Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1.
Externally added fibroblast growth factor-1 (FGF-1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF-1, and prenylation of an FGF-1 mutant that contains a C-terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF-1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF-1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na+/K+-ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked.
Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Montebello, Oslo, Norway.
Jedrzej
J
Małecki
Antoni
A
Wiedłocha
Jørgen
J
Wesche
Sjur
S
Olsnes
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D020126:Brefeldin A / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D011992:Endosomes / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D018942:Macrolides; D008564:Membrane Potentials; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000187:drug effects; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000502:physiology*; D014617:Vacuoles / Q000201:enzymology; D014634:Valinomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007476:Ionophores; D018942:Macrolides; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins; D000643:Ammonium Chloride; D016220:Fibroblast Growth Factor 1; D014634:Valinomycin; D020126:Brefeldin A; D015638:Cytochalasin D; C040929:bafilomycin A1; D008985:Monensin; D025262:Vacuolar Proton-Translocating ATPases; D009550:Nigericin; D015739:Nocodazole