Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.
Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.
Jones
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AT
Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA. wessling@hsph.harvard.edu
Spiro
D J
DJ
Kirchhausen
T
T
Melançon
P
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Wessling-Resnick
M
M
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D020755:Coat Protein Complex I / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D018942:Macrolides; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D014168:Transferrin / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002966:Clathrin; D020755:Coat Protein Complex I; D004791:Enzyme Inhibitors; D018942:Macrolides; D014168:Transferrin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; C040929:bafilomycin A1; D020727:ADP-Ribosylation Factors