Externally added aFGF mutants do not require extensive unfolding for transport to the cytosol and the nucleus in NIH/3T3 cells.
Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.
Wesche
J
J
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Wiedłocha
A
A
Falnes
P O
PO
Choe
S
S
Olsnes
S
S
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism; D004220:Disulfides; D016220:Fibroblast Growth Factor 1 / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021381:Protein Transport; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D004167:Diphtheria Toxin; D004220:Disulfides; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D016220:Fibroblast Growth Factor 1