The pleckstrin homology domain of the Arf6-specific exchange factor EFA6 localizes to the plasma membrane by interacting with phosphatidylinositol 4,5-bisphosphate and F-actin.
The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.
Macia
Eric
E
Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS-Université de Nice-Sophia Antipolis, 660, route des lucioles, 06560 Valbonne, France.
Partisani
Mariagrazia
M
Favard
Cyril
C
Mortier
Eva
E
Zimmermann
Pascale
P
Carlier
Marie-France
MF
Gounon
Pierre
P
Luton
Frédéric
F
Franco
Michel
M
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003599:Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding / Q000502:physiology; D020349:Surface Plasmon Resonance / Q000379:methods
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C109970:PSD protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D017830:Octoxynol; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human