Role of the Arf6 GDP/GTP cycle and Arf6 GTPase-activating proteins in actin remodeling and intracellular transport.
We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.
Klein
Stéphanie
S
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 660, route des Lucioles, 06560 Valbonne, France.
Franco
Michel
M
Chardin
Pierre
P
Luton
Frédéric
F
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism / Q000502:physiology*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D017607:Aluminum Compounds; D020690:GTPase-Activating Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D005459:Fluorides; C032311:aluminum fluoride