), anti-presenilin-1 (H-70), and anti-galectin-3 (D-20) antibodies were purchased from Santa Cruz Biotechnology. Anti-HA, anti-GP96, anti-clathrin, and anti-SEAP (Ab8188) were obtained from Babco, Stressgen, BD Biosciences, and Abcam, respectively. The anti-MHC class I (HC10 and HCA2) was obtained from ATCC and kindly provided by P. Benaroch. Secondary antibodies for immunofluorescence were purchased from Molecular Probes. All other reagents were purchased from ICN. HA-TSAP6 was cloned as described previously (3). The vector containing the secreted alkaline phosphatase (SEAP) was purchased from Clontech.

Secretion Assays—293T (5 x 106) cells cultured in Dulbecco's modified Eagle's media containing 10% fetal calf serum and 2 mM glutamine were washed twice in phosphate-buffered saline and resuspended in 8 ml of serum-free media for 3 h. Thereafter, 4 ml of supernatants were collected and centrifuged (300 x g) for 10 min to remove nonadherent cells; thereafter, supernatants were centrifuged a second time (5000 x g) for 10 min to remove cell debris and nuclei. Supernatants were concentrated (80x) by centrifugation using Amicon-Ultra, 4 units (10,000 molecular weight cut-off), and resuspended in a 2x SDS sample buffer. 15 µl of each sample was resolved by SDS-PAGE. Cells were solubilized in a lysis buffer containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA and a mixture of protease inhibitors, and 1 µg of total proteins were resolved by SDS-PAGE. After transfer, membranes were routinely blotted for TCTP, eEF1B{beta}, {beta}-tubulin, and actin to monitor the extent of cytosolic contamination because of either unspecific protein release or cell lysis. For cycloheximide (CHX) experiments, the indicated hours correspond to the total time of treatment with CHX including the 3-h secretion time. For all time points, CHX was added at 10 µg/ml, except for the time point 18 h where 1 µg/ml CHX was added. For each experiment, cell viability was assessed by trypan blue exclusion.

Transfections—For transfection experiments, 293T (2.5 x 106) or HepG2 (1 x 106) cells were plated the day before transfection. Cells were transfected by the Ca2PO4 precipitation method using a total of 40 µg of plasmid (HA-TSAP6:YFP-TCTP, 3:1) and secretion assays were performed the following day. For some experiments, brefeldin A (5 µM) or monensin (25 µM) was added 30 min or 2 h, respectively, prior to and during the 3-h secretion assay. K562 cells (4 x 106) were electroporated (GenePulser Xcel, Bio-Rad) (180 V, 20 ms, square-wave pulse) with a total of 16 µg of plasmid DNA (HA-TSAP6:YFP-TCTP, 3:1). Cells were allowed to recover in complete media for 2 h at 37 °C. Thereafter, cells were layered on a Ficoll-Paque (Amersham Biosciences) cushion, and viable cells were isolated from the interface after centrifugation at 900 x g for 20 min. Approximately 30% transfection efficiency was achieved as determined by transfection with a green fluorescent protein control plasmid. The following day, secretion assays were performed as described above.

Exosome Preparations—Exosomes were purified as described previously from supernatants of either the Dl cell line, bone marrow dendritic cells (24-h secretion assay), or 293T cells (3-h secretion assay) by 100,000 x g centrifugation followed by a wash of the exosome pellet in a large volume of phosphate-buffered saline (26, 27). For flotation on sucrose gradient, 50 µg of exosomes were resuspended in 2 ml of 2.5 M sucrose, 20 mM Hepes/NaOH, pH 7.2. A linear sucrose gradient (2–0.25 M) was layered on top of the exosome suspension and centrifuged for 15 h at 100,000 x g in a SW41 rotor. One-ml fractions were collected, and the sucrose density was measured with a refractometer. Fractions were diluted in 2 ml of Hepes buffer and centrifuged at 100,000 x g for 1 h in a TLA100.4 rotor, and pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE.

In Vivo Protein Labeling—For pulse-chase experiments, 8 x 107 293T cells cultured overnight under standard conditions were collected in a tube and subsequently starved of methionine for 1 h prior to metabolic labeling with 1 mCi of Redivue [35S]methionine (Amersham Biosciences). After labeling for 1 h, cells were extensively washed in phosphate-buffered saline and placed in 5.5 ml of serum-free media supplemented with 1 mM nonradioactive methionine for secretion assays that corresponded to the indicated times of chase. At each time point, 1 ml of the cell suspension (~1.5 x 107 cells) was centrifuged, and the cell pellet was lysed in lysis buffer. Precleared lysates and supernatants were immunoprecipitated with rabbit anti-TCTP followed by incubation with protein-G-agarose beads (Promega). For the IgG control, cell lysates were incubated with rabbit IgG plus protein-G-agarose beads. Immunoprecipitations were extensively washed in lysis buffer and resolved on a SDS-PAGE. Radioactive material was visualized by autoradiography.

Yeast Two-hybrid Analysis—Full-length human TSAP6 was fused with the LexA DNA-binding domain in pEG202. A cDNA library derived from the murine LTR6 cell line incubated at 32 °C for 4 h was cloned into the galactose-inducible pYESTrp2 vector (Invitrogen) containing the B42 activation domain. The yeast two-hybrid screen was performed as described previously (3, 28). Briefly, the yeast strain RFY231 (MAT{alpha} trp1{Delta}::hisGhis3ura3-1 leu2::3Lexop-LEU2) harboring pEG202-TSAP6 and the lacZ reporter plasmid pSH18-34 was transformed by the lithium acetate method with the LTR6 cDNA library. Approximately 1 x 106 yeast colonies were obtained. Yeast containing potential TSAP6 interactors, were identified by simultaneous growth and {beta}-galactosidase activity on galactose Ura-His-Trp-Leu- selection plates containing 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside. Yeast mating assays were performed as described (3, 28).

GST-Pull Down Experiments—TCTP and the negative control NKTR (29) were cloned into the pGEX-6P-1 vector (Amersham Biosciences). GST-TCTP and GST-NKTR fusion proteins were produced in the bacterial strain BL21 (DE3) (Stratagene) by inducing protein expression with isopropyl-1-thio-{beta}-D-galactopyranoside (1 mM) for 2 h at 37 °C. After lysis of bacteria, GST fusion proteins were subsequently captured on glutathione-agarose beads. [35S]Methionine/cysteine-labeled proteins were made in vitro by using a rabbit reticulocyte lysate transcription/translation (IVT) system (Promega) and added to the GST fusion proteins in lysis buffer. After incubation for 3 h at 4 °C, the bead-bound complexes were washed and resolved by SDS-PAGE, and the radiolabeled proteins were visualized by autoradiography.

Two-color Immunofluorescence Analysis—293T, HepG2, and HeLa cells fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.1%) were stained with chicken anti-TSAP6 and rabbit anti-TCTP antibodies followed by incubation with anti-chicken AlexaFluor-488 (green) and anti-rabbit AlexaFluor-594 (red) coupled secondary antibodies. Immunofluorescence staining imaging was performed sequentially for each wavelength by using a Leica TCS4D confocal microscope. In case of HeLa cells, immunofluorescence imaging was performed by wide-field deconvolution microscopy using an Olympus CellR system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous TCTP Is Exported via a Nonclassical Pathway from 293T Cells—Although it is well established that TCTP is a secreted protein, how it is exported is unknown. Thus, we initially carried out studies to characterize the route of export of TCTP. 293T cells were incubated in serum-free media either for 0, 0.5, 3, or 5 h, and supernatants were concentrated (see "Experimental Procedures"), resolved by SDS-PAGE, and immunoblotted for TCTP (Fig. 1A). By 3 h, TCTP was detected in 293T cell-derived supernatants at a level that was not further enhanced by 5 h (data not shown). The quantification of secreted TCTP was estimated by Western blot using a range of recombinant TCTP as a reference (supplemental Fig. S1). We estimated that the secreted TCTP in our 3-h assay represents ~0.3% of the total intracellular TCTP. To ensure that supernatants containing TCTP resulted from an active process of secretion rather than contamination because of cell lysis or unspecific release, supernatants were also immunoblotted against a panel of antibodies that recognize abundant cytosolic proteins. Immunoblot analysis clearly identified {beta}-tubulin, elongation factor-1 B{beta} (eEF1B{beta}), and actin (data not shown) in cell lysates, at a level of expression comparable with TCTP, but not in the corresponding supernatants. These data indicate that TCTP can be readily detected in supernatants of 293T cells using a relatively short assay period of secretion with minimal effects of cell lysis.



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  		FIG. 1.
Secretory properties of TCTP. A, TCTP is secreted by 3 h in 293T cells. Immunoblot analyses of the expression levels of TCTP were performed either on total lysates (TL)(left panel) or on supernatants (right panel). Blots were reprobed with anti-{beta}-tubulin ({beta}tub) and anti-eEF1B{beta} (B{beta}) to determine the extent of cytosolic contamination in the supernatant preparations as well as to normalize protein levels in cell extracts. Note that supernatants in each lane subjected to immunoblot analysis corresponded to ~400,000 cells. For total lysates, 1 µg of cell lysate (~30,000 cells) was loaded for each lane. B, TCTP secretion is resistant to brefeldin A (Bfa) and monensin (Mon) treatments of 293T cells. 293T cells were pretreated with either brefeldin A or monensin, and TCTP secretion was monitored. Note that brefeldin A and monensin completely inhibited the secretion of transfected SEAP, an ER/Golgi-dependently secreted protein. Immunoblotting with anti-eEF1B{beta} verified negligible cytosolic contamination in the supernatant preparations and confirmed equal loading of total lysates. C, pulse-chase experiments on TCTP in 293T cells. 293T cells were pulsed for 1 h and chased for the indicated times. Endogenous TCTP was immunoprecipitated (IP) from either total lysates (left panel) or supernatants (right panel), and radiolabeled TCTP was visualized by autoradiography. IgG was used as a negative immunoprecipitation control on total lysates. D, cycloheximide effects on TCTP secretion. 293T cells were treated with cycloheximide (CHX) for the indicated times and submitted to a 3-h secretion assay. Total lysates or supernatants were immunoblotted for TCTP. Equal loading of cell extracts was verified by immunoblotting with anti-eEF1B{beta} (B{beta}). Note that CHX appeared to affect the secretion of TCTP only by 18 h of treatment.

 
Soluble secreted proteins typically follow one of two routes of cellular export. The majority of secreted proteins translocates via an ER/Golgi-dependent pathway that can be blocked by brefeldin A or monensin, classical inhibitors of ER/Golgi trafficking (30). By contrast, a number of exported proteins are insensitive to these inhibitors and are consequently secreted independently of the ER/Golgi (31). To address whether TCTP is dependent on the ER/Golgi pathway for export, 293T cells were treated with either brefeldin A or monensin, cultured in serum-free media for 3 h, and their supernatants subsequently analyzed for the presence of TCTP. Fig. 1B shows that neither brefeldin A nor monensin perturbed the secretion of TCTP, whereas these inhibitors completely abrogated the secretion of overexpressed SEAP, an ER/Golgi-dependently secreted protein. Thus, these data indicate that the secretion of TCTP proceeds via an ER/Golgi-independent route.

Next, we addressed whether secreted TCTP in our assay represented newly synthesized proteins or, alternatively, a pre-existing pool. First, pulse-chase experiments were performed wherein 293T cells were metabolically radiolabeled for 1 h and chased in serum-free media for 0–16 h (Fig. 1C). At the end of each period, TCTP was immunoprecipitated from both the cell lysates and from the corresponding supernatants and analyzed by autoradiography. Although TCTP was detected in the lysates throughout the chase period, its appearance in the supernatants was clearly visible only by 16 h of chase. In cell lysates, the levels of TCTP appeared to diminish by 16 h of chase, which was accompanied by a concomitant accumulation of TCTP in the supernatants. Next, we repeated the 3-h secretion assay, but in the presence of cycloheximide (CHX) (Fig. 1D). Even by 8 h of treatment with CHX, the levels of secreted TCTP was not perturbed. Levels of secreted TCTP diminished when the time of treatment with CHX exceeded its half-life, which is about 16 h (see Fig. 1C). These results indicate that TCTP has a relatively long half-life and that the majority of secreted TCTP detected in our assay does not represent newly synthesized proteins but rather comes from a pre-existing pool of TCTP.

TSAP6 Interacts with TCTP and They Co-distribute—TCTP was identified in a yeast two-hybrid hunt using TSAP6 as a bait. An association between LexA-TSAP6 and B42-TCTP was verified by yeast mating assays (Fig. 2A). This analysis shows that by day 4, growth and {beta}-gal activity were observed on galactose but not glucose plates. A direct interaction between TSAP6 and TCTP was also tested by GST pull down assays. Fig. 2B shows that GST-TCTP bound to the IVT-radiolabeled TSAP6 but not to the negative control IVT AIP (32). The specificity of this interaction was further confirmed by showing that the negative control GST-NKTR did not bind IVT TSAP6.



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  		FIG. 2.
TSAP6 interacts with TCTP, and they co-distribute. A, yeast mating assays identified an interaction between LexA-TSAP6 and B42-TCTP. Yeast diploids carrying the indicated constructs were streaked either on glucose (Glu) or galactose (Gal) plates, and interactions were assessed either by growth or {beta}-galactosidase ({beta}-gal) activity after 4 days. RFHM1:Cdi3 and RFHM12:Cdi3 were used as internal negative and positive controls, respectively. ++ indicates a strong interaction; + indicates a weak interaction. Note that potential interactions are induced by the presence of Gal. B, GST-TCTP interacts with IVT-generated TSAP6. The indicated GST fusion proteins immobilized on beads were incubated with either AIP or TSAP6-radiolabeled products. Bound IVT products were visualized by autoradiography. Note that GST-NKTR and IVT AIP were used as negative controls. C, endogenous TSAP6 and TCTP co-distribute in 293T, HepG2, and HeLa cells to dotted like structures around the nucleus and at the plasma membrane. Cells stained with anti-TSAP6 (green) and anti-TCTP (red) were analyzed for co-distribution (yellow).

 
Immunofluorescence studies were initiated to determine whether TSAP6 and TCTP co-localize. 293T and HepG2 cells were stained for endogenous TSAP6 and TCTP and analyzed by confocal microscopy. Fig. 2C shows that the TSAP6 (green) and TCTP (red) staining appeared as dispersed dots with variable size around the nucleus and at the cell periphery, where TCTP and TSAP6 partially co-distributed. This co-distribution was also confirmed in HeLa cells using wide field deconvolution microscopy.

TSAP6 Facilitates the Secretion of TCTP—As TSAP6-related proteins STEAP, TIARP, and STAMP1 have been proposed to be involved in vesicular trafficking and protein secretion, we investigated the effects of overexpressing TSAP6 on the export of endogenously and exogenously expressed TCTP. 293T cells were transfected with increasing amounts of HA-TSAP6, and following 24 h, cells were placed in serum-free media, and their supernatants were assayed for the presence of TCTP. Prior to 3 h, TCTP was almost undetectable in the supernatants under conditions where cells were transfected with vector control (Fig. 3A). In the presence of overexpressed HA-TSAP6, supernatant-derived TCTP appeared to increase in a dose- and time-dependent manner. By 30 min of secretion, TCTP was detectable in the supernatants, and by 3 h, this effect was further augmented (Fig. 3, A and B). Immunoblotting with an anti-HA antibody resulted in no detectable signal of overexpressed TSAP6 in these supernatants (data not shown). Immunoblotting for {beta}-tubulin, eEF1B{beta}, and actin confirmed the absence of cytosolic contamination in the supernatants. As an additional precautionary measure, cell viability was routinely assessed by trypan blue exclusion. These analyses yielded similar rates of cell mortality regardless of the transfection conditions used.



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  		FIG. 3.
TSAP6 augments the secretion of TCTP. A, overexpression of TSAP6 enhances the secretion of endogenous TCTP in a dose- and time-dependent manner. 293T cells were transfected with the indicated amounts of pcDNA3-HA-TSAP6 (HA-TSAP6), and secretion assays were performed in serum-free media for up to 3 h. Supernatants (upper panel) and total lysates (TL) (lower panels) were analyzed by immunoblot analysis for TCTP. Note that TCTP was visualized as early as 0.5 h only in the presence of HA-TSAP6. Membranes were immunoblotted withanti-{beta}-tubulin ({beta}-tub), anti-eEF1B{beta} (B{beta}), and anti-actin to determine the extent of cytosolic contamination in the supernatant preparations as well as to verify equal loading of cell lysates. When necessary, DNA quantities were normalized for transfections with pcDNA3-HA. Anti-HA immunoblotting revealed the levels of expression of TSAP6. B, overexpressed TSAP6 promotes the export of ectopically expressed TCTP. 293T cells were transfected with the indicated constructs. Supernatants were analyzed by immunoblot analysis for endogenous and exogenous secreted TCTP. Exogenous TCTP migrates at ~49 kDa due to its COOH-terminal YFP tag. C, PS1, a 6–8 transmembrane protein, does not promote the secretion of TCTP. The effects of overexpressed HA-TSAP6 or PS1 either on endogenous or exogenous TCTP secretion were compared. Note that PS1 is a 43-kDa protein that migrates as a smear. D, TSAP6 promotes the secretion of TCTP in HepG2 cells. Cells were transfected with the indicated constructs, and secretions assays were performed as described for 293T cells. E, overexpression of TSAP6 enhances the secretion of both endogenously and exogenously expressed TCTP in K562 cells. Cells were electroporated with the indicated constructs. Twenty four hours after transfection, 3-h secretion assays were performed.

 
Similar experiments were performed with exogenously expressed YFP-TCTP (COOH-terminal tag) either in the presence or absence of HA-TSAP6. Fig. 3B shows that YFP-TCTP overexpressed alone was not detected in supernatants of 293T cells by 3 h, whereas co-expression of HA-TSAP6 resulted in a robust response, i.e. the export of exogenously expressed TCTP (Fig. 3B). In agreement with endogenous TCTP being released in a brefeldin A-independent fashion, YFP-TCTP secretion mediated by TSAP6 was not affected by brefeldin A treatment (Fig. 3B). These effects are likely due to specific release of YFP-TCTP because TSAP6 did not cause the release of two unrelated YFP fusion proteins (data not shown). It appears that YFP-TCTP is secreted only in the presence of TSAP6, whereas endogenous TCTP is constitutively secreted. We asked whether this is because of either the nature or the orientation of the tag by testing other tag-containing TCTP constructs in our secretion assay. This was not the case because FLAG-TCTP (NH2-terminal tag) and Fc-TCTP (COOH-terminal tag) were not constitutively secreted in the absence of overexpressed TSAP6 (supplemental Fig. S2). However, similar to YFP-TCTP, both appeared in the supernatants in the presence of overexpressed TSAP6.

To rule out the possibility of unspecific effects due to overexpressing a multipass membrane protein, such as TSAP6, we included in these analyses the familial Alzheimer's disease gene product Presenilin-1 (PS1) (Fig. 3C). PS1 is a 6–8 transmembrane protein implicated in promoting the secretion of amyloid {beta} protein (33). Our data show that by 3 h of secretion, overexpression of PS1 did not perturb the levels of either endogenous or overexpressed TCTP.

The effect of TSAP6 on TCTP secretion was also analyzed in the HepG2 hepatocyte cell line, a commonly used cell line to study secreted proteins (34). Fig. 3D illustrates that similar to the effects observed in 293T cells, co-transfection of HA-TSAP6 and YFP-TCTP in HepG2 cells resulted in the enhanced secretion of overexpressed TCTP. This was particularly striking in lieu of the fact that the transfection efficiency for HepG2 cells was estimated at 1–2% compared with 80% for 293T cells. The high levels of constitutive secretion of endogenous TCTP and the low transfection efficiency obtained may account for the absence of augmented secretion of endogenous TCTP by TSAP6 in this cell line. Finally, as TCTP was originally identified as a secreted factor in hematopoietic cells (18, 21), we addressed whether overexpression of TSAP6 could enhance the secretion of TCTP in the erythroleukemia cell line K562. Similar to the effects observed in 293T cells, overexpression of HA-TSAP6 increased the levels of both endogenous and exogenous TCTP in the supernatants of K562 cells (Fig. 3E).

TSAP6 and TCTP Are Found in Small Secreted Vesicles Called Exosomes—Several models have been proposed to describe mechanisms of nonclassical protein secretion, either by translocation across the membrane or by formation of vesicles carrying the protein (31). This latter model involves exosomes, which are small vesicles that form by inward budding from the limiting membrane into the lumen of endosomes, which are then called multivesicular bodies. Multivesicular bodies can fuse with the plasma membrane and release their content outside the cell, i.e. exosomes (35). They are secreted by many cell types and can be isolated from supernatants by a high speed centrifugation. During the course of these analyses, we found in a yeast two-hybrid hunt using TCTP as a bait, several proteins that have been described as exosomal resident proteins among potential TCTP binding partners. Thus, we addressed whether TCTP was also found in these secreted vesicles. We initially isolated exosomes from the supernatants of murine dendritic cells derived from either the D1 cell line or bone marrow primary cultures because their protein content has been well characterized (27, 36). Anti-TCTP immunoblotting revealed that indeed TCTP was present in both dendritic cell-derived exosome preparations (Fig. 4A). Immunoblotting for MHC class II, clathrin and TSG101, well characterized dendritic cell exosome markers, verified the quality of exosomes purified from these two cellular sources (26, 27). On the other hand, immunoblotting with anti-GP96, which recognizes an ER protein described previously (37) as being absent from exosomes, demonstrated the lack of cytosolic contamination in these preparations (Fig. 4A). Finally, we fractionated dendritic cell-derived exosome preparations on a sucrose gradient, and we analyzed their protein content by Western blot to further confirm the presence of TCTP in exosomes. Fig. 4B shows that both TCTP and MHC class II were specifically found at the characteristic density of exosomes, 1.15–1.19 g/cm3. Galectin-3, another protein described to be present in exosomes and secreted by a nonclassical route, was also co-purified in similar fractions. These analyses demonstrate for the first time that TCTP is present in exosomes.



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  		FIG. 4.
TCTP is found in exosomes, and overexpression of TSAP6 enhances its presence in these vesicles. A, identification of TCTP in exosomal preparations derived from murine dendritic cells. Exosomes isolated from supernatants of murine dendritic cells derived from either the splenic D1 cell line or bone marrow (BM) were immunoblotted with either anti-TCTP, -MHC class II, -clathrin, -GP96, or -TSG101. Note that the anti-GP96 antibody was used as a negative control for exosomes. Yield of purification of exosomes was 0.5 µg per 106 cells. Note that 10 µg of total lysates (TL) and 3 µg of exosomes (Exo) were loaded. B, TCTP and galectin-3 co-migrate with MHC class II at the expected exosome density on a sucrose gradient. Fractions collected from D1-derived exosomes on a continuous sucrose gradient were analyzed by immunoblotting with either anti-MHC class II, anti-galectin-3, or anti-TCTP antibodies. C, TCTP is present in 293T-derived exosomal preparations and is further enhanced by TSAP6. 293T cells transfected with the indicated combinations of YFP-TCTP and HA-TSAP6 were isolated and immunoblotted with either anti-TCTP, -HA, -GP96, -clathrin, -cyclin B1, or -MHC class I. Note that TSAP6 overexpression enhanced the presence of both TCTP and MHC class I but not of clathrin in exosomal preparations. Anti-GP96 and anti-cyclin B1 immunoblotting were used as negative controls for exosomes. Yield of purification of exosomes was 0.05 µg per 106 cells. 10 µg of TL and 2 µg of exosomes were loaded.

 
Next, the effect of TSAP6 on the presence of TCTP in exosomes was further investigated in 293T cells. Cells were transfected with YFP-TCTP either with or without HA-TSAP6, and secretion assays were performed in serum-free media for 3 h (Fig. 4C). To isolate exosomes, we performed a high speed centrifugation step, instead of the concentration of the supernatants. Immunoblot analysis on isolated exosome preparations clearly showed that endogenous TCTP as well as YFP-TCTP were detected in exosome preparations derived from 293T cell supernatants, and moreover, their presence was further enhanced by the overexpression of HA-TSAP6 (Fig. 4C). To verify the quality of exosome preparations, membranes were also immunoblotted with either anti-MHC class I or anti-clathrin antibodies. Most interestingly, the presence of MHC class I was increased in exosomes overexpressing HA-TSAP6, whereas the levels of clathrin remained unchanged. Anti-HA immunoblotting revealed that whereas TSAP6 was not visible in supernatants, it was detectable in exosomes. Altogether, these data indicate that TSAP6 increases TCTP secretion and its presence in exosomes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
While working on the functional characterization of TSAP6, we performed a yeast two-hybrid hunt using TSAP6 as a bait, and we identified TCTP as a potential binding partner. Concurrently, in a high throughput screening, comparing differentially expressed genes between tumor cells and their revertant counterparts, TCTP was identified as being appreciably down-modulated in the latter (17). As TSAP6 belongs to a family of 5–6 transmembrane molecules, potentially involved in vesicular trafficking, and TCTP is a secreted protein implicated in inflammatory responses, we addressed the involvement of TSAP6 on TCTP export.

The majority of secreted proteins exit the cell by an ER/Golgi-dependent pathway, whereas only a small number of proteins destined for secretion are exported independently of the ER/Golgi (31). This latter pathway is resistant to brefeldin A, and the proteins following it lack an NH2-terminal signal peptide. Interleukin-1{beta} and galectin-1 were among the first leaderless secretory proteins described to follow an ER/Golgi-independent route (38, 39). Our studies suggest that TCTP, a mediator of inflammatory responses that lacks an apparent signal peptide (40), is secreted independently of the ER/Golgi. Indeed, the secretion of either endogenous or overexpressed TCTP was insensitive to brefeldin A and monensin treatments. We further show that secreted TCTP in our assay system does not correspond to newly synthesized proteins but appears to originate from a pre-existing pool. This conclusion was based on the following observations: 1) newly synthesized, i.e. radiolabeled TCTP, was detected in the supernatants only by 16 h of chase, and 2) TCTP levels in the supernatants remained unchanged even after treating 293T cells for 8 h with CHX. Pre-existing pools of TCTP could represent easily accessible reserves that could be rapidly mobilized upon activation by an inflammatory signal. This scenario is reminiscent of myeloid granulocytes, which contain granules that act as a repository for the rapid mobilization of pre-packaged soluble mediators during inflammation.

Teshima. et al. (20) described TCTP in macrophages as being CHX-resistant, because they observed that the stimulating effects of macrophage colony-stimulating factor on TCTP expression were not diminished by 8 h of treatment with CHX. In accordance, we observed a similar CHX resistance of TCTP in 293T cells until 8 h of treatment with CHX in the absence of any stimuli. However, after 18 h of treatment with CHX (which is approximately the half-life of TCTP according to our pulse-chase experiments), we detected decreased levels of secreted TCTP. We also wanted to mention that the near-complete disappearance of secreted TCTP at 18 h cannot be explained exclusively by the small diminution of intracellular TCTP in the presence of CHX. It is likely that a nonsecretable pool of TCTP exists. This could explain the small estimated percentage of secreted TCTP as compared with its overall intracellular content and is in accordance with the previously described intracellular roles of TCTP (14, 16, 41).

The molecular components involved in processes related to nonclassical export are relatively unknown. Recently, some of these components have been identified. For example, the cytoplasmic domains of synaptotagmin (p40-Syt1), a component of synaptic vesicles, and the calcium-binding protein, S100A13, have been described as being essential components for the secretion of fibroblast growth factor-1, a pro-angiogenic polypeptide that is secreted in response to cellular stresses such as heat shock or serum starvation (42–45). In addition, the ATP-binding cassette transporters have also been associated with mechanisms of nonclassical export (46, 47). They are multipass membrane proteins with the characteristic feature of having an ATP-binding cassette, a domain that has not been found in TSAP6.

TSAP6-related family members have been localized to vesicular structures and have also been proposed to be involved in vesicular trafficking and/or secretory processes (8). In agreement, we found that TSAP6 and TCTP were also found in similar structures, where these proteins partially co-distributed near the plasma membrane and around the nucleus. A functional link between these two proteins was established by showing that TSAP6 overexpression consistently enhanced the secretion of both endogenous and exogenous TCTP. This was shown in both epithelial and hematopoietic cell lines. Although the source of secreted TCTP has been described previously to be in erythroid and leukemic cells, it has been also demonstrated that bronchial epithelial cells actively secrete TCTP (23). This suggests that other cell types besides hematopoietic cells can secrete TCTP. Our data identify a novel role for TSAP6 in the nonclassical export of TCTP and, moreover, suggest that TSAP6 could play a global role in the nonclassical export.

We further demonstrated that TCTP was present in exosome preparations derived from supernatants of dendritic cells. Similar to TCTP, galectin-3, another protein known to have both intracellular and extracellular activities and to be secreted by a nonclassical route, has also been found in exosome preparations from dendritic cells (27). We also found that galectin-3 co-purified with TCTP and MHC class II in the same sucrose gradient fraction of dendritic cell-derived exosomes. As exosomes have been implicated in intercellular communications (35), they could represent a means by which these two proteins could be secreted nonclassically and exert their extracellular activities on neighboring cells. Furthermore, we observed that TSAP6 enhanced the presence of TCTP in exosome preparations of 293T cells. This is in accordance with the observation that TSAP6 enhanced the overall secretion of TCTP in the supernatants. Moreover, TSAP6 also enhanced the presence of MHC class I in exosomes. This phenomenon appeared specific because TSAP6 did not augment the presence of clathrin, a previously described exosome resident protein, and may indicate a role for TSAP6 in the selective transport of proteins in exosomes. This observation is interesting in light of the fact that dendritic cell-derived exosomes have been described previously (27, 48, 49) to contain both MHC class I and II and have been shown to induce potent immune responses leading to regression of established tumors in mice. The finding that overexpressed TSAP6 was itself present in exosomes suggests the possibility of its role in exosome-associated activities. Overall, our studies identify a novel role for TSAP6 in the nonclassical export of TCTP and, furthermore, indicate that this multipass membrane protein could have a more general role in regulating vesicular trafficking and secretion.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2. Back

§ Both authors contributed equally to this work. Back

To whom correspondence should be addressed: Molecular Engines Laboratories, 20, Rue Bouvier, 75011 Paris, France. E-mail: atelerman{at}molecular-engines.com.

1 The abbreviations used are: TSAP6, tumor suppressor-activated pathway-6; STEAP, six transmembrane epithelial antigen of the prostate; TIARP, tumor necrosis factor {alpha}-induced adipose-related protein; STAMP1, six transmembrane protein of prostate 1; TCTP, translationally controlled tumor protein; HRF, histamine-releasing factor; AIP, ALG-2 interacting protein 1; IVT, in vitro transcribed/translated; GST, glutathione S-transferase; HA, hemagglutinin; CHX, cycloheximide; MHC, major histocompatibility complex; ER, endoplasmic reticulum; YFP, yellow fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank S. L. Shorte, P. Roux, E. Perret, and M. Marchand of the Plate-Forme Imagerie Dynamique of the Pasteur Institute for their expertise in confocal and wide-field deconvolution microscopies. We also thank C. Nizak for preliminary microscopy studies and L. D'Adamio, C. Cans, V. Nancy-Portebois, and P. Benaroch for helpful discussions.



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