The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes.
The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.
Paleotti
Olivia
O
Institut de Pharmacologie Moleculaire et Cellulaire, CNRS-Unité Mixte de Recherche 6097, 660 Route des Lucioles, 06560 Valbonne Sophia-Antipolis, France.
Macia
Eric
E
Luton
Frederic
F
Klein
Stephanie
S
Partisani
Mariagrazia
M
Chardin
Pierre
P
Kirchhausen
Tom
T
Franco
Michel
M
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D010716:Phosphatidylinositols / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000378:metabolism; D013997:Time Factors; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D004268:DNA-Binding Proteins; D008081:Liposomes; D010716:Phosphatidylinositols; D011990:Receptors, Transferrin; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D014168:Transferrin; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human