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Originally published In Press as doi:10.1074/jbc.M310500200 on November 27, 2003

J. Biol. Chem., Vol. 279, Issue 8, 6244-6251, February 20, 2004
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Unconventional Secretion of Fibroblast Growth Factor 2 Is Mediated by Direct Translocation across the Plasma Membrane of Mammalian Cells

Tobias Schäfer{ddagger}, Hanswalter Zentgraf§, Christoph Zehe{ddagger}, Britta Brügger{ddagger}, Jürgen Bernhagen¶, and Walter Nickel{ddagger}||

From the {ddagger}Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, §Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, and Unit of Biochemistry and Molecular Cell Biology, Institute of Biochemistry, University Hospital RWTH Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany

Received for publication, September 23, 2003 , and in revised form, November 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soluble proteins destined for secretion are usually characterized by an N-terminal signal peptide that promotes co- or post-translational import into the endoplasmic reticulum (ER)1 (1, 2). Once translocated into the lumen of the ER, secretory proteins are delivered to the cell surface by vesicular transport (3-7). By contrast, a growing number of secretory proteins are being identified that lack signal peptides for ER translocation, and yet they are exported to the extracellular space (8-10). This process has been termed unconventional protein secretion (also known as non-classical protein export or ER/Golgi-independent protein secretion). The group of unconventionally secreted proteins comprises critical factors such as the pro-angiogenic mediators FGF-1 and FGF-2 (11-20), inflammatory cytokines such as interleukin 1{beta} (21-23), and macrophage migration inhibitory factor (MIF; (24)), the galectins, a family of {beta}-galactoside-specific lectins of the extracellular matrix (25-32), viral proteins (33-36), and parasitic surface proteins potentially involved in the regulation of host cell infection (37, 38). Consistently, unconventional secretory processes have been shown to be gated in most cases (reviewed in Refs. 9 and 10); for example, interleukin 1{beta} secretion is induced upon activation of monocytes (22, 23); MIF secretion from monocytes is triggered by lipopolysaccharides (24, 39); secretion of galectin-1 is up-regulated during differentiation (29, 30); and externalization of fibroblast growth factor 1 (FGF-1) is triggered under stress conditions such as heat shock treatment (13, 17).

Intriguingly, several independent lines of evidence indicate that various kinds of mechanistically distinct non-classical export routes may exist (reviewed in Refs. 9 and 10). For example, interleukin 1{beta} has been reported to be imported into an endolysosomal compartment prior to secretion (23), whereas FGF-1 is likely to be translocated directly across the plasma membrane (40, 41). In case of FGF-2, Gal-1, and MIF, the intracellular site of membrane translocation is unknown. Based on various experimental observations, at least four independent pathways of unconventional protein secretion may exist (10). However, in all cases, the molecular machineries mediating ER/Golgi-independent protein secretion have not been revealed.

A key aim of the current study was to implement an experimental system that allows for a thorough biochemical analysis of FGF-2 export. Here we establish a novel assay employing affinity-purified plasma membrane inside-out vesicles to reconstitute FGF-2 secretion in vitro. The rationale for this system is to measure post-translational membrane translocation of FGF-2 into the lumen of inside-out vesicles, a process that mimics FGF-2 secretion, as the lumen of these vesicles is topologically equivalent to the extracellular space. FGF-2 membrane translocation is shown to require elevated temperature; however, it neither depends on exogenously added ATP nor is it sensitive to treatment with apyrase, an ATP-degrading enzyme (42). Interestingly, ouabain, an inhibitor of the sodium potassium ATPase (Na/K-ATPase), which has been suggested to play a role in FGF-2 export (18, 43), does not inhibit FGF-2 membrane translocation in vitro. Therefore, our data suggest that the partial inhibitory activity of ouabain on FGF-2 export in living cells is not due to a direct function of the Na/K-ATPase in FGF-2 membrane translocation.

Removal of peripheral membrane proteins blocks FGF-2 import into plasma membrane vesicles; however, FGF-2 can still bind to the cytoplasmic surface of these membranes. Translocation activity of salt-washed membranes can be restored by the addition of cytosol. These findings suggest that both integral membrane proteins and cytosolic factors are required for FGF-2 membrane translocation. When plasma membrane-derived inside-out vesicles were compared with right side-out vesicles, the latter ones were found not to be competent to import FGF-2, demonstrating that FGF-2 transport across the plasma membrane occurs in a unidirectional fashion. Intriguingly, when other unconventional secretory proteins such as MIF (24) and Gal-1 (25, 29) were tested, differential uptake into plasma membrane inside-out vesicles was observed in that MIF was found not to be transported, whereas Gal-1 did translocate as efficiently as FGF-2. These data are consistent with the postulation of multiple independent transport routes for secretory proteins exported by unconventional means (8-10). Our findings provide the first direct evidence that FGF-2 and Gal-1 are exported from mammalian cells by direct translocation across the plasma membrane.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Affinity-purified anti-FGF-2 antibodies, affinity-purified anti-galectin-1 antibodies, affinity-purified anti-GFP antibodies, and polyclonal anti-MIF antibodies were prepared as described (20, 32, 62). Polyclonal antibodies directed against the influenza HA epitope (YPYDVPDYA) were raised in rabbits according to standard procedures. Polyclonal anti-p30 antibodies (47) as well as the polyclonal antibodies directed against the Golgi protein p27 (49) were the kind gifts of Wilhelm Just (Biochemie-Zentrum Heidelberg) and Felix T. Wieland (Biochemie-Zentrum Heidelberg), respectively. Monoclonal antibodies against the Myc and the CD8 epitope were derived from the hybridoma cell lines 9E10 (63) and Okt8 cells (53), respectively. Monoclonal rat anti-HA antibodies were obtained from Roche Diagnostics, and polyclonal anti-calnexin antibodies were purchased from Stressgen. All secondary antibodies used for the detection of antigens by Western blotting were obtained from Bio-Rad.

Isolation of Plasma Membrane Vesicles—Genetically modified CHO cells stably expressing a modified form of the cell surface protein CD8 (CD8-LT) (44) were grown in spinner cultures according to standard procedures. Plasma membrane vesicles were prepared by a modification of the procedure described by Keppler and colleagues (45). CHOCD8-LT cells (3 x 109) were washed twice with 50 ml of ice-cold phosphate-buffered saline (PBS) and lysed by a 40-fold dilution in hypotonic buffer (0.5 mM NaPO4 buffer, pH 7.0; 0.1 mM EDTA supplemented with protease inhibitor mixture (Roche Applied Science)). Following incubation for 2 h at 4 °C, the cell lysate was centrifuged at 100,000 x gav for 40 min at 4 °C. The sediment was resuspended in 20 ml of hypotonic buffer and homogenized using a Potter-Elvehjem homogenizer. Following dilution in incubation buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose), the lysate was centrifuged for 10 min at 12,000 x gav at 4 °C. The sediment was resuspended and again subjected to the procedure described. Postnuclear supernatants from both steps were combined and subjected to ultracentrifugation at 100,000 x gav for 40 min at 4 °C. The resulting sediment was resuspended in 20 ml of incubation buffer and manually homogenized by 50 strokes using a tight-fitting Dounce B (glass/glass) homogenizer. The resulting membrane suspension was layered on top of a 6-ml 38% (w/v) sucrose solution (5 mM HEPES-KOH, pH 7.4). Following centrifugation at 280,000 x gav for 2 h in a Beckman SW41 swing-out rotor, membranes in the 0/38% interface were collected and diluted in 20 ml of incubation buffer. This suspension was again homogenized with a Dounce B homogenizer (30 strokes) followed by ultracentrifugation at 100,000 x gav for 40 min at 4 °C. The corresponding sediment was resuspended in 1 ml of incubation buffer and passed 20 times through a 27-gauge needle. The resulting membrane vesicle suspension was stored at -80 °C.

Affinity Purification of Inside-out and Right Side-out Vesicles— Plasma membrane vesicles were incubated with excess amounts of monoclonal anti-Myc antibodies for 30 min at 4 °C. After collecting the antibody-coated membranes by centrifugation (10 min, 7,600 x gav, 4 °C), the samples were washed with PBS, 0.1% BSA followed by incubation with 150 µl of a suspension of Dynabeads (M-280 sheep antimouse IgG) for 1 h at 4 °C. Membranes bound to magnetobeads were reisolated, washed as described above, and finally resuspended in PBS, 0.1% BSA. The same procedure was used to affinity-purify right side-out vesicles employing monoclonal antibodies directed against the luminal domain of CD8 instead of anti-Myc antibodies.

Electron Microscopy—Gradient-purified plasma membranes as well as affinity-depleted plasma membrane vesicles were placed onto a glow-discharged carbon-coated 300 mesh copper grid for 1 min. After rinsing the grid with water, it was placed face down onto a 100-µl droplet containing the antibodies indicated. Polyclonal anti-HA antibodies were used at a 1:25 dilution in PBS, 1% BSA. In case of double labeling experiments, the anti-HA antibody dilution was mixed 1:1 with Okt8 hybridoma cell supernatant. Following incubation for 1 h at room temperature, the grids were washed twice with 100 µl of PBS, 1% BSA. Afterward, the grids were placed onto a 100-µl droplet of a 1:1 mixture of 5 nm gold-labeled goat anti-rabbit IgG (Amersham Biosciences) and 10 nm gold-labeled goat anti-mouse IgG (Amersham Biosciences) each diluted 1:5 in PBS, 1% BSA. Following incubation for 1 h at room temperature in the dark, the grids were rinsed as described above and stained for 1 min with 20 µl of 2% aqueous uranyl acetate. Micrographs were taken with a Zeiss 10 A electron microscope at an acceleration voltage of 80 kV.

FGF-2 Membrane Translocation Assay—In standard incubations, about 7.5% of affinity-purified inside-out vesicles (prepared from 3 x 109 CHOCD8-LT cells) were incubated in a final volume of 100 µl containing transport assay buffer (2 mM MgCl2, 2 mM CaCl2, 50 mM NaCl, 25 mM sucrose, 10 mM dithiothreitol, 25 mM HEPES-KOH, pH 7.4, 10 µM GTP, protease inhibitor mixture (Roche Applied Science)), an ATP-regenerating system (8 units/ml creatine kinase, 5 mM creatine phosphate, 50 µM ATP), rat liver cytosol (5 mg/ml). Recombinant human FGF-2 (18-kDa isoform (20)), recombinant human Gal-1 (32), recombinant GFP (20), or recombinant MIF (64) were added at a final concentration of 50 µg/ml (5 µg of total protein per transport reaction). In some experiments the ATP-regenerating system and GTP were substituted for 25 mM EDTA. Where indicated apyrase (20 units/ml, Sigma) was added to degrade endogenous ATP. Transport mixtures were incubated for 4 h at 37 °C followed by the reisolation of magnetobead-bound plasma membrane vesicles. After two washing steps using PBS, 0.1% BSA, a potential luminal localization of the proteins in question was probed by protease protection experiments. The samples were treated for 2.5 h at 4 °C with 250 units of trypsin (Amersham Biosciences) in the presence or absence of Triton X-100 (1% w/v) in Tris buffer (0.05 M; pH 8.0). The reactions were terminated by adding protease inhibitor mixture (Roche Applies Science) followed by elution of proteins using SDS sample buffer. Proteins were separated on SDS gels and analyzed by Western blotting using the antibodies indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Affinity Purification of Plasma Membrane Inside-out Vesicles—In order to develop an FGF-2 export in vitro assay, we first established a procedure that allowed us to affinity-purify plasma membrane vesicles with homogenous inside-out topology. For this purpose, we made use of a CHO cell line stably expressing an engineered version of the cell surface molecule CD8 (44). In this fusion protein, which has been termed CD8-LT, the cytoplasmic tail was exchanged for an HA epitope, the cytoplasmic tail sequence of the adenoviral protein E19, and a Myc epitope at the extreme C terminus (Figs. 1 and 2). In terms of subcellular localization, CD8-LT was shown to behave identically to CD8wild-type as being a resident of the plasma membrane (44). The rationale of the affinity purification procedure described here was to isolate plasma membrane inside-out vesicles employing bead-immobilized anti-Myc antibodies directed against the cytoplasmic tail of CD8-LT. A subcellular fraction containing plasma membranes consisting of both inside-out and right side-out vesicles was prepared by conventional methods (for details see "Materials and Methods") based on the procedure described by Keppler and colleagues (45). In the second part of the protocol, inside-out vesicles were affinity-purified employing anti-Myc antibodies (Fig. 1A). As shown in Fig. 1B, the plasma membrane marker CD8-LT is strongly enriched in this fraction (lane 7) compared with the starting material (lane 1). By contrast, calnexin and p30, markers of the endoplasmic reticulum (46) and mitochondria (47), respectively, are not enriched in the inside-out vesicle fraction (calnexin) or are absent (p30). p27, a marker protein for Golgi membranes (48, 49), was found to be abundant in the fraction shown in lane 7; however, p27 was only enriched about 4-fold, whereas CD8-LT was enriched about 34-fold in affinity-purified vesicles compared with the starting material (lane 1) as analyzed by quantitation of the corresponding fluorograms using QuantityOne® software from Bio-Rad (data not shown). The presence of p27 in the fraction containing affinity-purified plasma membranes is likely to be due to a subpopulation of CD8-LT present in the Golgi en route to the cell surface. To verify these results using an independent method, we conducted a quantitative lipid analysis based on mass spectrometry (50, 51). For this purpose, we determined the ratio of phosphatidylcholine (PC), a bulk lipid present in all cellular membranes at similar concentrations, to sphingomyelin (SM), a phospholipid that is most abundant in plasma membranes (52). In the starting material, the SM to PC ratio was found to be 0.26 which increased to 0.48 for gradient-purified plasma membranes. The SM to PC ratio of affinity-purified plasma membranes was found to be similar to that of gradient-purified plasma membranes, which most likely indicates that both SM-poor (ER) and SM-enriched (endosomes/lysosomes) membranes are removed. These findings are consistent with the biochemical analysis of marker proteins demonstrating the efficacy of the purification procedure.



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  		FIG. 1.
Affinity purification of plasma membrane inside-out vesicles. A, schematic description of the purification procedure. For details see "Materials and Methods." B, Western blot analysis of marker proteins for the plasma membrane (CD8-LT), the endoplasmic reticulum (calnexin), mitochondria (p30), and the Golgi apparatus (p27). Lane 1, hypotonic suspension; lane 2, membrane fraction after the first homogenization; lane 3, membrane fraction after the second homogenization; lane 4, postnuclear supernatant; lane 5, gradient-purified plasma membranes; lane 6, supernatant of gradient-purified plasma membranes; lane 7, affinity-purified plasma membrane inside-out vesicles. In lanes 1-6, identical amounts of protein (10 µg/lane) were loaded. In lanes 5 and 7, 14 nmol of phosphatidylcholine (quantified by mass spectrometry; (50)) were loaded in order to ensure that equal amounts of membranes are compared.

 



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  		FIG. 2.
Biochemical analysis of the membrane topology of affinity-purified inside-out and right side-out vesicles. A, schematic description of the membrane topology and specific features of CD8-LT. B, affinity-purified inside-out and right side-out vesicles were treated with thrombin in the presence and absence of detergent as indicated. The samples were separated on SDS gels followed by Western blotting using monoclonal antibodies directed against the influenza HA epitope in order to detect CD8-LT. As indicated, the slow migrating form represents full-length CD8-LT, whereas the fast migrating form represents thrombin-cleaved CD8-LT (44). Lanes 1-4, inside-out vesicles; lanes 5-8, right side-out vesicles. TX-100, Triton X-100.

 
Biochemical Determination of the Topology of Plasma Membrane Inside-out and Right-side Out Vesicles—To confirm the inside-out topology of plasma membrane vesicles purified according to the protocol depicted in Fig. 1, we conducted a biochemical analysis based on protease protection experiments (Fig. 2). Because CD8-LT contains a thrombin cleavage site in its cytoplasmic tail (Fig. 2A), the Myc tag can be cleaved off under native conditions in case plasma membrane vesicles do have an inside-out topology. As shown in Fig. 2B, CD8-LT derived from affinity-purified inside-out vesicles (lanes 1-4) treated with thrombin is quantitatively digested under native conditions (compare lane 1 with lane 3). In addition to plasma membrane-derived inside-out vesicles, we affinity-purified right side-out vesicles employing monoclonal antibodies directed against the luminal domain of CD8 (Okt8) (53). As shown in Fig. 2B, lanes 5-8, thrombin cleavage of CD8-LT did not occur under native conditions (compare lane 5 with lane 7) but rather required membrane solubilization with detergent (lane 8). Thus, the experimental approach described in Fig. 1 allowed us to efficiently separate inside-out from right side-out vesicles derived from a mixture of gradient-purified plasma membrane vesicles.

Ultrastructural Analysis of Inside-out and Right-side Out Vesicles—To evaluate the biochemical analysis of inside-out and right side-out vesicles shown in Fig. 2 by using an independent method, we conducted an ultrastructural analysis based on negative staining (Fig. 3). The majority of the plasma membrane vesicles was characterized by a diameter between 200 and 400 nm, consistent with a quantitative analysis employing dynamic light scattering (54) (number mean = 295 nm; data not shown). Three types of antibodies were used for immunostaining, two of which are directed against the cytoplasmic domain of CD8-LT (anti-HA and anti-Myc) used to detect inside-out vesicles. The third antibody is directed against the luminal domain of CD8 (Okt8) and was used to identify right side-out vesicles. In a first set of experiments, gradient-purified plasma membrane vesicles were processed for immunostaining. As shown in Fig. 3, A-C, for each kind of antibody labeled vesicles could be observed demonstrating that gradient-purified plasma membranes represent a mixture of inside-out and right side-out vesicles. In Fig. 3D, double labeling was performed combining anti-Myc and anti-HA antibodies. As expected, both sizes of gold particles were observed on one and the same vesicle which therefore has inside-out topology. In Fig. 3E, double labeling was performed combining anti-CD8 and anti-HA antibodies. Importantly, vesicles were labeled with either small (anti-HA; inside-out topology) or large gold particles (anti-CD8; right side-out topology). In order to analyze vesicular structures following affinity purification, we conducted experiments in which gradient-purified vesicles were affinity-depleted using either anti-Myc or anti-CD8 antibodies followed by double staining employing anti-CD8 and anti-HA antibodies. In Fig. 3F, a vesicular structure is shown that is derived from the supernatant of a vesicle mixture that was treated with anti-Myc antibody-coated beads. Consistently, this structure is positive for the luminal domain of CD8 (large gold particles) and, therefore, has right side-out topology. In Fig. 3G, a vesicular structure is shown that is derived from the supernatant of a vesicle mixture that was treated with anti-CD8 antibody-coated beads. As expected, this vesicle is exclusively labeled with small gold particles demonstrating the availability of the HA epitope. In both types of experiments, more than 95% of the vesicular structures were characterized by either inside-out or right side-out topology, depending on the antibody used for affinity depletion. These findings confirm that the affinity purification protocol depicted in Fig. 1 effectively yields homogeneous preparations of inside-out and right side-out vesicles.



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  		FIG. 3.
Ultrastructural analysis of inside-out and right side-out vesicles. A-E, analysis of gradient-purified plasma membrane vesicles based on negative staining; F and G, analysis of affinity-depleted plasma membrane inside-out and right side-out vesicles based on negative staining. Membranes were processed for immunostaining as described under "Materials and Methods." A, gradient-purified plasma membrane vesicle stained with polyclonal anti-HA antibodies (5 nm gold; inside-out topology). B, gradient-purified plasma membrane vesicle stained with monoclonal anti-Myc antibodies (5 nm gold; inside-out topology). C, gradient-purified plasma membrane vesicle stained with monoclonal anti-CD8 antibodies (5 nm gold; right side-out topology). D, gradient-purified plasma membrane vesicle stained with both polyclonal anti-HA antibodies and monoclonal anti-Myc antibodies (5 nm gold, anti-HA; 10 nm gold, anti-Myc; inside-out topology). E, double labeling of gradient-purified plasma membrane vesicles using both polyclonal anti-HA antibodies and monoclonal anti-CD8 antibodies demonstrating a mixture of inside-out and right side-out vesicles (5 nm gold, anti-HA; 10 nm gold, anti-CD8). F, an example for a right side-out vesicle derived from a supernatant of anti-Myc-depleted vesicles treated with both polyclonal anti-HA antibodies and monoclonal anti-CD8 antibodies (5 nm gold, anti-HA; 10 nm gold, anti-CD8). More than 95% of the vesicles present in this supernatant was positive with regard to CD8 staining (10 nm gold). G, an example for an inside-out vesicle derived from a supernatant of anti-CD8-depleted vesicles treated with both polyclonal anti-HA antibodies and monoclonal anti-CD8 antibodies (5 nm gold, anti-HA; 10 nm gold, anti-CD8). In this case, more than 95% of the vesicles present in this supernatant were positive with regard to anti-HA staining (5 nm gold). White arrows in C and G point to 5-nm gold particles that are difficult to be observed. The bar in each panel corresponds to 100 nm.

 
Membrane Translocation of FGF-2 in Vitro—Based on affinity-purified inside-out and right side-out plasma membrane vesicle preparations characterized in the experiments shown in Figs. 2 and 3, we analyzed whether recombinant human FGF-2 (18-kDa isoform) is capable of translocating into the lumen of these vesicles (Fig. 4). In this experimental setup, FGF-2 was added to vesicles that were still attached to magnetic beads. Under standard conditions, an ATP-regenerating system, GTP, as well as cytosol were added (Fig. 4). The samples were incubated for 4 h at either 37 or 4 °C followed by reisolation of bead-bound vesicles using a magnetic device. Membrane translocation of FGF-2 was then analyzed by protease protection experiments. As shown in Fig. 4A, FGF-2 was readily detectable in the fraction containing reisolated inside-out vesicles (lanes 1 and 2). When protease was added, a major part of FGF-2 associated with these vesicles was not degraded (Fig. 4A, lane 3); however, upon addition of detergent, the majority of FGF-2 was proteolyzed (Fig. 4A, lane 4). As shown in Fig. 4, B and C, following incubation at 37 °C, FGF-2 could neither be extracted from these vesicles by high salt nor by heparin treatment. Furthermore, sonication of the vesicle preparation in the presence of trypsin resulted in degradation of FGF-2 in the absence of detergent (Fig. 4D). These results demonstrate the existence of a significant population of FGF-2 that was not only bound to inside-out vesicles but rather was transported into the lumen of these structures as it could neither be digested with protease nor extracted with high concentrations of salt under native conditions, for example.



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  		FIG. 4.
Membrane translocation of FGF-2 across the plasma membrane of inside-out vesicles reconstituted in vitro. Affinity-purified inside-out vesicles attached to magnetobeads were incubated with human recombinant FGF-2 under the conditions described under "Materials and Methods." Following incubation for 4 h at 4 or 37 °C as indicated, the samples were treated with trypsin in the presence and absence of detergent (A-C and E and F). Alternatively, detergent treatment was substituted by sonication as indicated (D). Proteolysis was terminated by adding protease inhibitors followed by the addition of SDS sample buffer. Proteins were separated on SDS gels and transferred to a polyvinylidene difluoride membrane in order to detect FGF-2 by Western blotting. Lane 1, untreated sample of reisolated membranes; lane 2, reisolated membranes treated with detergent or sonication as indicated; lane 3, reisolated membranes treated with trypsin in the absence of detergent/sonication; lane 4, reisolated membranes treated with trypsin in the presence of detergent or sonication as indicated. A, incubation at 37 °C. B, incubation at 37 °C followed by treatment with 3 M NaCl. C, incubation at 37 °C followed by treatment with heparin (125 µg/ml). D, incubation at 37 °C, protease protection experiment applying sonication as indicated. E, incubation at 4 °C. F, right side-out vesicles were used instead of inside-out vesicles, incubation at 37 °C.

 
As a negative control, FGF-2 translocation experiments were carried out at 4 °C. As shown in Fig. 4E, FGF-2 was still found to be able to bind to inside-out vesicles (lane 1); however, transport into the lumen could not be observed as demonstrated by a lack of protease protection under native versus non-native conditions (compare lane 3 with lane 4). Thus, FGF-2 membrane translocation requires an elevated temperature in order to occur at an appreciable rate. A kinetic analysis of FGF-2 membrane translocation is shown in Fig. 5. At 37 °C (•), significant amounts of imported FGF-2 are already detectable after 15 min of incubation. FGF-2 import proceeds in a linear fashion for up to 60 min followed by a significant drop in membrane translocation activity. At 4 °C ({diamondsuit}), significant amounts of FGF-2 membrane translocation cannot be observed over the whole time course of the experiment.



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  		FIG. 5.
Kinetics of FGF-2 membrane translocation in vitro. Affinity-purified inside-out vesicles attached to magnetobeads were incubated at either 37 (•) or 4 °C ({diamondsuit}) with human recombinant FGF-2 under the conditions described under "Materials and Methods." Samples were taken at 0, 5, 15, 30, 60, and 240 min of incubation. Further processing of the samples including SDS-PAGE and Western blotting using anti-FGF-2 antibodies was performed as described in the legend of Fig. 4. For each time point, the FGF-2 signals corresponding to those shown in lanes 3 and 4 of Fig. 4 were quantified using QuantityOne® software from Bio-Rad. FGF-2 membrane translocation is expressed as the amount of the protease-protected FGF-2 signal detected in the absence of detergent (lane 3 of Fig. 4) relative to the protease-protected FGF-2 signal detected in the presence of detergent (lane 4 of Fig. 4). Curve fitting is based on nonlinear regression (f = y0 + a (1 - e-bx)).

 
In order to analyze a potential topological restriction of FGF-2 membrane translocation, we incubated FGF-2 with right side-out vesicles characterized in Figs. 2 and 3. As demonstrated in Fig. 4F, FGF-2 was found to bind to these vesicles (lanes 1 and 2), which was not unexpected because right side-out vesicles contain heparan sulfate proteoglycans on their surface. Strikingly, however, vesicle-bound FGF-2 does not translocate into the lumen of right side-out vesicles because it is accessible to trypsin cleavage without the addition of detergent (lanes 3 and 4). These results demonstrate that, under the conditions used, FGF-2 membrane translocation occurs in a unidirectional manner mediated by a machinery with asymmetric membrane topology.

In another set of experiments shown in Fig. 6, we analyzed a potential role of the Na/K-ATPase as this protein has been suggested to play a role in FGF-2 export (18, 43). Therefore, we tested whether ATP is required for FGF-2 translocation across the membrane of inside-out vesicles. As compared with standard conditions in the presence of an ATP-regenerating system (Fig. 6A), both omission of the ATP-regenerating system combined with EDTA treatment (Fig. 6B) and apyrase treatment to degrade endogenous ATP (Fig. 6C) did not result in a block of FGF-2 transport. Strikingly, ouabain, a drug targeted against the Na/K-ATPase that has also been shown to partially inhibit FGF-2 export from living cells (18, 20, 43), did not inhibit FGF-2 membrane translocation (Fig. 6D). In contrast to previous conclusions (18, 43), these results suggest that the Na/K-ATPase is not directly involved in the non-classical export from eukaryotic cells of FGF-2.



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  		FIG. 6.
Biochemical characterization of membrane translocation of FGF-2 across the plasma membrane of inside-out vesicles. Affinity-purified inside-out vesicles attached to magnetobeads were incubated with human recombinant FGF-2 under the conditions described under "Materials and Methods." Afterward, the samples were treated with trypsin in the presence or absence of detergent. Trypsin-mediated proteolysis was terminated by adding protease inhibitors followed by the addition of SDS sample buffer. Proteins were separated on SDS gels and transferred to a polyvinylidene difluoride membrane in order to detect FGF-2 by Western blotting. Lane 1, untreated sample of reisolated membranes; lane 2, reisolated membranes treated with detergent; lane 3, reisolated membranes treated with trypsin in the absence of detergent; lane 4, reisolated membranes treated with trypsin in the presence of detergent. A, incubation at 37 °C in the presence of an ATP-regenerating system. B, incubation at 37 °C in the absence of GTP and the ATP-regenerating system but in the presence of 25 mM EDTA. C, incubation at 37 °C in the absence of an ATP-regenerating system combined with the addition of apyrase. D, incubation at 37 °C in the presence of 10 µM ouabain. E, incubation at 37 °C in the absence of cytosol. F, plasma membrane inside-out vesicles were first treated with 3 M NaCl to remove peripheral membrane proteins followed by incubation at 37 °C. G, salt-treated plasma membrane inside-out vesicles as used in the experiment shown in F were supplemented with cytosol to add back cytosolic proteins followed by incubation at 37 °C.

 
Similar to the ATP depletion experiments, the omission of cytosol from the reaction did not cause a translocation defect (Fig. 6E). However, when inside-out vesicles were treated with high concentrations of salt in order to remove peripheral membrane proteins (Fig. 6F), FGF-2 was no longer capable of entering the lumen of these vesicles. By contrast, binding to salt-washed inside-out vesicles was not affected significantly, suggesting that FGF-2 recruitment to inside-out vesicles is mediated by integral membrane components. Most strikingly, translocation activity of salt-washed inside-out vesicles could be restored when cytosol was added back during the incubation as demonstrated in Fig. 6G. Thus, these results suggest that the overall process of FGF-2 membrane translocation requires both integral and peripheral membrane proteins.

In some cases such as the one shown in Fig. 6A, lane 3, translocated FGF-2 appeared to run slightly faster on SDS gels compared with the input material (lane 1). When the supernatant of FGF-2 translocation experiments was analyzed for FGF-2, the migration behavior on SDS gels was identical compared with the input material (data not shown). These findings may suggest that FGF-2 membrane translocation was not complete leaving a small part of the protein accessible to protease even under native conditions. Similar observations have been made in in vitro studies reconstituting protein import into mitochondria (55). In any case, the various controls described in Figs. 4, 5, 6 establish that protease protection of FGF-2 following incubation with inside-out vesicles at 37 °C is due to membrane translocation into the lumen of these vesicles.

Differential Import of Unconventional Secretory Proteins into Plasma Membrane-derived Inside-out Vesicles—To compare FGF-2 import into inside-out vesicles with other unconventionally secreted proteins as well as unrelated proteins, we conducted import assays using recombinant Gal-1 and MIF as well as GFP as a negative control (Fig. 7). For each protein, 5 µg was added to the transport reaction in order to ensure conditions that allow a direct comparison of binding and import efficiencies. Because different antibodies were used to detect the various reporter proteins, the absolute signals shown in Fig. 7 cannot be compared directly. However, we have quantified the relative amounts of binding compared with the input material based on the analysis of fluorograms employing QuantityOne® software from Bio-Rad. This analysis revealed that about 1% of FGF-2 added to inside-out vesicles could be reisolated in the membrane-bound fraction (data not shown). As already shown in Figs. 4 and 6, efficient import of FGF-2 was observed under standard conditions (Fig. 7A) as indicated by protease protection following incubation of FGF-2 with inside-out vesicles (compare lanes 3 and 4). Similarly, following incubation of Gal-1 with inside-out vesicles, efficient import could be detected (Fig. 7B). Intriguingly, under the same experimental conditions, MIF import into inside-out vesicles did not occur (Fig. 7C) as no protease protection of MIF could be observed (lanes 3 and 4). Surprisingly, GFP used as a negative control was observed to bind to inside-out vesicles (Fig. 7D, lanes 1 and 2) but was not found to be imported under conditions that allow efficient membrane translocation of FGF-2 and Gal-1. These findings demonstrate differential uptake of unconventional secretory proteins into plasma membrane-derived inside-out vesicles, suggesting that FGF-2 and Gal-1 externalization occurs by a pathway that is distinct as that for MIF.



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  		FIG. 7.
Specific translocation of FGF-2 and Gal-1 across the plasma membrane of inside-out vesicles. A, standard FGF-2 translocation assay as described in the legend of Fig. 4. B, translocation assay conducted under identical conditions as those used in A but substituting FGF-2 for galectin-1. C, translocation assay conducted under identical conditions as those used in A but substituting FGF-2 for MIF. D, translocation assay conducted under identical conditions as those used in A but substituting FGF-2 for GFP. In each experiment, 5 µg of recombinant protein were added to the incubation mixture. Sample processing was performed as described in the legend to Fig. 4. Lane 1, untreated sample of reisolated membranes; lane 2, reisolated membranes treated with detergent; lane 3, reisolated membranes treated with trypsin in the absence of detergent; lane 4, reisolated membranes treated with trypsin in the presence of detergent.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study we introduce a novel experimental system that allows us to functionally reconstitute FGF-2 secretion in vitro. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate specific membrane translocation of FGF-2. This process mimics FGF-2 secretion because the lumen of inside-out vesicles is topologically equivalent to the extracellular space. The experimental system described is unique in the field and, for the first time, allows a thorough biochemical analysis of FGF-2 membrane translocation. FGF-2 import into inside-out vesicles is shown to depend on an elevated temperature; however, this process apparently does not require ATP as an energy source. The machinery that mediates FGF-2 membrane translocation is embedded in the plasma membrane based on an asymmetric orientation as FGF-2 only enters inside-out vesicles with right side-out vesicles not being competent to import FGF-2. We demonstrate a two-step process that involves FGF-2 binding to integral membrane components followed by membrane translocation. The latter process is shown to require cytosolic factors.

Our finding that FGF-2 membrane translocation apparently occurs in an ATP-independent fashion was unexpected because the overall process of FGF-2 secretion has been shown to depend on ATP in vivo (15). Moreover, the Na/K-ATPase has been implicated in the process of FGF-2 externalization, and the need for ATP was attributed to a potential direct role of the Na/K-ATPase in FGF-2 membrane translocation (18). This conclusion was supported by the fact that ouabain, a cardiac glycoside that inhibits the Na/K-ATPase, also partially interferes with FGF-2 export in vivo (18, 20, 43). Our findings now suggest that the molecular export mechanism itself does not require ATP. Moreover, ouabain does not inhibit FGF-2 membrane translocation in vitro, and therefore, it appears questionable whether the Na/K-ATPase is essential for FGF-2 membrane translocation.

When FGF-2 was compared with other unconventional secretory proteins, it turned out that Gal-1, a {beta}-galactoside-specific lectin of the extracellular matrix (9, 25, 26, 29), is also capable of translocating across the membrane of plasma membrane-derived inside-out vesicles. Intriguingly, macrophage MIF, an inflammatory cytokine (56-59) secreted by unconventional means (24), was not found to be imported by inside-out vesicles demonstrating distinct requirements for FGF-2 and MIF membrane translocation. Our findings suggest that non-classical export of FGF-2 and Gal-1 from mammalian cells occurs by direct translocation across the plasma membrane, whereas MIF might be imported into some sort of intracellular secretory organelle that, upon fusion with the plasma membrane, releases MIF into the extracellular space. This kind of mechanism has been proposed for non-classical export of inter-leukin 1{beta} (IL1{beta}), another example of unconventional protein secretion, as import into an endolysosomal compartment was shown to precede IL1{beta} release from activated human monocytes (23). Consistently, IL1{beta} secretion has been demonstrated to be inhibited in the presence of glyburide and probenicide (60, 61), drugs targeted against ABC1 transporters, that also block MIF secretion (24).

Even though FGF-2 and Gal-1 are capable of translocating across the membrane of inside-out vesicles, it is far from clear whether they also use the same molecular machinery and mechanism to reach the extracellular space. Reports from Hughes and co-workers suggest that members of the galectin family are released by a process termed membrane blebbing (9, 31). Gal-3 has been shown to accumulate below the plasma membrane followed by packaging into exosomes that bud from the cell surface (31). However, it is not yet clear whether Gal-1 follows a similar pathway; therefore, at the current time it remains an open question how Gal-1 is released from mammalian cells. In any case, our data establish that the molecular machineries mediating FGF-2 and Gal-1 export are residents of the plasma membrane and are composed of both integral membrane components and peripheral membrane proteins.


   FOOTNOTES
 
* This work was supported by German Research Foundation Grant DFG Ni 423 and the Ministry of Science, Research, and the Arts of the State of Baden-Württemberg. 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

|| To whom correspondence should be addressed. E-mail: walter.nickel{at}urz.uni-heidelberg.de.

1 The abbreviations used are: ER, endoplasmic reticulum; FGF-2, fibroblast growth factor 2; MIF, macrophage migration inhibitory factor; Gal-1, galectin-1; GFP, green fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SM, sphingomyelin; IL1{beta}, interleukin 1{beta}; CHO, Chinese hamster ovary; PC, phosphatidylcholine. Back


   ACKNOWLEDGMENTS
 
We thank Christian Ungermann (Biochemie-Zentrum Heidelberg) for advice throughout the course of this study as well as Jörg Moelleken (Biochemie-Zentrum Heidelberg) for experimental help in the acquisition of light scattering data. Polyclonal anti-p30 antibodies and polyclonal antibodies directed against the Golgi protein p27 were kind gifts of Wilhelm Just (Biochemie-Zentrum Heidelberg) and Felix T. Wieland (Biochemie-Zentrum Heidelberg), respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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