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

Abstract

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)P 2 ) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of ϳ0.5 M EFA6-PH for PI(4,5)P 2 . Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P 2 . These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P 2 , 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.The ADP-ribosylation factor (Arf) family, which includes six isoforms, plays a key role in the intracellular vesicular transport Likewise, the binding of GDP-bound Arfs to their guanine nucleotide exchange factors (GEFs) 2 is critical to localize the Arf proteins to their proper membrane (for review, see Ref. 4). The Arf specific GEF family, also referred to as the Sec7 family, comprises 15 members divided in two classes, the large and the small GEFs, which are responsible for the activation of 6 Arf isoforms. This larger number of GEFs suggests that each Arf is under the control of several ArfGEFs. Growing evidence indicates that the ArfGEFs are targeted to specific compartments where they will activate a cognate Arf. Little is known about how the large GEFs, GBF1, Big1, and Big2 are targeted to their working compartments, respectively cis-Golgi, endoplasmic reticulum-Golgi intermediate (5), and trans-Golgi network (6). In contrast, the small Arf-GEF family members (Arno/Cytohe-* This work was supported by grants from Association pour la Recherche contre le Cancer, Cancé ropô le PACA (Provence-Alpes-Cô te d'Azur), and Agence Nationale de la Recherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indi...

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

“…They are recruited to the membranes through an interaction with PIP 2 or PIP 3 , depending on the different affinities of pleckstrin homology domains for these phospholipids (83,84). The synthesis of PIP 2 and/or PIP 3 may be one of the major mechanisms through which ARF6 is activated at the membrane in response to agonist stimulation of cells (85). Suzuki et al (86) reported the presence of an ARF6-GEF in the male reproductive tract demonstrating that EFA6 is localized in spermatogenic cells of adult mice testes.…”

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…56 Neither the PH nor the PH-Ct domains are autoinhibitory; yet, PIP 2 -containing membranes increase activity by 2 orders of magnitude 48,49 (Table 1). Remarkably, although EFA6 is strictly specific for Arf6 in solution, it activates both Arf1 and Arf6 efficiently on membranes, highlighting the fact that membranes can modulate the specificity of GEFs for their small GTPase substrates.…”

“…While the function of N-terminal regions has remained poorly understood, the regulatory mechanisms of PH domains have been extensively studied and this revealed an unexpected diversity of regulatory regimes. Although they display minimal homology to each other, the PH domains of cytohesins, 5 BRAG 29,46,47 and EFA6 48,49 all bind PI(4,5)P 2 phosphoinositides. Cytohesins also interact with PI(3,4,5)P 3 , with splice variants with a di-glycine instead of a tri-glycine in the b1-b2 loop of the PH domain showing a marked preference for this rare phosphoinositide.…”

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes

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