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EFA6, a sec7 domain‐containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization

EFA6, a sec7 domain‐containing exchange factor for ARF6, coordinates membrane recycling and actin... The EMBO Journal Vol.18 No.6 pp.1480–1491, 1999 EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization 1 2 ated in the control of various steps of endocytic trafficking Michel Franco , Peter J.Peters , (Novick and Zerial, 1997). More recently, small G proteins Joe ¨ lle Boretto, Elly van Donselaar , 3 4 belonging to the Rho subgroup have also been implicated Antonino Neri , Crislyn D’Souza-Schorey 5 in endocytic trafficking and are thought to coordinate the and Philippe Chavrier dynamics of the peripheral membrane system and the Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, cortical actin cytoskeleton (Lamaze et al., 1996; Murphy Case 906, 13288 Marseille Cedex 9, France, Department of et al., 1996). Cell Biology, University of Utrecht Medical School, 3584 Utrecht, In addition to Rab and Rho proteins, members of the The Netherlands, Servizio Ematologia, Ospedale Maggiore IRCCS, 4 ADP-ribosylation factor (ARF) subfamily of small G Milan, Italy and Department of Biological Sciences and the Walther Cancer Institute, University of Notre Dame, IN 46556, USA proteins are thought to function as regulators of membrane 1 trafficking. Several studies have demonstrated that activ- Present address: Institut de Pharmacologie Mole´culaire et Cellulaire ation of ARF1 is required for recruitment of the clathrin- du CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France coat adaptor AP1 and the non-clathrin coat COPI to Golgi membranes (Serafini et al., 1991; Orci et al., 1993; Corresponding author e-mail: chavrier@ciml.univ-mrs.fr Stamnes and Rothman, 1993; Traub et al., 1993), as well as recruitment of AP3 adaptor complex to endosomal We have identified a human cDNA encoding a novel membrane structures (Ooi et al., 1998). Hydrolysis of protein, exchange factor for ARF6 (EFA6), which bound GTP triggers coat disassembly allowing vesicle contains Sec7 and pleckstrin homology domains. EFA6 fusion with the acceptor membrane (Tanigawa et al., promotes efficient guanine nucleotide exchange on 1993). These observations, together with the fact that ARF6 and is distinct from the ARNO family of ARF1 ARF activation is coupled with phospholipase D (PLD) exchange factors. The protein localizes to a dense stimulation (Brown et al., 1993; Cockcroft et al., 1994) matrix on the cytoplasmic face of plasma membrane have led to the proposal that ARF1 is central to the process invaginations, induced on its expression. We show that of vesicle budding by coordinating coat recruitment and EFA6 regulates endosomal membrane recycling and membrane vesiculation with coat disassembly and vesicle promotes the redistribution of transferrin receptors to fusion (Bednarek et al., 1996). ARF6, on the other hand, the cell surface. Furthermore, expression of EFA6 the least conserved ARF protein, is associated with and induces actin-based membrane ruffles that are inhibi- controls the integrity of peripheral membranes and appears ted by co-expression of dominant-inhibitory mutant to cycle between the plasma membrane and a recycling forms of ARF6 or Rac1. Our results demonstrate that endosomal compartment depending on its nucleotide status by catalyzing nucleotide exchange on ARF6 at the (D’Souza-Schorey et al., 1995, 1998; Peters et al., 1995; plasma membrane and by regulating Rac1 activation, Radhakrishna and Donaldson, 1997). In Chinese hamster EFA6 coordinates endocytosis with cytoskeletal ovary (CHO) cells, ARF6Q67L, a GTP-bound and con- rearrangements. stitutively activated mutant of ARF6, is localized to the Keywords: actin/ARF6/endocytosis/guanine nucleotide plasma membrane where it induces extensive membrane exchange factor/Rac1 invaginations, decreases the rate of transferrin (Tfn) internalization, and triggers a redistribution of Tfn recep- tors (Tfn-Rs) to the cell surface (D’Souza-Schorey et al., 1995, 1998). In contrast, ARF6T27N is associated with a Introduction pericentriolar tubulovesicular compartment that contains Tfn-Rs and cellubrevin, and is morphologically reminis- A variety of macromolecules such as ligand-bound recep- cent of the recycling endosomal compartment (D’Souza- tors, plasma membrane proteins and solutes are internal- Schorey et al., 1995, 1998; Peters et al., 1995). ARF6 ized by clathrin-dependent or -independent endocytic has also been implicated in cortical actin cytoskeleton pathways and are delivered to sorting endosomes rearrangements. Expression of ARF6Q67L induces the (reviewed in Gruenberg and Maxfield, 1995). In the formation of actin-rich surface protrusions (Radhakrishna sorting compartment, ligands and receptors destined for et al., 1996; D’Souza-Schorey et al., 1997) that can be degradation are segregated from those that recycle back inhibited by co-expression of deletion mutants of POR1 to the plasma membrane (Ghosh et al., 1994; Hopkins (D’Souza-Schorey et al., 1997), a protein initially identi- et al., 1994). Recycling to the plasma membrane may fied as a partner of the Rho GTP-binding Rac1 (Van Aelst occur directly from the sorting endosome (Hopkins et al., et al., 1996), and which has been shown to interact with 1994; Daro et al., 1996) or via a pericentriolar recycling endosomal compartment (Hopkins et al., 1994). Several activated ARF6. The relationship between ARF6 function Ras-related small G proteins of the Rab subgroup that and actin cytoskeleton organization is further illustrated localize to early endocytic compartments have been implic- by the finding that inhibitors of actin polymerization, such 1480 © European Molecular Biology Organization EFA6, an exchange factor for ARF6 as cytochalasin D, block ARF6-mediated actin rearrange- observed that ARNO promotes exchange on ARF6 with ments and induce a redistribution of ARF6 from the a much lower efficiency compared with ARF1 (Frank plasma membrane to recycling endosomes (Radhakrishna et al., 1998). Finally, we and others have observed and Donaldson, 1997). The above observations have led that overexpression of ARNO and ARNO3 results in a to the speculation that ARF6 activation by GDP/GTP fragmentation of the Golgi apparatus and an inhibition of exchange takes place intracellularly on the recycling Golgi function without affecting the endocytic pathway endosome and drives the recycling of membrane to the (Franco et al., 1998; Monier et al., 1998). Therefore, cell surface. It has also been postulated that ARF6 controls the regulatory function of ARNO-like GEFs on ARF1 plasma membrane remodeling by coordinating membrane activation appears critical for maintaining the integrity of flow from a pericentriolar recycling compartment with Golgi structure and for vesicle transport within the Golgi cortical actin organization (Radhakrishna et al., 1996; complex. The above findings support the contention that D’Souza-Schorey et al., 1997; Radhakrishna and ARNO proteins function as GEFs preferentially for ARF1. Donaldson, 1997). There are several observations that In order to identify GEFs involved in ARF6 activation, support the conclusion that exit sites of recycling mem- we sought to isolate and characterize new Sec7 domain- branes at the cell surface are polarized and correlate with containing ARF GEFs that could promote nucleotide the formation of actin-based structures. For instance, exchange preferentially on ARF6. By analysis of the recycling Tfn-Rs are targeted to specialized regions of the database, we have identified a human cDNA encoding a plasma membrane at the leading lamellae of migrating protein that we have designated EFA6 (exchange factor fibroblasts and in membrane ruffling areas (Hopkins et al., for ARF6), which contains Sec7 and PH domains. EFA6 1994; Bretscher and Aguado-Velasco, 1998b). Whether promotes efficient guanine nucleotide exchange on ARF6 and how ARF6 regulation of membrane traffic is coordin- in comparison with ARF1. We have analyzed the subcellu- ated with its effect on actin rearrangements is still lar localization of EFA6 in transfected cells and investi- unknown. In addition, the site of activation of ARF6 by gated its effect on the peripheral plasma membrane/ nucleotide exchange has not yet been determined. endosome system. Our results suggest that EFA6 regulates Activation of the low molecular weight GTP-binding two coordinated activities that remodel the cell periphery: proteins is mediated by guanine nucleotide exchange (i) the outward flow of membrane from a recycling factors (GEFs), which catalyze the replacement of bound endosomal compartment; and (ii) a redistribution of the GDP with GTP. An important breakthrough toward the cortical actin cytoskeleton that may facilitate endosomal identification of GEFs acting on ARF proteins was the membrane delivery to the cell surface. Furthermore, EFA6 cloning of two related ARF1 GEFs encoding genes in expression had no effect on the integrity of the biosynthetic yeast, GEA1 and GEA2 (Peyroche et al., 1996). This and secretory apparatus. made possible the cloning of a human cDNA encoding ARNO (ARF nucleotide-binding-site opener), which pro- Results motes guanine nucleotide exchange on human ARF1 in vitro (Chardin et al., 1996). These three proteins share EFA6 is a GEF for ARF6 a conserved region of ~200 amino acids which bears 42% We searched the database for new Sec7 domain-related of sequence identity with a portion of the Saccharomyces sequences that might code for ARF6-specific GEF(s). cerevisiae SEC7 gene product (Franzusoff and Schekman, Using the amino acid sequence of the ARNO Sec7 domain 1989) and is essential for GEF activity (Chardin et al., as bait, a BLAST search (Altschul et al., 1990) identified 1996). Two additional Sec7 domain-containing GEFs have several sequences homologous to the Sec7 domain of been identified: cytohesin-1 and GRP-1/ARNO3, which ARNO, including a novel open reading frame encoding a show extensive homology with the predicted sequence of 645 amino acid protein (data not shown). This cDNA ARNO (Liu and Pohajdak, 1992; Klarlund et al., 1997; clone, originally called PSD and expressed in brain tissues Franco et al., 1998). Similarities between the three ARNO- (Perletti et al., 1997), encodes an ARF6 GEF (see below) family members are not restricted to the conserved Sec7 which we have named EFA6. Adjacent to the Sec7 domain, domain but extend to an N-terminal coiled-coil sequence the predicted sequence of EFA6 contains a PH domain as well as to a C-terminal pleckstrin homology (PH) (Figure 1A). Except for the conserved Sec7 and PH domain. This PH domain encodes the high affinity of the domains, EFA6 and ARNO-related GEFs are divergent in GEF for phosphoinositide-3,4,5-trisphosphates [PtdIns- primary sequence. As shown in Figure 1A, EFA6 contains (3,4,5)P ] (Klarlund et al., 1997) and mediates PtdIns- proline-rich regions at N- and C-terminal ends and a (4,5)P -dependent stimulation of ARNO GEF activity putative coiled-coil forming sequence at the C-terminus. (Chardin et al., 1996). Recently, we analyzed the substrate Amino acid sequence comparison revealed that the Sec7 specificity of the ARNO family members and showed that domain of EFA6 exhibits 28 and 30% amino acid identity all three proteins stimulate guanine nucleotide exchange with the Sec7 domains of Sec7p and ARNO, respectively, on ARF1 but are inefficient on ARF6, in vitro (Franco compared with 42% amino acid identity between the Sec7 et al., 1998). Klarlund et al. (1998) also reported that domain of ARNO and Sec7p. The three-dimensional GRP-1, the mouse homolog of human ARNO3, does not structure of the Sec7 domain of ARNO (Cherfils et al., promote exchange on ARF6. In addition, cytohesin-1 1998; Mossessova et al., 1998) has demonstrated the catalyzes nucleotide exchange on ARF1 and ARF3 but importance of two conserved motifs within the protein not on ARF5 (Meacci et al., 1997). These findings from sequence, motifs 1 and 2, which fold to form a hydrophobic different laboratories are in contrast with those of Frank groove that has been proposed to interact with the switch et al., who reported that ARNO can catalyze GDP/ regions of ARF1 (Be´raud-Dufour et al., 1998; Cherfils GTP exchange on ARF6 in vitro, although these authors et al., 1998; Mossessova et al., 1998). Interestingly, motifs 1481 M.Franco et al. (Figure 1C, closed triangles), whereas ARNO3 was unable to catalyze nucleotide exchange on myrARF6 under the same conditions. Conversely, nucleotide exchange on myrARF1 was rapid and reached a maximal rate within 5 min in the presence of ARNO3, while it was only weakly accelerated in the presence of EFA6 (Figure 1D). Analogous to ARNO-stimulated exchange on myrARF1 (Chardin et al., 1996), EFA6-catalyzed exchange on myrARF6 was unaffected by the addition of brefeldin A (data not shown). Motif 1 of all Sec7 domains identified so far has a conserved glutamate residue (Figure 1B, arrow). Substitution of this glutamate residue by a lysine in the Sec7 domain of ARNO (ARNO ) reduces its E156K nucleotide exchange activity by at least 1000-fold (Be´raud- Dufour et al., 1998; Mossessova et al., 1998). Similarly, EFA6 has lost GEF activity indicating that Glu242 E242K of EFA6 is essential for catalytic activity (Figure 1C). Finally, and as already shown for ARNO (Chardin et al., 1996; Franco et al., 1998), the isolated EFA6 Sec7 domain was sufficient to promote nucleotide exchange on ARF6 (data not shown). Therefore, we conclude that EFA6 belongs to the family of Sec7 domain containing ARF GEFs, and is the first ARF6-specific GEF identified so far. EFA6 localizes to the plasma membrane and induces invaginations To determine the cellular localization and function of EFA6, we epitope tagged the N-terminal end of EFA6 with the vesicular stomatitis virus glycoprotein (VSV-G) and transiently expressed the protein in TRVb-1 cells [a CHO-derived cell line overexpressing the human Tfn-R (McGraw et al., 1987)]. Confocal immunofluorescence microscopy analysis with anti-VSV-G-tag antibody showed that, analogous to the distribution of ARF6 (Figure 2A), EFA6 localized to membrane ruffles (arrows) and microvilli-like structures (arrowheads) that were induced at the periphery of TRVb-1 cells (Figure 2B). Noticeably, membrane ruffles were more enhanced in EFA6-expressing Fig. 1. EFA6 catalyzes guanine nucleotide exchange preferentially on ARF6. (A) Schematic representation of ARNO and EFA6. Proline-rich cells compared with ARF6-transfected cells. Membrane regions (Pro), Sec7 domains, PH domains and coiled-coil regions (c.c.) association of overexpressed ARF6 and EFA6 was con- are shown. Structure of deletion mutants EFA6ΔC and ΔSec7 is firmed by immunoblot analysis of membranes and cytosol depicted. (B) Alignment of sequences of two highly conserved motifs of transfected cells, fractionated by high-speed centrifug- of Sec7 domains from human EFA6 (Perletti et al., 1997), ARNO ation. As seen in Figure 2C, immunoblotting with anti- (Chardin et al., 1996), cytohesin 1 (Ch-1) (Liu and Pohajdak, 1992), ARNO3 (Franco et al., 1998), Arabidopsis GNOM (Shevell et al., hemagglutinin (HA) tag and anti-EFA6 antibodies revealed 1994), yeast Gea1 (Peyroche et al., 1996) and Sec7 (Achstetter et al., protein species of ~22 kDa (ARF6-HA) and ~70 kDa 1988) proteins. These motifs are thought to form a hydrophobic (VSV-G-EFA6), respectively. Both ARF6 and EFA6 were groove that comes in contact with the switch regions of ARF to form predominantly membrane-bound while only a small frac- the active site of the GEF (Be´raud-Dufour et al., 1998; Mossessova tion of these proteins was present in the soluble fraction. et al., 1998). The invariant glutamate residue (ARNO/Glu156 and EFA6/Glu242) which is involved in nucleotide exchange, is indicated In contrast, deletion of the 241 C-terminal amino acids of with an arrowhead. (C and D) Measurement of [ S]GTPγS binding to EFA6, including the PH domain, led to a protein that was myrARF6 (C) or myrARF1 (D) in the absence (circles) or the mostly detected in the cytosolic fraction (Figure 1C, VSV- presence of purified recombinant EFA6 (squares), EFA6 E242K G-EFA6ΔPH), suggesting that the PH domain is required (diamonds) or ARNO3 (triangles). Data shown are representative of three independent experiments. for membrane association of EFA6. We have studied the peripheral rearrangements induced 1 and 2 of the Sec7 domain of EFA6 are the least conserved on EFA6 expression at the ultrastructural level. Cryo- (Figure 1B), suggesting that its substrate specificity may immunogold electron microscopy using antibodies directed be distinct from ARNO-related Sec7 domains. against EFA6 revealed that the protein was localized to To determine the substrate specificity of EFA6, we the plasma membrane, on folds and invaginations, that in measured the ability of recombinant EFA6 to catalyze the cross sections appeared as membrane enclosed structures binding of guanosine 5-[γ-thio]triphosphate ([ S]GTPγS) with an electroluescent lumen (Figure 3A). These on recombinant myristoylated ARF6 (myrARF6) and structures resembled membrane alterations previously myrARF1. As shown in Figure 1C, EFA6-catalyzed bind- observed in ARF6Q67L-expressing cells (Peters et al., ing of [ S]GTPγS to myrARF6 was maximal after 10 min 1995) and that have been shown to be continuous with 1482 EFA6, an exchange factor for ARF6 Fig. 3. Localization of EFA6 by electron microscopy. TRVb-1 cells expressing VSV-G-EFA6 were fixed and processed for cryoimmuno- gold labeling. Sections were labeled with anti-EFA6 antibodies followed by protein A–10 nm gold. (A) In TRVb-1 cells overexpressing EFA6, the plasma membrane exhibited extensive membrane invaginations (i). Most of the cytoplasmic face of the plasma membrane was coated by an electron-dense matrix. EFA6 localizes exclusively to this dense matrix (arrowhead) and regions of the plasma membrane with no EFA6 are devoid of matrix (large arrow). Small arrows depict cross sections across tubular structures consisting of a sheath of electron-dense material surrounding a membrane-bound electroluescent lumen. Bar, 200 nm. (B) Regions of plasma membrane with little EFA6 labeling show a thinner matrix (large arrowheads). EFA6 labeling is excluded from coated pits (small arrowhead) and caveolae (arrows) as well as from the Golgi region of the cell. m, mitochondria; i, invaginations; n, nucleus; asterisk, untransfected cell. Bar, 500 nm. the extracellular milieu (D’Souza-Schorey et al., 1998). Strikingly, the cytoplasmic face of the plasma membrane and of EFA6-induced invaginations exhibited a homo- genous electron-dense matrix 50 nm thick. This dense Fig. 2. Localization of overexpressed EFA6 by indirect immuno- material was decorated with anti-EFA6 antibodies indicat- fluorescence microscopy. TRVb-1 cells were grown on coverslips and ing that EFA6 was present on this matrix. Regions of transfected with expression plasmids encoding HA-tagged ARF6 (A) plasma membrane showing high levels of labeling with or VSV-G-tagged EFA6 (B). Cells were fixed and labeled with either anti-HA (A) or anti-VSV-G (B) epitope antibody. Arrows indicate anti-EFA6 antibodies had a thicker matrix (Figure 3A, labeling of plasma membrane ruffles at cell edges. Arrowheads in B arrowheads). On the contrary, regions with little EFA6 point to labeling for EFA6 on microvilli at the dorsal cell surface. Bar, labeling had only a thin matrix (Figure 3B, large arrow- 10 μm. (C) The post-nuclear supernatants (PNS) of TRVb-1 cells heads) and regions with no labeling were devoid of matrix expressing either ARF6-HA, VSV-G-EFA6 or VSV-G-EFA6ΔPH were (Figure 3A, large arrow). Some of the EFA6-positive centrifuged at 100 000 g to give a membrane (P100) and a soluble fraction (S100). Fractions prepared from ARF6-HA-expressing cells structures appeared as cross-sections across sheathed were immunoblotted using anti-HA mouse monoclonal antibody cables with an electron-dense sheath surrounding a core (upper panel). Fractions from VSV-G-EFA6 or VSV-G-EFA6ΔPH- electroluescent lumen enclosed within a membrane expressing cells were incubated with rabbit anti-EFA6 polyclonal (Figure 3A, small arrows). This electron-dense matrix antibodies (lower panel). Molecular weights (kDa) of protein markers are indicated. M.Franco et al. Overexpression of EFA6 inhibits Tfn uptake and affects the distribution of the Tfn-R Overexpression of ARF6Q67L, a mutant locked in the GTP-bound conformation decreases the rate of Tfn uptake in TRVb-1 cells (D’Souza-Schorey et al., 1995). Since overexpression of EFA6 should increase the pool of ARF6 in a GTP-bound state, we anticipated that the Tfn cycle should be similarly affected in EFA6-transfected cells. To this end, we examined the internalization of fluoresceinated human Tfn in TRVb-1 cells expressing HA-tagged ARF6Q67L. In untransfected cells, Tfn internalized for 20 min at 37°C, accumulated in a single discrete patch in the pericentriolar region of the cells as well as in vesicular structures spread throughout the cytoplasm. Similar to that observed in cells expressing ARF6(Q67L) (Figure 5A), uptake of Tfn was dramatically inhibited in EFA6- expressing cells with little or no accumulation of Tfn after a 20 min incubation (Figure 5B). Conversely, expression of myc-ARNO3, an ARF1 GEF, had no effect on Tfn uptake (Figure 5C), indicating that the inhibitory effect of EFA6 on the Tfn cycle is mediated by activation of Fig. 4. Plasma membrane association of nucleotide exchange-defective ARF6-in vivo. Cryoimmunogold labeling with antibodies mutant of EFA6. TRVb-1 cells expressing EFA6ΔSec7, which presents directed against Tfn-R revealed that in mock-transfected a deletion of the entire Sec7 nucleotide exchange domain, were fixed TRVb-1 cells most of the Tfn-R label (74%) resides and processed for cryoimmunogold labeling as described in the legend of Figure 3. (A) In low-level expressor, EFA6ΔSec7 is localized to the intracellularly in a tubular and vesicular compartment in plasma membrane that does not show morphological alteration and is the pericentriolar region (Table I). On the contrary, a devoid of matrix. Labeling is also detected on intracellular endocytic majority of the Tfn-Rs (79%) were detected on the plasma structures (e). (B) In high level expressing cell, EFA6ΔSec7 is membrane and on plasma membrane folds and vaginations associated with the plasma membrane on thin and long extensions. of TRVb-1 cells expressing EFA6 (Table I). Taken together, Plasma membrane appears uncoated. EFA6ΔSec7 is also detected in the cytosol. e, endosome; G, Golgi apparatus; m, mitochondria; these findings indicate that expression of EFA6 induces a n, nucleus; p, plasma membrane. Bar, 200 nm. redistribution of Tfn-Rs from the perinuclear endosomal recycling compartment to plasma membrane. In order to investigate whether the effects of EFA6 may represent polymerized actin filaments beneath the expression were restricted to the peripheral plasma mem- surface of plasma membrane invaginations (see below). brane/endosomal system, we analyzed the intracellular As shown in Figure 3B, EFA6 label was excluded from distribution of markers of the secretory and late endocytic clathrin coated pit and caveolae profiles at the plasma pathways in EFA6-expressing cells. The distribution of membrane and from the Golgi. p23 [a cis-Golgi network-associated protein (Rojo et al., In order to investigate whether these morphological 1997)] and of sec61β (a resident ER marker) was not effects were dependent on guanine nucleotide exchange modified by EFA6 overexpression (data not shown). catalyzed by EFA6, TRVb-1 cells overexpressing a GEF- Furthermore, the distribution of the lysosomal marker defective mutant form of EFA6 were analyzed by cryo- LAMP-1 remained unperturbed in EFA6-expressing cells immunogold electron microscopy. Figure 4 shows labeling (not shown). Thus, expression of EFA6 results in a for EFA6 in TRVb-1 cells expressing low (Figure 4A) or specific and profound remodeling of the peripheral plasma high levels (Figure 4B) of EFA6ΔSec7, a Sec7 domain membrane/endosomal system. deletion mutant. In low-level expressors, most of the EFA6 labeling was detected at the plasma membrane, which EFA6 overexpression induces rearrangements of exhibited normal unperturbed morphology and was devoid the actin cytoskeleton of matrix (Figure 4A). Occasionally, EFA6ΔSec7 was also In addition to the regulation of peripheral membrane associated with intracellular endocytic structures but was trafficking, ARF6 has also been demonstrated to regulate absent from Golgi, nucleus and ER profiles. At a higher the assembly and organization of the cortical actin cyto- level of expression (Figure 4B), EFA6ΔSec7 was localized skeleton (Radhakrishna et al., 1996; D’Souza-Schorey to the plasma membrane on thin and long extensions that et al., 1997). These studies, together with our observation were morphologically distinct from the invaginations seen that a dense cytoskeletal-like matrix accumulated at the in wild-type EFA6-expressing cells. The plasma membrane cell surface upon EFA6 expression, prompted us to investi- of EFA6ΔSec7-expressing cells was devoid of matrix. gate the consequences of EFA6 expression on the actin Some EFA6ΔSec7 label was also detected in the cytosol. cytoskeleton in HeLa cells. We have found that cells Taken together, these findings indicate that EFA6 localizes expressing wild-type EFA6 showed a marked accumulation predominantly to the plasma membrane and that this of F-actin in peripheral membrane ruffles (Figure 6A). distribution is independent of its activity as an ARF6- EFA6-induced ruffles at the peripheral edges of the cell specific GEF. Plasma membrane association of cata- were more prominent compared with actin rearrangements lytically active EFA6 induces membrane invaginations observed in ARF6Q67L-expressing cells (not shown). and the formation of a dense matrix. Double labeling for EFA6 with anti-VSV-G tag antibody 1484 EFA6, an exchange factor for ARF6 Table I. Quantitation of the intracellular distribution of transferrin receptors in control and EFA6-overexpressing TRVb-1 cells TRVb-1 cells Plasma membrane Pericentriolar vesicles and invaginations and tubules Mock-transfected 26% 74% EFA6-transfected 79% 21% TRVb-1 cells on monolayers were mock-transfected or transfected with EFA6-expressing vector, fixed and processed for cryoimmunogold labeling and labeled with anti-EFA6 antiserum followed by protein A– 10 nm gold. The data presented represent percentages of 10 nm gold particle count of the total number of gold particles per cell, obtained from 40 random cross-sections of cells. First, expression of the GEF-defective mutant, EFA6 E242K (see Figure 1C), did not induce membrane ruffling, but instead led to the formation of numerous actin-rich mem- brane extensions (Figure 6C, arrowheads). Similarly, expression of EFA6ΔSec7 also resulted in plasma mem- brane extensions (Figure 6E and F). EFA6ΔSec7 and EFA6 localized predominantly at the cell surface, E242K confirming the ultrastructural analysis (Figure 4). Expres- sion of a C-terminal deletion mutant, EFA6ΔC, generated by deletion of the 124 C-terminal amino acids of EFA6, including a putative coiled-coil sequence and proline-rich region (Figure 1A), abolished the capacity of EFA6 to trigger F-actin reorganization (Figure 6G and H). Furthermore, deletion of this C-terminal region in EFA6 (EFA6 ΔC) also abolished membrane E242K E242K extension formation (Figure 6I and J), suggesting that the C-terminal domain may be required for interaction with other factors required for cytoskeletal rearrangements. Altogether, these results indicate that EFA6-induced mem- brane ruffling depends on a functional Sec7 domain and an intact C-terminal end. The Rho GTP-binding protein, Rac1, is involved in EFA6-induced cytoskeletal rearrangements Co-expression of EFA6 and ARF6T27N abolished EFA6- mediated membrane ruffling in doubly transfected HeLa cells (Figure 7A and B). Interestingly, these cells exhibited membrane extensions similar to those observed in cells expressing EFA6 mutants defective in GEF activity. These extensions were absent in HeLa cells expressing only ARF6T27N (not shown). Since the Rho-family proteins Rac1 and CDC42 regulate the organization of actin filaments at the cell cortex to Fig. 5. Transferrin accumulation is blocked in EFA6-expressing cells. induce membrane ruffling and filopodium formation, TRVb-1 cells expressing ARF6Q67L-HA (A), VSV-G-EFA6 (B)orthe respectively, we investigated the effects of the dominant- ARF1-GEF myc-ARNO3 (C) were incubated at 37°C with iron- negative mutants of these proteins on EFA6-induced saturated fluoresceinated human transferrin (FITC–Tfn) for 20 min, ruffling. When EFA6 was co-expressed with GDP-bound fixed and processed for immunofluorescence microscopy. ARF6Q67L, EFA6 and ARNO3 were detected with anti-HA, anti-VSV-G and with Rac1T17N, membrane ruffling was inhibited and co- anti-myc tag antibodies, respectively. Panels show superimposition of transfected cells exhibited numerous F-actin rich mem- FITC–Tfn with epitope-tagged protein labeling. In untransfected cells, brane extensions (Figure 7C and D) that were not FITC–Tfn is predominantly present in the pericentriolar recycling detected in cells transfected with Rac1N17 only (not compartment. In ARF6Q67L- (A) or EFA6-expressing (B) cells there is no accumulation of FITC–Tfn. In contrast, overexpressed ARNO3 shown). On the contrary, co-expression of EFA6 with (C) has no effect on Tfn distribution. Bar, 10 μm. GDP-bound CDC42T17N had no effect on EFA6- induced membrane ruffling (Figure 7E and F, arrows). In addition, expression of CDC42N17 had no effect on revealed that EFA6 co-localized with F-actin in the mem- membrane extensions induced upon expression of the brane ruffles (Figure 6B). To determine structural domains GEF-defective mutants, EFA6 and EFA6ΔSec7 (data E242K of EFA6 involved in peripheral rearrangements, various not shown). These results suggest that EFA6-induced mutants of EFA6 were transiently expressed in HeLa cells. membrane ruffling is independent of CDC42 and that 1485 M.Franco et al. on Rac1 in vitro, as shown using the nucleotide exchange assay described above (data not shown). The effect of EFA6 on Tfn cycling is independent of EFA6-mediated cytoskeletal reorganization We were interested in determining whether the regulatory activity of EFA6 on membrane dynamics was dependent on EFA6-induced actin cytoskeleton reorganization. To this end, TRVb-1 cells expressing EFA6ΔC, which does not affect F-actin distribution, were incubated with fluoresceinated Tfn for 20 min. As shown above for wild-type EFA6, overexpression of EFA6ΔC prevented intracellular accumulation of Tfn (compare Figure 5A with 8A). In contrast, cells expressing EFA6 ΔC, a E242K double mutant defective for GEF activity, did not perturb Tfn endocytosis and the distribution of internalized Tfn was similar to that observed in untransfected cells (Figure 8B). Thus, the effects of EFA6 on Tfn cycling requires ARF6 activation by a functional Sec7 domain but is independent of EFA6-induced actin cytoskeleton remodeling which appears to require the C-terminal end of EFA6. Discussion In contrast to the other ARF family members that are associated with the secretory pathway (Donaldson and Klausner, 1994; P.J.Peters, unpublished observations), ARF6 is associated with the peripheral plasma membrane/ endosome system where it regulates membrane trafficking from an internal fusion competent recycling compartment to the plasma membrane (D’Souza-Schorey et al., 1995, 1998; Peters et al., 1995; Radhakrishna and Donaldson, 1997). Overexpression studies have led to the proposal that ARF6 activation by GDP/GTP exchange is required for the delivery of membrane to the cell surface (Radhakrishna and Donaldson, 1997; D’Souza-Schorey et al., 1998). Recycling to the surface would be facilitated by remodeling of the underlying actin cytoskeleton that is also dependent on ARF6 activation (D’Souza-Schorey et al., 1997; Radhakrishna and Donaldson, 1997). Since ARF6T27N, a mutant thought to be locked in the GDP- bound conformation, localized to the recycling compart- ment, it was speculated that ARF6 activation takes place intracellularly, on the recycling compartment itself or on putative recycling vesicle intermediates en route to the cell surface (Radhakrishna and Donaldson, 1997; D’Souza- Fig. 6. Effects of EFA6 and EFA6 mutants on actin cytoskeleton rearrangements. HeLa cells were transfected VSV-G-tagged wild-type Schorey et al., 1998). However, this model was still EFA6 (A, B), the nucleotide exchange-defective mutants EFA6 E242K speculative due to the lack of an ARF6-GEF. In the (C, D) and EFA6ΔSec7 (E, F), the C-terminal deletion mutant present study, we describe a new Sec7 domain containing EFA6ΔC(G, H) and the double mutant EFA6 ΔC(I, J). After E242K GEF, EFA6, which promotes efficient GDP/GTP exchange 40 h, cells were fixed and labeled with Texas Red-conjugated phalloidin to visualize actin rearrangements (left panels). Right panels on ARF6. In addition to EFA6, mammalian cells express show the superimposition of the phalloidin labeling (red) and anti- three highly related GEFs, ARNO, cytohesin-1 and VSV-G staining (green). Cells overexpressing EFA6 exhibit increased ARNO3/GRP-1, and a 200-kDa GEF, all of which contain staining for F-actin in membrane ruffles (A). EFA6 colocalizes with a Sec7 domain and promote preferential GDP/GTP F-actin in these ruffles (B). Overexpression of GEF-defective mutant exchange on ARF1 (and ARF3) (Chardin et al., 1996; forms, EFA6 (C, D) and EFA6ΔSec7 (E, F), induces the E242K extension of filopodia. Deletion of the C-terminal region of EFA6 Meacci et al., 1997; Morinaga et al., 1997; Franco et al., abolishes EFA6-induced membrane ruffling (G, H) and EFA6 - E242K 1998; Klarlund et al., 1998). Thus, EFA6 is the first mediated filopodia extension (I, J). Bar, 10 μm. ARF6-specific GEF reported to date. We show that exo- genous EFA6 is associated with a dense matrix underlying EFA6 triggers the activation of Rac1 which is required plasma membrane domains that are extensively invagin- for EFA6-induced membrane ruffling. However, it should ated. Plasma membrane alterations induced on EFA6 be noted that EFA6 did not catalyze nucleotide exchange expression are accompanied by a reorganization of cortical 1486 EFA6, an exchange factor for ARF6 Fig. 7. Rac1 is involved in EFA6-induced actin rearrangements. HeLa cells were co-transfected with constructs expressing VSV-G-EFA6 in combination with ARF6T27N-HA (A, B), myc-Rac1T17N (C, D) or myc-CDC42T17N (E, F). Left panels show Texas Red–phalloidin staining of F-actin. Right panels show superimposition of Texas Red–phalloidin labeling and anti-VSV-G tag labeling. In contrast to EFA6 which induces membrane ruffling (see Figure 9A and B), coexpression of EFA6 together with ARF6T27N (A, B) or Rac1T17N (C, D) leads only to filopodia extension. Cdc42T17N does not affect EFA6-induced membrane ruffling (E and F, arrows). Bar, 10 μm. actin, with the formation of membrane ruffles. The latter remodeling by catalyzing GDP/GTP exchange on ARF6 response appears to involve activation of the Rho protein, and by activating Rac1, respectively. Rac1. Consistent with previous observations in cells At the ultrastructural level, overexpressed EFA6 was expressing a constitutively activated mutant of ARF6, found exclusively on the cytoplasmic side of the plasma (ARFQ67L), EFA6 expression affects the Tfn cycle and membrane and on numerous plasma membrane invagin- is accompanied by a redistribution of Tfn-R from the ations that were induced on EFA6 expression. Furthermore, recycling compartment to the cell periphery. These results the effects of EFA6 on membrane traffic and organelle suggest that EFA6 coordinates membrane and actin structure were restricted to the endosomal and plasma 1487 M.Franco et al. intermediates or the recycling endosome) are incompetent for fusion with the plasma membrane until EFA6 converts ARF6 into its GTP-bound active conformation. The initial step of this cascade requires (i) the close proximity of the two membrane systems and (ii) the activation of ARF6 by EFA6 at the interface between these two membrane systems. Regulation of this step could be achieved by controlling EFA6 recruitment to the cell periphery. The PH domain of EFA6 may play an important role in this process since PH domains have been shown to be required for translocation of protein to the plasma membrane (Lemmon et al., 1997). In agreement with this hypothesis, we have observed that deletion of the PH domain of EFA6 leads to a protein which is mostly cytosolic. Overexpres- sion of EFA6 should induce a massive accumulation of ARF6-GTP at the plasma membrane. Normally, GTP hydrolysis that probably involves a plasma membrane- associated GAP should allow the return of GDP-bound ARF6 to recycling endosomes via the endocytic pathway (D’Souza-Schorey et al., 1998), and EFA6 may be released from the membrane into the cytosol. However, in EFA6- expressing cells, GAP-stimulated GTP hydrolysis may become limiting, preventing membrane internalization and thereby blocking Tfn-R internalization and Tfn uptake as also observed in ARF6Q67L-expressing cells. An interesting finding from the ultrastructural analysis is that EFA6 induces the formation of and is associated with a dense matrix that coats the cytoplasmic face of plasma membrane invaginations. We have also observed that EFA6 induces the projection of actin-based membrane ruffles in which F-actin and EFA6 co-localize. It is possible Fig. 8. EFA6-mediated inhibition of Tfn accumulation does not require that the dense matrix observed by electron microscopy is EFA6-induced actin cytoskeleton reorganization but requires a composed of actin filaments polymerized beneath the functional Sec7 domain. TRVb-1 cells expressing VSV-G-EFA6ΔC(A) plasma membrane. Plasma membrane-associated matrix or VSV-G-EFA6 ΔC(B) were incubated for 20 min with E242K and invaginations, as well as membrane ruffling formation fluoresceinated transferrin as described in Figure 5. Panels show the superimposition of fluoresceinated Tfn with VSV-G labeling. In depends on a functional sec7 domain. In contrast, GEF- EFA6ΔC-expressing cells (A), there is no accumulation of FITC–Tfn. defective mutant forms of EFA6 lead to the projection of In contrast, overexpression of GEF-defective EFA6 ΔC (B) has no E242K long actin-rich membrane extensions. Consistent with this effect on Tfn uptake. Bar, 10 μm. finding, extensions are also induced upon co-expression of EFA6 and ARF6T27N that should result in dominant inhibition of EFA6 exchange activity. Moreover, co- membrane system. Indeed, we observed that EFA6 perturbs expression of EFA6 with Rac1T17N, a dominant-inhibi- the Tfn cycle and redistributes TfnR-containing compart- tory mutant of Rac1, also results in membrane extension. ments to the cell surface without affecting the morphology Finally, deletion of the carboxy end of EFA6 abolished of the Golgi apparatus, endoplasmic reticulum and EFA6-induced actin cytoskeleton reorganization. Based LAMP-1-positive lysosomes. The observation that the on these observations, it is tempting to speculate how distribution of EFA6 is at the plasma membrane has some EFA6 could control actin cytoskeleton reorganization via important implications for the GDP/GTP cycle of ARF6. the coordinated activation of ARF6 and Rac1 (Figure One possibility was that EFA6-catalyzed exchange takes 9). We propose that Sec7 domain-catalyzed nucleotide place on the recycling compartment where GDP-bound exchange on ARF6 allows the engagement of specific ARF6 accumulates (Peters et al., 1995; D’Souza-Schorey effectors such as PLD, which is stimulated by ARF6 et al., 1998). However, according to this proposal, GEF- in vitro (Massenburg et al., 1994) and in vivo during defective mutants such as EFA6 or EFA6ΔSec7 exocytosis in chromaffin cells (Caumont et al., 1998). E242K should accumulate on recycling endosome together with PLD may be involved in membrane dynamics at the GDP-bound ARF6. Cryoimmunogold electron microscopy interface between plasma membrane and recycling endo- and confocal microscopy analyses contradict this predic- some by stimulating the production of fusogenic lipids. tion, since overexpressed EFA6 and EFA6ΔSec7 have On the other hand, GTP-bound ARF6 has been shown to E242K the same distribution as wild-type EFA6 at the plasma interact with POR1 (D’Souza-Schorey et al., 1997), a membrane, demonstrating that plasma membrane localiz- protein initially identified as a Rac1 effector involved in ation of EFA6 does not require GDP/GTP exchange on membrane ruffling (Van Aelst et al., 1996). Since ARF6. Therefore, our data favor the alternative possibility Rac1T17N does not inhibit ARFQ67L-induced actin that EFA6 activates ARF6 at the plasma membrane. In rearrangement (D’Souza-Schorey et al., 1997), it is likely this model, recycling membranes (recycling transport that ARF6 and Rac1 are not engaged in a linear pathway 1488 EFA6, an exchange factor for ARF6 et al., 1997), and together with the Rac1-specific GEF, Tiam-1, it affects neuronal morphology (Luo et al., 1996; Kozma et al., 1997; Leeuwen et al., 1997). While ARF6 is ubiquitously expressed (Tsuchiya et al., 1991; Yang et al., 1998), the human PSD gene coding for EFA6 is mainly expressed in brain (Perletti et al., 1997). Whether EFA6, by its coordinated action on ARF6 and Rac1, may be involved in neuronal secretion or axonal elongation is presently unknown, but would be an interesting subject of investigation. By screening for EFA6-like genes, we have isolated a cDNA encoding a Sec7 and PH domain- containing protein showing high similarity with the corresponding domains of EFA6 (M.Franco and B.Chavrier, unpublished). Interestingly, this EFA6-like gene is expressed in a wide range of tissues. The availabil- ity of these cDNAs will help in clarifying the function of EFA6-like GEFs in various cell types. Fig. 9. Model of EFA6-regulated actin reorganization. We propose that targeting of the PH domain of EFA6 to the plasma membrane allows Sec7 domain-catalyzed nucleotide exchange on ARF6. GTP-bound ARF6 interacts with and activates specific downstream effectors at the Materials and methods plasma membrane. Activation of PLD leads to the generation of fusogenic lipids involved in the fusion of recycling membranes with Cells and antibodies the plasma membrane. In parallel, EFA6 allows Rac1 activation by TRVb-1 cells, a CHO-derived cell line overexpressing the human recruiting a Rac1-specific GEF. The ARF6 and Rac1 pathways transferrin receptor (McGraw et al., 1987), were grown in Ham’s F-12 converge at the level of POR1 that interacts with both GTP-bound medium, 5% fetal calf serum (FCS), penicillin/streptomycin, and 100 ARF6 and Rac1 to control membrane ruffling. Recycling of membrane μg/ml G418. HeLa cells were grown in Dulbecco’s modified essential occurs at discrete sites of the plasma membrane that coincide with medium supplemented with 10% FCS and antibiotics. areas of membrane ruffling. The different domains of EFA6 are The following antibodies were used for the studies described: mouse schematically represented as in Figure 1A. monoclonal antibody (mAb) against VSV-G epitope (clone P5D4, Boehringer Mannheim) and rat mAb against HA epitope (clone 3F10, Boehringer Mannheim); mouse mAb against myc epitope (clone 9E10; (ARF6-GTP promoting Rac1 activation), but are rather Evan et al., 1985); mouse mAb anti-Tfn-R (clone H68.4; Zymed, CA); functioning on parallel pathways that may converge at the rabbit antiserum against p23 (Rojo et al., 1997); rabbit antiserum against level of POR1. Noticeably, ARF6Q67L stimulates actin Sec61β (provided by R.Hendriks, ZMBH, Heidelberg, Germany); rabbit antiserum against LAMP-1 (provided by S.Meresse, CIML, Marseille, reorganization but does not induce the formation of the France); and anti-EFA6 rabbit antiserum raised against the purified dense matrix we observed in EFA6-expressing cells (Peters recombinant protein. et al., 1995; D’Souza-Schorey et al., 1998). An interpret- ation of these remarkable phenotypic differences may be Construction of EFA6 expression plasmids that the mere engagement of the ARF6-dependent pathway EFA6 was cloned from a human brain cDNA library (Clontech) by high fidelity PCR using two oligonucleotide primers; 5EFA6 primer by ARF6Q67L, in the absence of Rac1 engagement, leads (5-GATCGATCCATATGCCTCTCAAGTCACCTG-3), which con- only to a partial response. Since EFA6 does not catalyze tains a NdeI linker, the ATG start codon and 5 nucleotides of EFA6, nucleotide exchange on the Rho-family proteins (M.Franco and 3EFA6 primer (5-GGGCGGCGGAAGCCCTGAGTCAACGGAT- and P.Chavrier, unpublished observations), we propose CCTAAGTACA-3), which contains a BamHI linker, TAG stop codon and 3 complementary nucleotide residues of EFA6. Primer sequences that EFA6-dependent Rac1 activation is mediated by a encoding EFA6 were designed from the published human PSD sequence Rac1-specific GEF. A possibility is that the C-terminus of (Perletti et al., 1997). The PCR product was inserted into pGEM1 and EFA6 which contains coiled-coil and Pro-rich regions pSRα expression vectors with a N-terminal VSV-G tag using NdeI and known to participate in protein–protein interactions, regu- BamHI restriction sites. lates this Rac1 GEF. Hence, deletion of the C-terminus EFA6 was constructed by two-stage overlap-extension PCR. E242K The first-stage reaction products were generated using complementary of EFA6 by prevention of Rac1 engagement would inhibit overlapping end primers containing an oligonucleotide-directed point the actin-based ruffling response. Exocytosis of recycling mutation; the N-terminal product was generated with the 5EFA6 primer membrane occurs at the leading edge of migrating cells described above and the primer 5-CCTGGGTCTTACCCATTAAG-3 (Hopkins et al., 1994; for a review see Bretscher and (EFA6E242K-1). The C-terminal product was generated with the primer 5-CTTAATGGGTAAGACCCAGG-3 (EFA6E242K-2) and the Aguado-Velasco, 1998a). Recently, EGF treatment of KB 3EFA6 primer. EFA6 cDNA was used as template. Second-stage PCR cells has been shown to induce the formation of membrane was carried out with first-stage reaction products to anneal the overlapping ruffles that are enriched in Tfn-Rs, and Tfn-Rs are also ends, followed by amplification. The full-length EFA6E242K PCR targeted to Rac1-induced membrane ruffles in these cells product was cloned in the NdeI and BamHI restriction sites of (Bretscher and Aguado-Velasco, 1998b). We believe that, pGEM1VSV-G and pSRαVSV-G expression vectors. EFA6ΔC and EFA6 ΔC were constructed by PCR amplification by its coordinated actions on both Rac1- and ARF6-driven E242K of corresponding EFA6 and EFA6 cDNAs in pGEM1VSV-G using E242K pathways, EFA6 allows to couple membrane ruffling and the 5EFA6 primer and the primer 5-TGTACTAAAGCTTCTACTGGG- membrane fusion at discrete sites of the plasma membrane AGAGGCGGGTGG-3 (EFA6ΔC) obtained by substituting glutamate in a polarized fashion. 522 of EFA6 with a stop codon. Similarly, EFA6ΔPH was obtained by PCR using the 5EFA6 primer and the primer 5-TGTCTAAAGCTTTC- ARF6 has been shown to co-purify along with AGCCCCGCTTGCCCCGAGG-3 (EFA6ΔPH) substituting tryptophane chromaffin granules (Galas et al., 1997) and controls 404 of EFA6 with a stop codon. EFA6Δ was constructed by Sec7 exocytosis during regulated secretion in chromaffin cells two-stage PCR amplification of EFA6 with the 5EFA6 primer and (Caumont et al., 1998). Rac1 is also implicated in regulated the primer 5-GTCGGCCAACTCAGACAGGCTGTCCAGCTCTGA- exocytosis in mast cells (Norman et al., 1996; Guillemot GTC-3 (EFA6-S156), and with the primer 5-GACTCAGAGCTGGAC- 1489 M.Franco et al. AGCCTGTCTGAGTTGGCCGAC-3 (EFA6-S340) and the 3EFA6 Acknowledgements primer. These PCR products were then amplified with the 5 and 3 We are indebted to J.Gruenberg, R.Hendriks and S.Me´resse for generously primers of EFA6 to create an internal deletion from amino acids 157 to providing antibodies for this study. We are grateful for the skillful work 341. The resulting PCR product was subcloned into the NdeI and BamHI of the dark room assistance at Utrecht University. P.Chardin, M.Chabre restriction sites of pGEM1VSV-G. and M.Zerial are thanked for helpful comments on this manuscript. We Expression plasmids used in this study encoding ARF6-HA, thank B.Antonny for sharing results prior to publication. This work was ARF6Q67L-HA, ARF6T27N-HA (Peters et al., 1995), mycRac1N17, supported by INSERM and CNRS institutional funding and specific mycCDC42N17 (Dutartre et al., 1996) and mycARNO3 (Franco et al., grants from La Ligue Nationale contre le Cancer and Le Groupement 1998) were as described previously. des Entreprises Franc¸aises dans la Lutte contre le Cancer (to P.C.), by grants from the Dutch Cancer Society (to P.J.P.) and a grant from the [ S]GTPγS binding assay Associazione Italiana Ricerca sul Cancro (to A.N.). M.F. was supported Myristoylated ARF1 and ARF6 were produced and purified as described by a postdoctoral fellowship from the Fondation pour la Recherche previously (Franco et al., 1995). Recombinant EFA6 and ARNO3 were Me´dicale. 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Received September 7, 1998; revised January 19, 1999; Meacci,E., Tsai,S.C., Adamik,R., Moss,J. and Vaughan,M. (1997) accepted January 20, 1999 Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor. Proc. Natl Acad. Sci. USA, 94, 1745–1748. Monier,S., Chardin,P., Robineau,S. and Goud,B. (1998) Overexpression of the ARF1 exchange factor ARNO inhibits the early secretory http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

EFA6, a sec7 domain‐containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization

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Springer Journals
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Copyright © European Molecular Biology Organization 1999
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0261-4189
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10.1093/emboj/18.6.1480
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Abstract

The EMBO Journal Vol.18 No.6 pp.1480–1491, 1999 EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization 1 2 ated in the control of various steps of endocytic trafficking Michel Franco , Peter J.Peters , (Novick and Zerial, 1997). More recently, small G proteins Joe ¨ lle Boretto, Elly van Donselaar , 3 4 belonging to the Rho subgroup have also been implicated Antonino Neri , Crislyn D’Souza-Schorey 5 in endocytic trafficking and are thought to coordinate the and Philippe Chavrier dynamics of the peripheral membrane system and the Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, cortical actin cytoskeleton (Lamaze et al., 1996; Murphy Case 906, 13288 Marseille Cedex 9, France, Department of et al., 1996). Cell Biology, University of Utrecht Medical School, 3584 Utrecht, In addition to Rab and Rho proteins, members of the The Netherlands, Servizio Ematologia, Ospedale Maggiore IRCCS, 4 ADP-ribosylation factor (ARF) subfamily of small G Milan, Italy and Department of Biological Sciences and the Walther Cancer Institute, University of Notre Dame, IN 46556, USA proteins are thought to function as regulators of membrane 1 trafficking. Several studies have demonstrated that activ- Present address: Institut de Pharmacologie Mole´culaire et Cellulaire ation of ARF1 is required for recruitment of the clathrin- du CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France coat adaptor AP1 and the non-clathrin coat COPI to Golgi membranes (Serafini et al., 1991; Orci et al., 1993; Corresponding author e-mail: chavrier@ciml.univ-mrs.fr Stamnes and Rothman, 1993; Traub et al., 1993), as well as recruitment of AP3 adaptor complex to endosomal We have identified a human cDNA encoding a novel membrane structures (Ooi et al., 1998). Hydrolysis of protein, exchange factor for ARF6 (EFA6), which bound GTP triggers coat disassembly allowing vesicle contains Sec7 and pleckstrin homology domains. EFA6 fusion with the acceptor membrane (Tanigawa et al., promotes efficient guanine nucleotide exchange on 1993). These observations, together with the fact that ARF6 and is distinct from the ARNO family of ARF1 ARF activation is coupled with phospholipase D (PLD) exchange factors. The protein localizes to a dense stimulation (Brown et al., 1993; Cockcroft et al., 1994) matrix on the cytoplasmic face of plasma membrane have led to the proposal that ARF1 is central to the process invaginations, induced on its expression. We show that of vesicle budding by coordinating coat recruitment and EFA6 regulates endosomal membrane recycling and membrane vesiculation with coat disassembly and vesicle promotes the redistribution of transferrin receptors to fusion (Bednarek et al., 1996). ARF6, on the other hand, the cell surface. Furthermore, expression of EFA6 the least conserved ARF protein, is associated with and induces actin-based membrane ruffles that are inhibi- controls the integrity of peripheral membranes and appears ted by co-expression of dominant-inhibitory mutant to cycle between the plasma membrane and a recycling forms of ARF6 or Rac1. Our results demonstrate that endosomal compartment depending on its nucleotide status by catalyzing nucleotide exchange on ARF6 at the (D’Souza-Schorey et al., 1995, 1998; Peters et al., 1995; plasma membrane and by regulating Rac1 activation, Radhakrishna and Donaldson, 1997). In Chinese hamster EFA6 coordinates endocytosis with cytoskeletal ovary (CHO) cells, ARF6Q67L, a GTP-bound and con- rearrangements. stitutively activated mutant of ARF6, is localized to the Keywords: actin/ARF6/endocytosis/guanine nucleotide plasma membrane where it induces extensive membrane exchange factor/Rac1 invaginations, decreases the rate of transferrin (Tfn) internalization, and triggers a redistribution of Tfn recep- tors (Tfn-Rs) to the cell surface (D’Souza-Schorey et al., 1995, 1998). In contrast, ARF6T27N is associated with a Introduction pericentriolar tubulovesicular compartment that contains Tfn-Rs and cellubrevin, and is morphologically reminis- A variety of macromolecules such as ligand-bound recep- cent of the recycling endosomal compartment (D’Souza- tors, plasma membrane proteins and solutes are internal- Schorey et al., 1995, 1998; Peters et al., 1995). ARF6 ized by clathrin-dependent or -independent endocytic has also been implicated in cortical actin cytoskeleton pathways and are delivered to sorting endosomes rearrangements. Expression of ARF6Q67L induces the (reviewed in Gruenberg and Maxfield, 1995). In the formation of actin-rich surface protrusions (Radhakrishna sorting compartment, ligands and receptors destined for et al., 1996; D’Souza-Schorey et al., 1997) that can be degradation are segregated from those that recycle back inhibited by co-expression of deletion mutants of POR1 to the plasma membrane (Ghosh et al., 1994; Hopkins (D’Souza-Schorey et al., 1997), a protein initially identi- et al., 1994). Recycling to the plasma membrane may fied as a partner of the Rho GTP-binding Rac1 (Van Aelst occur directly from the sorting endosome (Hopkins et al., et al., 1996), and which has been shown to interact with 1994; Daro et al., 1996) or via a pericentriolar recycling endosomal compartment (Hopkins et al., 1994). Several activated ARF6. The relationship between ARF6 function Ras-related small G proteins of the Rab subgroup that and actin cytoskeleton organization is further illustrated localize to early endocytic compartments have been implic- by the finding that inhibitors of actin polymerization, such 1480 © European Molecular Biology Organization EFA6, an exchange factor for ARF6 as cytochalasin D, block ARF6-mediated actin rearrange- observed that ARNO promotes exchange on ARF6 with ments and induce a redistribution of ARF6 from the a much lower efficiency compared with ARF1 (Frank plasma membrane to recycling endosomes (Radhakrishna et al., 1998). Finally, we and others have observed and Donaldson, 1997). The above observations have led that overexpression of ARNO and ARNO3 results in a to the speculation that ARF6 activation by GDP/GTP fragmentation of the Golgi apparatus and an inhibition of exchange takes place intracellularly on the recycling Golgi function without affecting the endocytic pathway endosome and drives the recycling of membrane to the (Franco et al., 1998; Monier et al., 1998). Therefore, cell surface. It has also been postulated that ARF6 controls the regulatory function of ARNO-like GEFs on ARF1 plasma membrane remodeling by coordinating membrane activation appears critical for maintaining the integrity of flow from a pericentriolar recycling compartment with Golgi structure and for vesicle transport within the Golgi cortical actin organization (Radhakrishna et al., 1996; complex. The above findings support the contention that D’Souza-Schorey et al., 1997; Radhakrishna and ARNO proteins function as GEFs preferentially for ARF1. Donaldson, 1997). There are several observations that In order to identify GEFs involved in ARF6 activation, support the conclusion that exit sites of recycling mem- we sought to isolate and characterize new Sec7 domain- branes at the cell surface are polarized and correlate with containing ARF GEFs that could promote nucleotide the formation of actin-based structures. For instance, exchange preferentially on ARF6. By analysis of the recycling Tfn-Rs are targeted to specialized regions of the database, we have identified a human cDNA encoding a plasma membrane at the leading lamellae of migrating protein that we have designated EFA6 (exchange factor fibroblasts and in membrane ruffling areas (Hopkins et al., for ARF6), which contains Sec7 and PH domains. EFA6 1994; Bretscher and Aguado-Velasco, 1998b). Whether promotes efficient guanine nucleotide exchange on ARF6 and how ARF6 regulation of membrane traffic is coordin- in comparison with ARF1. We have analyzed the subcellu- ated with its effect on actin rearrangements is still lar localization of EFA6 in transfected cells and investi- unknown. In addition, the site of activation of ARF6 by gated its effect on the peripheral plasma membrane/ nucleotide exchange has not yet been determined. endosome system. Our results suggest that EFA6 regulates Activation of the low molecular weight GTP-binding two coordinated activities that remodel the cell periphery: proteins is mediated by guanine nucleotide exchange (i) the outward flow of membrane from a recycling factors (GEFs), which catalyze the replacement of bound endosomal compartment; and (ii) a redistribution of the GDP with GTP. An important breakthrough toward the cortical actin cytoskeleton that may facilitate endosomal identification of GEFs acting on ARF proteins was the membrane delivery to the cell surface. Furthermore, EFA6 cloning of two related ARF1 GEFs encoding genes in expression had no effect on the integrity of the biosynthetic yeast, GEA1 and GEA2 (Peyroche et al., 1996). This and secretory apparatus. made possible the cloning of a human cDNA encoding ARNO (ARF nucleotide-binding-site opener), which pro- Results motes guanine nucleotide exchange on human ARF1 in vitro (Chardin et al., 1996). These three proteins share EFA6 is a GEF for ARF6 a conserved region of ~200 amino acids which bears 42% We searched the database for new Sec7 domain-related of sequence identity with a portion of the Saccharomyces sequences that might code for ARF6-specific GEF(s). cerevisiae SEC7 gene product (Franzusoff and Schekman, Using the amino acid sequence of the ARNO Sec7 domain 1989) and is essential for GEF activity (Chardin et al., as bait, a BLAST search (Altschul et al., 1990) identified 1996). Two additional Sec7 domain-containing GEFs have several sequences homologous to the Sec7 domain of been identified: cytohesin-1 and GRP-1/ARNO3, which ARNO, including a novel open reading frame encoding a show extensive homology with the predicted sequence of 645 amino acid protein (data not shown). This cDNA ARNO (Liu and Pohajdak, 1992; Klarlund et al., 1997; clone, originally called PSD and expressed in brain tissues Franco et al., 1998). Similarities between the three ARNO- (Perletti et al., 1997), encodes an ARF6 GEF (see below) family members are not restricted to the conserved Sec7 which we have named EFA6. Adjacent to the Sec7 domain, domain but extend to an N-terminal coiled-coil sequence the predicted sequence of EFA6 contains a PH domain as well as to a C-terminal pleckstrin homology (PH) (Figure 1A). Except for the conserved Sec7 and PH domain. This PH domain encodes the high affinity of the domains, EFA6 and ARNO-related GEFs are divergent in GEF for phosphoinositide-3,4,5-trisphosphates [PtdIns- primary sequence. As shown in Figure 1A, EFA6 contains (3,4,5)P ] (Klarlund et al., 1997) and mediates PtdIns- proline-rich regions at N- and C-terminal ends and a (4,5)P -dependent stimulation of ARNO GEF activity putative coiled-coil forming sequence at the C-terminus. (Chardin et al., 1996). Recently, we analyzed the substrate Amino acid sequence comparison revealed that the Sec7 specificity of the ARNO family members and showed that domain of EFA6 exhibits 28 and 30% amino acid identity all three proteins stimulate guanine nucleotide exchange with the Sec7 domains of Sec7p and ARNO, respectively, on ARF1 but are inefficient on ARF6, in vitro (Franco compared with 42% amino acid identity between the Sec7 et al., 1998). Klarlund et al. (1998) also reported that domain of ARNO and Sec7p. The three-dimensional GRP-1, the mouse homolog of human ARNO3, does not structure of the Sec7 domain of ARNO (Cherfils et al., promote exchange on ARF6. In addition, cytohesin-1 1998; Mossessova et al., 1998) has demonstrated the catalyzes nucleotide exchange on ARF1 and ARF3 but importance of two conserved motifs within the protein not on ARF5 (Meacci et al., 1997). These findings from sequence, motifs 1 and 2, which fold to form a hydrophobic different laboratories are in contrast with those of Frank groove that has been proposed to interact with the switch et al., who reported that ARNO can catalyze GDP/ regions of ARF1 (Be´raud-Dufour et al., 1998; Cherfils GTP exchange on ARF6 in vitro, although these authors et al., 1998; Mossessova et al., 1998). Interestingly, motifs 1481 M.Franco et al. (Figure 1C, closed triangles), whereas ARNO3 was unable to catalyze nucleotide exchange on myrARF6 under the same conditions. Conversely, nucleotide exchange on myrARF1 was rapid and reached a maximal rate within 5 min in the presence of ARNO3, while it was only weakly accelerated in the presence of EFA6 (Figure 1D). Analogous to ARNO-stimulated exchange on myrARF1 (Chardin et al., 1996), EFA6-catalyzed exchange on myrARF6 was unaffected by the addition of brefeldin A (data not shown). Motif 1 of all Sec7 domains identified so far has a conserved glutamate residue (Figure 1B, arrow). Substitution of this glutamate residue by a lysine in the Sec7 domain of ARNO (ARNO ) reduces its E156K nucleotide exchange activity by at least 1000-fold (Be´raud- Dufour et al., 1998; Mossessova et al., 1998). Similarly, EFA6 has lost GEF activity indicating that Glu242 E242K of EFA6 is essential for catalytic activity (Figure 1C). Finally, and as already shown for ARNO (Chardin et al., 1996; Franco et al., 1998), the isolated EFA6 Sec7 domain was sufficient to promote nucleotide exchange on ARF6 (data not shown). Therefore, we conclude that EFA6 belongs to the family of Sec7 domain containing ARF GEFs, and is the first ARF6-specific GEF identified so far. EFA6 localizes to the plasma membrane and induces invaginations To determine the cellular localization and function of EFA6, we epitope tagged the N-terminal end of EFA6 with the vesicular stomatitis virus glycoprotein (VSV-G) and transiently expressed the protein in TRVb-1 cells [a CHO-derived cell line overexpressing the human Tfn-R (McGraw et al., 1987)]. Confocal immunofluorescence microscopy analysis with anti-VSV-G-tag antibody showed that, analogous to the distribution of ARF6 (Figure 2A), EFA6 localized to membrane ruffles (arrows) and microvilli-like structures (arrowheads) that were induced at the periphery of TRVb-1 cells (Figure 2B). Noticeably, membrane ruffles were more enhanced in EFA6-expressing Fig. 1. EFA6 catalyzes guanine nucleotide exchange preferentially on ARF6. (A) Schematic representation of ARNO and EFA6. Proline-rich cells compared with ARF6-transfected cells. Membrane regions (Pro), Sec7 domains, PH domains and coiled-coil regions (c.c.) association of overexpressed ARF6 and EFA6 was con- are shown. Structure of deletion mutants EFA6ΔC and ΔSec7 is firmed by immunoblot analysis of membranes and cytosol depicted. (B) Alignment of sequences of two highly conserved motifs of transfected cells, fractionated by high-speed centrifug- of Sec7 domains from human EFA6 (Perletti et al., 1997), ARNO ation. As seen in Figure 2C, immunoblotting with anti- (Chardin et al., 1996), cytohesin 1 (Ch-1) (Liu and Pohajdak, 1992), ARNO3 (Franco et al., 1998), Arabidopsis GNOM (Shevell et al., hemagglutinin (HA) tag and anti-EFA6 antibodies revealed 1994), yeast Gea1 (Peyroche et al., 1996) and Sec7 (Achstetter et al., protein species of ~22 kDa (ARF6-HA) and ~70 kDa 1988) proteins. These motifs are thought to form a hydrophobic (VSV-G-EFA6), respectively. Both ARF6 and EFA6 were groove that comes in contact with the switch regions of ARF to form predominantly membrane-bound while only a small frac- the active site of the GEF (Be´raud-Dufour et al., 1998; Mossessova tion of these proteins was present in the soluble fraction. et al., 1998). The invariant glutamate residue (ARNO/Glu156 and EFA6/Glu242) which is involved in nucleotide exchange, is indicated In contrast, deletion of the 241 C-terminal amino acids of with an arrowhead. (C and D) Measurement of [ S]GTPγS binding to EFA6, including the PH domain, led to a protein that was myrARF6 (C) or myrARF1 (D) in the absence (circles) or the mostly detected in the cytosolic fraction (Figure 1C, VSV- presence of purified recombinant EFA6 (squares), EFA6 E242K G-EFA6ΔPH), suggesting that the PH domain is required (diamonds) or ARNO3 (triangles). Data shown are representative of three independent experiments. for membrane association of EFA6. We have studied the peripheral rearrangements induced 1 and 2 of the Sec7 domain of EFA6 are the least conserved on EFA6 expression at the ultrastructural level. Cryo- (Figure 1B), suggesting that its substrate specificity may immunogold electron microscopy using antibodies directed be distinct from ARNO-related Sec7 domains. against EFA6 revealed that the protein was localized to To determine the substrate specificity of EFA6, we the plasma membrane, on folds and invaginations, that in measured the ability of recombinant EFA6 to catalyze the cross sections appeared as membrane enclosed structures binding of guanosine 5-[γ-thio]triphosphate ([ S]GTPγS) with an electroluescent lumen (Figure 3A). These on recombinant myristoylated ARF6 (myrARF6) and structures resembled membrane alterations previously myrARF1. As shown in Figure 1C, EFA6-catalyzed bind- observed in ARF6Q67L-expressing cells (Peters et al., ing of [ S]GTPγS to myrARF6 was maximal after 10 min 1995) and that have been shown to be continuous with 1482 EFA6, an exchange factor for ARF6 Fig. 3. Localization of EFA6 by electron microscopy. TRVb-1 cells expressing VSV-G-EFA6 were fixed and processed for cryoimmuno- gold labeling. Sections were labeled with anti-EFA6 antibodies followed by protein A–10 nm gold. (A) In TRVb-1 cells overexpressing EFA6, the plasma membrane exhibited extensive membrane invaginations (i). Most of the cytoplasmic face of the plasma membrane was coated by an electron-dense matrix. EFA6 localizes exclusively to this dense matrix (arrowhead) and regions of the plasma membrane with no EFA6 are devoid of matrix (large arrow). Small arrows depict cross sections across tubular structures consisting of a sheath of electron-dense material surrounding a membrane-bound electroluescent lumen. Bar, 200 nm. (B) Regions of plasma membrane with little EFA6 labeling show a thinner matrix (large arrowheads). EFA6 labeling is excluded from coated pits (small arrowhead) and caveolae (arrows) as well as from the Golgi region of the cell. m, mitochondria; i, invaginations; n, nucleus; asterisk, untransfected cell. Bar, 500 nm. the extracellular milieu (D’Souza-Schorey et al., 1998). Strikingly, the cytoplasmic face of the plasma membrane and of EFA6-induced invaginations exhibited a homo- genous electron-dense matrix 50 nm thick. This dense Fig. 2. Localization of overexpressed EFA6 by indirect immuno- material was decorated with anti-EFA6 antibodies indicat- fluorescence microscopy. TRVb-1 cells were grown on coverslips and ing that EFA6 was present on this matrix. Regions of transfected with expression plasmids encoding HA-tagged ARF6 (A) plasma membrane showing high levels of labeling with or VSV-G-tagged EFA6 (B). Cells were fixed and labeled with either anti-HA (A) or anti-VSV-G (B) epitope antibody. Arrows indicate anti-EFA6 antibodies had a thicker matrix (Figure 3A, labeling of plasma membrane ruffles at cell edges. Arrowheads in B arrowheads). On the contrary, regions with little EFA6 point to labeling for EFA6 on microvilli at the dorsal cell surface. Bar, labeling had only a thin matrix (Figure 3B, large arrow- 10 μm. (C) The post-nuclear supernatants (PNS) of TRVb-1 cells heads) and regions with no labeling were devoid of matrix expressing either ARF6-HA, VSV-G-EFA6 or VSV-G-EFA6ΔPH were (Figure 3A, large arrow). Some of the EFA6-positive centrifuged at 100 000 g to give a membrane (P100) and a soluble fraction (S100). Fractions prepared from ARF6-HA-expressing cells structures appeared as cross-sections across sheathed were immunoblotted using anti-HA mouse monoclonal antibody cables with an electron-dense sheath surrounding a core (upper panel). Fractions from VSV-G-EFA6 or VSV-G-EFA6ΔPH- electroluescent lumen enclosed within a membrane expressing cells were incubated with rabbit anti-EFA6 polyclonal (Figure 3A, small arrows). This electron-dense matrix antibodies (lower panel). Molecular weights (kDa) of protein markers are indicated. M.Franco et al. Overexpression of EFA6 inhibits Tfn uptake and affects the distribution of the Tfn-R Overexpression of ARF6Q67L, a mutant locked in the GTP-bound conformation decreases the rate of Tfn uptake in TRVb-1 cells (D’Souza-Schorey et al., 1995). Since overexpression of EFA6 should increase the pool of ARF6 in a GTP-bound state, we anticipated that the Tfn cycle should be similarly affected in EFA6-transfected cells. To this end, we examined the internalization of fluoresceinated human Tfn in TRVb-1 cells expressing HA-tagged ARF6Q67L. In untransfected cells, Tfn internalized for 20 min at 37°C, accumulated in a single discrete patch in the pericentriolar region of the cells as well as in vesicular structures spread throughout the cytoplasm. Similar to that observed in cells expressing ARF6(Q67L) (Figure 5A), uptake of Tfn was dramatically inhibited in EFA6- expressing cells with little or no accumulation of Tfn after a 20 min incubation (Figure 5B). Conversely, expression of myc-ARNO3, an ARF1 GEF, had no effect on Tfn uptake (Figure 5C), indicating that the inhibitory effect of EFA6 on the Tfn cycle is mediated by activation of Fig. 4. Plasma membrane association of nucleotide exchange-defective ARF6-in vivo. Cryoimmunogold labeling with antibodies mutant of EFA6. TRVb-1 cells expressing EFA6ΔSec7, which presents directed against Tfn-R revealed that in mock-transfected a deletion of the entire Sec7 nucleotide exchange domain, were fixed TRVb-1 cells most of the Tfn-R label (74%) resides and processed for cryoimmunogold labeling as described in the legend of Figure 3. (A) In low-level expressor, EFA6ΔSec7 is localized to the intracellularly in a tubular and vesicular compartment in plasma membrane that does not show morphological alteration and is the pericentriolar region (Table I). On the contrary, a devoid of matrix. Labeling is also detected on intracellular endocytic majority of the Tfn-Rs (79%) were detected on the plasma structures (e). (B) In high level expressing cell, EFA6ΔSec7 is membrane and on plasma membrane folds and vaginations associated with the plasma membrane on thin and long extensions. of TRVb-1 cells expressing EFA6 (Table I). Taken together, Plasma membrane appears uncoated. EFA6ΔSec7 is also detected in the cytosol. e, endosome; G, Golgi apparatus; m, mitochondria; these findings indicate that expression of EFA6 induces a n, nucleus; p, plasma membrane. Bar, 200 nm. redistribution of Tfn-Rs from the perinuclear endosomal recycling compartment to plasma membrane. In order to investigate whether the effects of EFA6 may represent polymerized actin filaments beneath the expression were restricted to the peripheral plasma mem- surface of plasma membrane invaginations (see below). brane/endosomal system, we analyzed the intracellular As shown in Figure 3B, EFA6 label was excluded from distribution of markers of the secretory and late endocytic clathrin coated pit and caveolae profiles at the plasma pathways in EFA6-expressing cells. The distribution of membrane and from the Golgi. p23 [a cis-Golgi network-associated protein (Rojo et al., In order to investigate whether these morphological 1997)] and of sec61β (a resident ER marker) was not effects were dependent on guanine nucleotide exchange modified by EFA6 overexpression (data not shown). catalyzed by EFA6, TRVb-1 cells overexpressing a GEF- Furthermore, the distribution of the lysosomal marker defective mutant form of EFA6 were analyzed by cryo- LAMP-1 remained unperturbed in EFA6-expressing cells immunogold electron microscopy. Figure 4 shows labeling (not shown). Thus, expression of EFA6 results in a for EFA6 in TRVb-1 cells expressing low (Figure 4A) or specific and profound remodeling of the peripheral plasma high levels (Figure 4B) of EFA6ΔSec7, a Sec7 domain membrane/endosomal system. deletion mutant. In low-level expressors, most of the EFA6 labeling was detected at the plasma membrane, which EFA6 overexpression induces rearrangements of exhibited normal unperturbed morphology and was devoid the actin cytoskeleton of matrix (Figure 4A). Occasionally, EFA6ΔSec7 was also In addition to the regulation of peripheral membrane associated with intracellular endocytic structures but was trafficking, ARF6 has also been demonstrated to regulate absent from Golgi, nucleus and ER profiles. At a higher the assembly and organization of the cortical actin cyto- level of expression (Figure 4B), EFA6ΔSec7 was localized skeleton (Radhakrishna et al., 1996; D’Souza-Schorey to the plasma membrane on thin and long extensions that et al., 1997). These studies, together with our observation were morphologically distinct from the invaginations seen that a dense cytoskeletal-like matrix accumulated at the in wild-type EFA6-expressing cells. The plasma membrane cell surface upon EFA6 expression, prompted us to investi- of EFA6ΔSec7-expressing cells was devoid of matrix. gate the consequences of EFA6 expression on the actin Some EFA6ΔSec7 label was also detected in the cytosol. cytoskeleton in HeLa cells. We have found that cells Taken together, these findings indicate that EFA6 localizes expressing wild-type EFA6 showed a marked accumulation predominantly to the plasma membrane and that this of F-actin in peripheral membrane ruffles (Figure 6A). distribution is independent of its activity as an ARF6- EFA6-induced ruffles at the peripheral edges of the cell specific GEF. Plasma membrane association of cata- were more prominent compared with actin rearrangements lytically active EFA6 induces membrane invaginations observed in ARF6Q67L-expressing cells (not shown). and the formation of a dense matrix. Double labeling for EFA6 with anti-VSV-G tag antibody 1484 EFA6, an exchange factor for ARF6 Table I. Quantitation of the intracellular distribution of transferrin receptors in control and EFA6-overexpressing TRVb-1 cells TRVb-1 cells Plasma membrane Pericentriolar vesicles and invaginations and tubules Mock-transfected 26% 74% EFA6-transfected 79% 21% TRVb-1 cells on monolayers were mock-transfected or transfected with EFA6-expressing vector, fixed and processed for cryoimmunogold labeling and labeled with anti-EFA6 antiserum followed by protein A– 10 nm gold. The data presented represent percentages of 10 nm gold particle count of the total number of gold particles per cell, obtained from 40 random cross-sections of cells. First, expression of the GEF-defective mutant, EFA6 E242K (see Figure 1C), did not induce membrane ruffling, but instead led to the formation of numerous actin-rich mem- brane extensions (Figure 6C, arrowheads). Similarly, expression of EFA6ΔSec7 also resulted in plasma mem- brane extensions (Figure 6E and F). EFA6ΔSec7 and EFA6 localized predominantly at the cell surface, E242K confirming the ultrastructural analysis (Figure 4). Expres- sion of a C-terminal deletion mutant, EFA6ΔC, generated by deletion of the 124 C-terminal amino acids of EFA6, including a putative coiled-coil sequence and proline-rich region (Figure 1A), abolished the capacity of EFA6 to trigger F-actin reorganization (Figure 6G and H). Furthermore, deletion of this C-terminal region in EFA6 (EFA6 ΔC) also abolished membrane E242K E242K extension formation (Figure 6I and J), suggesting that the C-terminal domain may be required for interaction with other factors required for cytoskeletal rearrangements. Altogether, these results indicate that EFA6-induced mem- brane ruffling depends on a functional Sec7 domain and an intact C-terminal end. The Rho GTP-binding protein, Rac1, is involved in EFA6-induced cytoskeletal rearrangements Co-expression of EFA6 and ARF6T27N abolished EFA6- mediated membrane ruffling in doubly transfected HeLa cells (Figure 7A and B). Interestingly, these cells exhibited membrane extensions similar to those observed in cells expressing EFA6 mutants defective in GEF activity. These extensions were absent in HeLa cells expressing only ARF6T27N (not shown). Since the Rho-family proteins Rac1 and CDC42 regulate the organization of actin filaments at the cell cortex to Fig. 5. Transferrin accumulation is blocked in EFA6-expressing cells. induce membrane ruffling and filopodium formation, TRVb-1 cells expressing ARF6Q67L-HA (A), VSV-G-EFA6 (B)orthe respectively, we investigated the effects of the dominant- ARF1-GEF myc-ARNO3 (C) were incubated at 37°C with iron- negative mutants of these proteins on EFA6-induced saturated fluoresceinated human transferrin (FITC–Tfn) for 20 min, ruffling. When EFA6 was co-expressed with GDP-bound fixed and processed for immunofluorescence microscopy. ARF6Q67L, EFA6 and ARNO3 were detected with anti-HA, anti-VSV-G and with Rac1T17N, membrane ruffling was inhibited and co- anti-myc tag antibodies, respectively. Panels show superimposition of transfected cells exhibited numerous F-actin rich mem- FITC–Tfn with epitope-tagged protein labeling. In untransfected cells, brane extensions (Figure 7C and D) that were not FITC–Tfn is predominantly present in the pericentriolar recycling detected in cells transfected with Rac1N17 only (not compartment. In ARF6Q67L- (A) or EFA6-expressing (B) cells there is no accumulation of FITC–Tfn. In contrast, overexpressed ARNO3 shown). On the contrary, co-expression of EFA6 with (C) has no effect on Tfn distribution. Bar, 10 μm. GDP-bound CDC42T17N had no effect on EFA6- induced membrane ruffling (Figure 7E and F, arrows). In addition, expression of CDC42N17 had no effect on revealed that EFA6 co-localized with F-actin in the mem- membrane extensions induced upon expression of the brane ruffles (Figure 6B). To determine structural domains GEF-defective mutants, EFA6 and EFA6ΔSec7 (data E242K of EFA6 involved in peripheral rearrangements, various not shown). These results suggest that EFA6-induced mutants of EFA6 were transiently expressed in HeLa cells. membrane ruffling is independent of CDC42 and that 1485 M.Franco et al. on Rac1 in vitro, as shown using the nucleotide exchange assay described above (data not shown). The effect of EFA6 on Tfn cycling is independent of EFA6-mediated cytoskeletal reorganization We were interested in determining whether the regulatory activity of EFA6 on membrane dynamics was dependent on EFA6-induced actin cytoskeleton reorganization. To this end, TRVb-1 cells expressing EFA6ΔC, which does not affect F-actin distribution, were incubated with fluoresceinated Tfn for 20 min. As shown above for wild-type EFA6, overexpression of EFA6ΔC prevented intracellular accumulation of Tfn (compare Figure 5A with 8A). In contrast, cells expressing EFA6 ΔC, a E242K double mutant defective for GEF activity, did not perturb Tfn endocytosis and the distribution of internalized Tfn was similar to that observed in untransfected cells (Figure 8B). Thus, the effects of EFA6 on Tfn cycling requires ARF6 activation by a functional Sec7 domain but is independent of EFA6-induced actin cytoskeleton remodeling which appears to require the C-terminal end of EFA6. Discussion In contrast to the other ARF family members that are associated with the secretory pathway (Donaldson and Klausner, 1994; P.J.Peters, unpublished observations), ARF6 is associated with the peripheral plasma membrane/ endosome system where it regulates membrane trafficking from an internal fusion competent recycling compartment to the plasma membrane (D’Souza-Schorey et al., 1995, 1998; Peters et al., 1995; Radhakrishna and Donaldson, 1997). Overexpression studies have led to the proposal that ARF6 activation by GDP/GTP exchange is required for the delivery of membrane to the cell surface (Radhakrishna and Donaldson, 1997; D’Souza-Schorey et al., 1998). Recycling to the surface would be facilitated by remodeling of the underlying actin cytoskeleton that is also dependent on ARF6 activation (D’Souza-Schorey et al., 1997; Radhakrishna and Donaldson, 1997). Since ARF6T27N, a mutant thought to be locked in the GDP- bound conformation, localized to the recycling compart- ment, it was speculated that ARF6 activation takes place intracellularly, on the recycling compartment itself or on putative recycling vesicle intermediates en route to the cell surface (Radhakrishna and Donaldson, 1997; D’Souza- Fig. 6. Effects of EFA6 and EFA6 mutants on actin cytoskeleton rearrangements. HeLa cells were transfected VSV-G-tagged wild-type Schorey et al., 1998). However, this model was still EFA6 (A, B), the nucleotide exchange-defective mutants EFA6 E242K speculative due to the lack of an ARF6-GEF. In the (C, D) and EFA6ΔSec7 (E, F), the C-terminal deletion mutant present study, we describe a new Sec7 domain containing EFA6ΔC(G, H) and the double mutant EFA6 ΔC(I, J). After E242K GEF, EFA6, which promotes efficient GDP/GTP exchange 40 h, cells were fixed and labeled with Texas Red-conjugated phalloidin to visualize actin rearrangements (left panels). Right panels on ARF6. In addition to EFA6, mammalian cells express show the superimposition of the phalloidin labeling (red) and anti- three highly related GEFs, ARNO, cytohesin-1 and VSV-G staining (green). Cells overexpressing EFA6 exhibit increased ARNO3/GRP-1, and a 200-kDa GEF, all of which contain staining for F-actin in membrane ruffles (A). EFA6 colocalizes with a Sec7 domain and promote preferential GDP/GTP F-actin in these ruffles (B). Overexpression of GEF-defective mutant exchange on ARF1 (and ARF3) (Chardin et al., 1996; forms, EFA6 (C, D) and EFA6ΔSec7 (E, F), induces the E242K extension of filopodia. Deletion of the C-terminal region of EFA6 Meacci et al., 1997; Morinaga et al., 1997; Franco et al., abolishes EFA6-induced membrane ruffling (G, H) and EFA6 - E242K 1998; Klarlund et al., 1998). Thus, EFA6 is the first mediated filopodia extension (I, J). Bar, 10 μm. ARF6-specific GEF reported to date. We show that exo- genous EFA6 is associated with a dense matrix underlying EFA6 triggers the activation of Rac1 which is required plasma membrane domains that are extensively invagin- for EFA6-induced membrane ruffling. However, it should ated. Plasma membrane alterations induced on EFA6 be noted that EFA6 did not catalyze nucleotide exchange expression are accompanied by a reorganization of cortical 1486 EFA6, an exchange factor for ARF6 Fig. 7. Rac1 is involved in EFA6-induced actin rearrangements. HeLa cells were co-transfected with constructs expressing VSV-G-EFA6 in combination with ARF6T27N-HA (A, B), myc-Rac1T17N (C, D) or myc-CDC42T17N (E, F). Left panels show Texas Red–phalloidin staining of F-actin. Right panels show superimposition of Texas Red–phalloidin labeling and anti-VSV-G tag labeling. In contrast to EFA6 which induces membrane ruffling (see Figure 9A and B), coexpression of EFA6 together with ARF6T27N (A, B) or Rac1T17N (C, D) leads only to filopodia extension. Cdc42T17N does not affect EFA6-induced membrane ruffling (E and F, arrows). Bar, 10 μm. actin, with the formation of membrane ruffles. The latter remodeling by catalyzing GDP/GTP exchange on ARF6 response appears to involve activation of the Rho protein, and by activating Rac1, respectively. Rac1. Consistent with previous observations in cells At the ultrastructural level, overexpressed EFA6 was expressing a constitutively activated mutant of ARF6, found exclusively on the cytoplasmic side of the plasma (ARFQ67L), EFA6 expression affects the Tfn cycle and membrane and on numerous plasma membrane invagin- is accompanied by a redistribution of Tfn-R from the ations that were induced on EFA6 expression. Furthermore, recycling compartment to the cell periphery. These results the effects of EFA6 on membrane traffic and organelle suggest that EFA6 coordinates membrane and actin structure were restricted to the endosomal and plasma 1487 M.Franco et al. intermediates or the recycling endosome) are incompetent for fusion with the plasma membrane until EFA6 converts ARF6 into its GTP-bound active conformation. The initial step of this cascade requires (i) the close proximity of the two membrane systems and (ii) the activation of ARF6 by EFA6 at the interface between these two membrane systems. Regulation of this step could be achieved by controlling EFA6 recruitment to the cell periphery. The PH domain of EFA6 may play an important role in this process since PH domains have been shown to be required for translocation of protein to the plasma membrane (Lemmon et al., 1997). In agreement with this hypothesis, we have observed that deletion of the PH domain of EFA6 leads to a protein which is mostly cytosolic. Overexpres- sion of EFA6 should induce a massive accumulation of ARF6-GTP at the plasma membrane. Normally, GTP hydrolysis that probably involves a plasma membrane- associated GAP should allow the return of GDP-bound ARF6 to recycling endosomes via the endocytic pathway (D’Souza-Schorey et al., 1998), and EFA6 may be released from the membrane into the cytosol. However, in EFA6- expressing cells, GAP-stimulated GTP hydrolysis may become limiting, preventing membrane internalization and thereby blocking Tfn-R internalization and Tfn uptake as also observed in ARF6Q67L-expressing cells. An interesting finding from the ultrastructural analysis is that EFA6 induces the formation of and is associated with a dense matrix that coats the cytoplasmic face of plasma membrane invaginations. We have also observed that EFA6 induces the projection of actin-based membrane ruffles in which F-actin and EFA6 co-localize. It is possible Fig. 8. EFA6-mediated inhibition of Tfn accumulation does not require that the dense matrix observed by electron microscopy is EFA6-induced actin cytoskeleton reorganization but requires a composed of actin filaments polymerized beneath the functional Sec7 domain. TRVb-1 cells expressing VSV-G-EFA6ΔC(A) plasma membrane. Plasma membrane-associated matrix or VSV-G-EFA6 ΔC(B) were incubated for 20 min with E242K and invaginations, as well as membrane ruffling formation fluoresceinated transferrin as described in Figure 5. Panels show the superimposition of fluoresceinated Tfn with VSV-G labeling. In depends on a functional sec7 domain. In contrast, GEF- EFA6ΔC-expressing cells (A), there is no accumulation of FITC–Tfn. defective mutant forms of EFA6 lead to the projection of In contrast, overexpression of GEF-defective EFA6 ΔC (B) has no E242K long actin-rich membrane extensions. Consistent with this effect on Tfn uptake. Bar, 10 μm. finding, extensions are also induced upon co-expression of EFA6 and ARF6T27N that should result in dominant inhibition of EFA6 exchange activity. Moreover, co- membrane system. Indeed, we observed that EFA6 perturbs expression of EFA6 with Rac1T17N, a dominant-inhibi- the Tfn cycle and redistributes TfnR-containing compart- tory mutant of Rac1, also results in membrane extension. ments to the cell surface without affecting the morphology Finally, deletion of the carboxy end of EFA6 abolished of the Golgi apparatus, endoplasmic reticulum and EFA6-induced actin cytoskeleton reorganization. Based LAMP-1-positive lysosomes. The observation that the on these observations, it is tempting to speculate how distribution of EFA6 is at the plasma membrane has some EFA6 could control actin cytoskeleton reorganization via important implications for the GDP/GTP cycle of ARF6. the coordinated activation of ARF6 and Rac1 (Figure One possibility was that EFA6-catalyzed exchange takes 9). We propose that Sec7 domain-catalyzed nucleotide place on the recycling compartment where GDP-bound exchange on ARF6 allows the engagement of specific ARF6 accumulates (Peters et al., 1995; D’Souza-Schorey effectors such as PLD, which is stimulated by ARF6 et al., 1998). However, according to this proposal, GEF- in vitro (Massenburg et al., 1994) and in vivo during defective mutants such as EFA6 or EFA6ΔSec7 exocytosis in chromaffin cells (Caumont et al., 1998). E242K should accumulate on recycling endosome together with PLD may be involved in membrane dynamics at the GDP-bound ARF6. Cryoimmunogold electron microscopy interface between plasma membrane and recycling endo- and confocal microscopy analyses contradict this predic- some by stimulating the production of fusogenic lipids. tion, since overexpressed EFA6 and EFA6ΔSec7 have On the other hand, GTP-bound ARF6 has been shown to E242K the same distribution as wild-type EFA6 at the plasma interact with POR1 (D’Souza-Schorey et al., 1997), a membrane, demonstrating that plasma membrane localiz- protein initially identified as a Rac1 effector involved in ation of EFA6 does not require GDP/GTP exchange on membrane ruffling (Van Aelst et al., 1996). Since ARF6. Therefore, our data favor the alternative possibility Rac1T17N does not inhibit ARFQ67L-induced actin that EFA6 activates ARF6 at the plasma membrane. In rearrangement (D’Souza-Schorey et al., 1997), it is likely this model, recycling membranes (recycling transport that ARF6 and Rac1 are not engaged in a linear pathway 1488 EFA6, an exchange factor for ARF6 et al., 1997), and together with the Rac1-specific GEF, Tiam-1, it affects neuronal morphology (Luo et al., 1996; Kozma et al., 1997; Leeuwen et al., 1997). While ARF6 is ubiquitously expressed (Tsuchiya et al., 1991; Yang et al., 1998), the human PSD gene coding for EFA6 is mainly expressed in brain (Perletti et al., 1997). Whether EFA6, by its coordinated action on ARF6 and Rac1, may be involved in neuronal secretion or axonal elongation is presently unknown, but would be an interesting subject of investigation. By screening for EFA6-like genes, we have isolated a cDNA encoding a Sec7 and PH domain- containing protein showing high similarity with the corresponding domains of EFA6 (M.Franco and B.Chavrier, unpublished). Interestingly, this EFA6-like gene is expressed in a wide range of tissues. The availabil- ity of these cDNAs will help in clarifying the function of EFA6-like GEFs in various cell types. Fig. 9. Model of EFA6-regulated actin reorganization. We propose that targeting of the PH domain of EFA6 to the plasma membrane allows Sec7 domain-catalyzed nucleotide exchange on ARF6. GTP-bound ARF6 interacts with and activates specific downstream effectors at the Materials and methods plasma membrane. Activation of PLD leads to the generation of fusogenic lipids involved in the fusion of recycling membranes with Cells and antibodies the plasma membrane. In parallel, EFA6 allows Rac1 activation by TRVb-1 cells, a CHO-derived cell line overexpressing the human recruiting a Rac1-specific GEF. The ARF6 and Rac1 pathways transferrin receptor (McGraw et al., 1987), were grown in Ham’s F-12 converge at the level of POR1 that interacts with both GTP-bound medium, 5% fetal calf serum (FCS), penicillin/streptomycin, and 100 ARF6 and Rac1 to control membrane ruffling. Recycling of membrane μg/ml G418. HeLa cells were grown in Dulbecco’s modified essential occurs at discrete sites of the plasma membrane that coincide with medium supplemented with 10% FCS and antibiotics. areas of membrane ruffling. The different domains of EFA6 are The following antibodies were used for the studies described: mouse schematically represented as in Figure 1A. monoclonal antibody (mAb) against VSV-G epitope (clone P5D4, Boehringer Mannheim) and rat mAb against HA epitope (clone 3F10, Boehringer Mannheim); mouse mAb against myc epitope (clone 9E10; (ARF6-GTP promoting Rac1 activation), but are rather Evan et al., 1985); mouse mAb anti-Tfn-R (clone H68.4; Zymed, CA); functioning on parallel pathways that may converge at the rabbit antiserum against p23 (Rojo et al., 1997); rabbit antiserum against level of POR1. Noticeably, ARF6Q67L stimulates actin Sec61β (provided by R.Hendriks, ZMBH, Heidelberg, Germany); rabbit antiserum against LAMP-1 (provided by S.Meresse, CIML, Marseille, reorganization but does not induce the formation of the France); and anti-EFA6 rabbit antiserum raised against the purified dense matrix we observed in EFA6-expressing cells (Peters recombinant protein. et al., 1995; D’Souza-Schorey et al., 1998). An interpret- ation of these remarkable phenotypic differences may be Construction of EFA6 expression plasmids that the mere engagement of the ARF6-dependent pathway EFA6 was cloned from a human brain cDNA library (Clontech) by high fidelity PCR using two oligonucleotide primers; 5EFA6 primer by ARF6Q67L, in the absence of Rac1 engagement, leads (5-GATCGATCCATATGCCTCTCAAGTCACCTG-3), which con- only to a partial response. Since EFA6 does not catalyze tains a NdeI linker, the ATG start codon and 5 nucleotides of EFA6, nucleotide exchange on the Rho-family proteins (M.Franco and 3EFA6 primer (5-GGGCGGCGGAAGCCCTGAGTCAACGGAT- and P.Chavrier, unpublished observations), we propose CCTAAGTACA-3), which contains a BamHI linker, TAG stop codon and 3 complementary nucleotide residues of EFA6. Primer sequences that EFA6-dependent Rac1 activation is mediated by a encoding EFA6 were designed from the published human PSD sequence Rac1-specific GEF. A possibility is that the C-terminus of (Perletti et al., 1997). The PCR product was inserted into pGEM1 and EFA6 which contains coiled-coil and Pro-rich regions pSRα expression vectors with a N-terminal VSV-G tag using NdeI and known to participate in protein–protein interactions, regu- BamHI restriction sites. lates this Rac1 GEF. Hence, deletion of the C-terminus EFA6 was constructed by two-stage overlap-extension PCR. E242K The first-stage reaction products were generated using complementary of EFA6 by prevention of Rac1 engagement would inhibit overlapping end primers containing an oligonucleotide-directed point the actin-based ruffling response. Exocytosis of recycling mutation; the N-terminal product was generated with the 5EFA6 primer membrane occurs at the leading edge of migrating cells described above and the primer 5-CCTGGGTCTTACCCATTAAG-3 (Hopkins et al., 1994; for a review see Bretscher and (EFA6E242K-1). The C-terminal product was generated with the primer 5-CTTAATGGGTAAGACCCAGG-3 (EFA6E242K-2) and the Aguado-Velasco, 1998a). Recently, EGF treatment of KB 3EFA6 primer. EFA6 cDNA was used as template. Second-stage PCR cells has been shown to induce the formation of membrane was carried out with first-stage reaction products to anneal the overlapping ruffles that are enriched in Tfn-Rs, and Tfn-Rs are also ends, followed by amplification. The full-length EFA6E242K PCR targeted to Rac1-induced membrane ruffles in these cells product was cloned in the NdeI and BamHI restriction sites of (Bretscher and Aguado-Velasco, 1998b). We believe that, pGEM1VSV-G and pSRαVSV-G expression vectors. EFA6ΔC and EFA6 ΔC were constructed by PCR amplification by its coordinated actions on both Rac1- and ARF6-driven E242K of corresponding EFA6 and EFA6 cDNAs in pGEM1VSV-G using E242K pathways, EFA6 allows to couple membrane ruffling and the 5EFA6 primer and the primer 5-TGTACTAAAGCTTCTACTGGG- membrane fusion at discrete sites of the plasma membrane AGAGGCGGGTGG-3 (EFA6ΔC) obtained by substituting glutamate in a polarized fashion. 522 of EFA6 with a stop codon. Similarly, EFA6ΔPH was obtained by PCR using the 5EFA6 primer and the primer 5-TGTCTAAAGCTTTC- ARF6 has been shown to co-purify along with AGCCCCGCTTGCCCCGAGG-3 (EFA6ΔPH) substituting tryptophane chromaffin granules (Galas et al., 1997) and controls 404 of EFA6 with a stop codon. EFA6Δ was constructed by Sec7 exocytosis during regulated secretion in chromaffin cells two-stage PCR amplification of EFA6 with the 5EFA6 primer and (Caumont et al., 1998). Rac1 is also implicated in regulated the primer 5-GTCGGCCAACTCAGACAGGCTGTCCAGCTCTGA- exocytosis in mast cells (Norman et al., 1996; Guillemot GTC-3 (EFA6-S156), and with the primer 5-GACTCAGAGCTGGAC- 1489 M.Franco et al. AGCCTGTCTGAGTTGGCCGAC-3 (EFA6-S340) and the 3EFA6 Acknowledgements primer. These PCR products were then amplified with the 5 and 3 We are indebted to J.Gruenberg, R.Hendriks and S.Me´resse for generously primers of EFA6 to create an internal deletion from amino acids 157 to providing antibodies for this study. We are grateful for the skillful work 341. The resulting PCR product was subcloned into the NdeI and BamHI of the dark room assistance at Utrecht University. P.Chardin, M.Chabre restriction sites of pGEM1VSV-G. and M.Zerial are thanked for helpful comments on this manuscript. We Expression plasmids used in this study encoding ARF6-HA, thank B.Antonny for sharing results prior to publication. This work was ARF6Q67L-HA, ARF6T27N-HA (Peters et al., 1995), mycRac1N17, supported by INSERM and CNRS institutional funding and specific mycCDC42N17 (Dutartre et al., 1996) and mycARNO3 (Franco et al., grants from La Ligue Nationale contre le Cancer and Le Groupement 1998) were as described previously. des Entreprises Franc¸aises dans la Lutte contre le Cancer (to P.C.), by grants from the Dutch Cancer Society (to P.J.P.) and a grant from the [ S]GTPγS binding assay Associazione Italiana Ricerca sul Cancro (to A.N.). M.F. was supported Myristoylated ARF1 and ARF6 were produced and purified as described by a postdoctoral fellowship from the Fondation pour la Recherche previously (Franco et al., 1995). Recombinant EFA6 and ARNO3 were Me´dicale. C.D.-S. is a Leukemia Society of America Special Fellow. produced from pET3a-transformed BL21 (DE3) Escherichia coli as described previously (Franco et al., 1998). For [ S]GTPγS binding, measurements were carried out as follows: MyrARF1 or myrARF6 (1 μM) were incubated at 37°C in 50 mM HEPES–NaOH pH 7.5, 1 mM References MgCl , 1 mM dithiothreitol (DTT), 100 mM KCl, 10 μM[ S]GTPγS Achstetter,T., Franzusoff,A., Field,C. and Schekman,R. (1988) SEC7 (~1000 c.p.m./pmol, NEN) supplemented with azolectin vesicles encodes an unusual, high molecular weight protein required for (1.5 g/l, Sigma). Soluble bacterial protein lysates containing EFA6 or membrane traffic from the yeast Golgi apparatus. J. Biol. Chem., 263, ARNO3 were added to a final concentration of 40 μg/ml of total proteins. 11711–11717. The estimated GEF concentration in the assay was ~100–200 nM. At Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990) indicated time intervals, aliquots of of 25 μl were measured for Basic local alignment search tool. J. Mol. Biol., 215, 403–410. radioactivity as described previously (Franco et al., 1995). Bednarek,S.Y., Orci,L. and Schekman,R. (1996) Traffic COPs and the formation of vesicle coats. Trends Cell Biol., 6, 468–473. Internalization of FITC-conjugated transferrin Be´raud-Dufour,S., Robineau,S., Chardin,P., Paris,S., Chabre,M., CHO cells plated on 11 mm round glass coverslips were transiently Cherfils,J. and Antonny,B. (1998) A glutamic finger in the guanine transfected with pSRα constructs using the Fugene 6 transfection reagent nucleotide exchange factor ARNO displaces Mg and the β-phosphate as described by the manufacturer (Boehringer Mannheim). Forty hours to destabilize GDP on ARF1. EMBO J., 17, 3651–3659. after transfection, cells were pre-incubated in serum-free medium con- Bretscher,M.S. and Aguado-Velasco,C. 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Received September 7, 1998; revised January 19, 1999; Meacci,E., Tsai,S.C., Adamik,R., Moss,J. and Vaughan,M. (1997) accepted January 20, 1999 Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor. Proc. Natl Acad. Sci. USA, 94, 1745–1748. Monier,S., Chardin,P., Robineau,S. and Goud,B. (1998) Overexpression of the ARF1 exchange factor ARNO inhibits the early secretory

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The EMBO Journal – Springer Journals

Published: Mar 15, 1999

Keywords: actin; ARF6; endocytosis; guanine nucleotide exchange factor; Rac1

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