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First published online 9 March 2004
doi: 10.1242/jcs.01027

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Unconventional protein secretion: membrane translocation of FGF-2 does not require protein unfolding

¹ Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
² Zentrum für Molekulare Biologie Heidelberg, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

View larger version (27K): [in a new window]   Fig. 1. Generation of model cell lines designed to study protein unfolding aspects during unconventional secretion of FGF-2. (A) cDNA constructs used in this study. CHOMCAT-TAM2 cells (Engling et al., 2002) were transduced with retroviral particles carrying the cDNA constructs shown. Clonal cell lines were obtained by FACS sorting. For details, see Materials and Methods. (B) Western blot analysis of model cell lines grown in the absence or presence of doxicycline. Cell lysates were separated on 10% SDS gels followed by protein transfer to a PVDF membrane. The constructs depicted in A were detected using affinity-purified anti-GFP antibodies (Engling et al., 2002). In the case of FGF-2-GFP, the material derived from about 10,000 cells was loaded; in the case of FGF-2-GFP-DHFR and MTS-GFP-DHFR, the material derived from about 20,000 cells was loaded.

View larger version (142K): [in a new window]   Fig. 2. Characterization of the various model cell lines based on confocal microscopy. CHOFGF-2-GFP (A-C), CHOFGF-2-GFP-DHFR (D-F) and CHOMTS-GFP-DHFR cells (G-I) were grown on coverslips in the absence (panels A,D,G) or presence of doxicycline (panels B,C,E,F,H,I). Cells were fixed without permeabilization using paraformaldehyde followed by immunostaining using affinity-purified anti-GFP antibodies. In B, E and H total GFP fluorescence is shown, whereas in panels C, F and I cell-surface staining is shown.

View larger version (24K): [in a new window]   Fig. 3. Quantitative analysis of unconventional secretion of FGF-2-GFP and FGF-2-GFP-DHFR fusion proteins in living cells based on FACS. CHOFGF-GFP (A,B), CHOFGF-2-GFP-DHFR (C,D) and CHOMTS-GFP-DHFR cells (E,F) were grown in the absence (grey curves) or presence (green curves) of doxicycline. Cells were dissociated from the culture plates using a protease-free protocol followed by immunostaining using either affinity-purified anti-GFP (A,C,E) or monoclonal anti-His-tag antibodies (B,D,F). Living cells were analysed simultaneously for both total GFP fluorescence and cell-surface staining employing a BD FACSCalibur system.

View larger version (121K): [in a new window]   Fig. 4. MTS-GFP-DHFR import into mitochondria is inhibited in the presence of aminopterin. (A-C) CHOMTS-GFP-DHFR cells were grown on coverslips in the absence (A) or presence (B and C) of doxicycline. Cells shown in C were treated with aminopterin (50 mM) throughout induction of protein synthesis. Cells were fixed using paraformaldehyde followed by the analysis of GFP-derived fluorescence using a Zeiss LSM 510 confocal microscope. (D) Biochemical analysis of MTS-GFP-DHFR import into mitochondria in the presence and absence of aminopterin. CHOMTS-GFP-DHFR cells were grown in the presence of doxicycline. Where indicated, aminopterin (ao; 50 mM) was added throughout induction of protein synthesis. The various cell lysates were analysed by SDS-PAGE and western blotting using affinity-purified anti-GFP antibodies. Three species of MTS-GFP-DHFR are detected, the precursor (*) as well as an intermediate () and the fully processed form ().

View larger version (49K): [in a new window]   Fig. 5. Stabilization of the DHFR domain of MTS-GFP-DHFR and FGF-2-GFP-DHFR in the presence of aminopterin. Doxicycline-induced CHOMTS-GFP-DHFR cells and CHOFGF-2-GFP-DHFR cells were lysed in a detergent-containing buffer. The lysates were cleared by centrifugation and subjected to protease treatment (200 mg trypsin/ml) in the presence or absence of aminopterin (50 mM). Lanes 1-3: MTS-GFP-DHFR; lanes 4-6: FGF-2-GFP-DHFR. The various samples were subjected to SDS-PAGE and western blotting followed by antigen detection using monoclonal anti-DHFR antibodies. As can be seen in lanes 1 (MTS-GFP-DHFR) and 4 (FGF-2-GFP-DHFR), post-lysis degradation results in the appearance of the DHFR fragment without adding exogenous protease.

View larger version (54K): [in a new window]   Fig. 6. Qualitative analysis of FGF-2 export in the absence and presence of aminopterin. Doxicycline-induced CHOFGF-2-GFP-DHFR cells were grown on coverslips in the absence (A,B) or presence (C-F) of aminopterin. Cells were fixed without permeabilization using paraformaldehyde followed by immunostaining using either affinity-purified anti-GFP antibodies (B,D) or monoclonal anti-His-tag antibodies (F). Total GFP fluorescence (A,C,E) as well as cell-surface staining (B,D,F) were analysed using a Zeiss LSM 510 confocal microscope.

View larger version (18K): [in a new window]   Fig. 7. Quantitative analysis of FGF-2 export in the absence or presence of aminopterin employing FACS. (A) Noninduced (grey curves) and doxicycline-induced (green curves) CHOFGF-2-GFP-DHFR cells were grown in the absence (a-d) or presence (e-h) of aminopterin. Following detachment cells were processed for FACS analysis using either affinity-purified anti-GFP antibodies (a,c,e,g) or monoclonal anti-His-tag antibodies (b,d,f,h). Total GFP fluorescence (a,b,e,f), as well as cell-surface staining (c,d,g,h), were measured simultaneously using a Becton Dickinson FACSCalibur system. (B) Statistical analysis of FGF-2-GFP-DHFR export in the absence and presence of aminopterin based on expression-corrected cell-surface staining. Because aminopterin causes a significant increase of FGF-2-GFP-DHFR expression, the amount of doxicycline added to aminopterin-treated cells was titrated until an expression level was reached similar to that of cells grown in the absence of aminopterin. Under these conditions, FGF-2-GFP-DHFR export was determined on the basis of cell-surface staining. Cell-surface staining measured in the absence of aminopterin was set to 100% and compared with cell-surface staining in the presence of aminopterin (n=3).

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