Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.     

ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation

We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.     

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.     

An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration

We describe a novel role for the ARF6 GTPase in the regulation of adherens junction (AJ) turnover in MDCK epithelial cells. Expression of a GTPase-defective ARF6 mutant, ARF6(Q67L), led to a loss of AJs and ruffling of the lateral plasma membrane via mechanisms that were mutually exclusive. ARF6-GTP-induced AJ disassembly did not require actin remodeling, but was dependent on the internalization of E-cadherin into the cytoplasm via vesicle transport. ARF6 activation was accompanied by increased migratory potential, and treatment of cells with hepatocyte growth factor (HGF) induced the activation of endogenous ARF6. The effect of ARF6(Q67L) on AJs was specific since ARF6 activation did not perturb tight junction assembly or cell polarity. In contrast, dominant-negative ARF6, ARF6(T27N), localized to AJs and its expression blocked cell migration and HGF-induced internalization of cadherin-based junctional components into the cytoplasm. Finally, we show that ARF6 exerts its role downstream of v-Src activation during the disassembly of AJs. These findings document an essential role for ARF6- regulated membrane traffic in AJ disassembly and epithelial cell migration.     

A Role for ARF6 and ARNO in the regulation of endosomal dynamics in neurons

During development, neuronal processes extend, branch and navigate to ultimately synapse with target tissue. We have shown a regulatory role for ARNO and ARF6 in dendritic branching and axonal elongation and branching during neuritogenesis, particularly with respect to cytoskeletal dynamics. Here, we have examined the role of ARF6 and the ARF GEF ARNO in endosomal dynamics during neurite elongation in hippocampal neurons. Axonal and dendritic endosomes were labeled by expression of the endosomal marker, endotubin. Expression of endotubin-green fluorescent protein resulted in targeting to tubular-vesicular structures throughout the somatodendritic and axonal domains. These endosomal structures did not colocalize with conventional early or late endosomal markers or with the synaptic vesicle marker, SV2. However, they did label with internalized lectin, indicating that they are endosomal structures. Expression of catalytically inactive ARNO (ARNO-E156K) or inactive ARF6 (ARF6-T27N) caused a redistribution of endotubin to the cell surface of the axons and dendrites. In contrast, expression of these constructs had no effect upon the distribution of SV2-positive structures. Furthermore, expression of inactive ARF1 (ARF1-T31N) did not change endotubin distribution. These results suggest that endotubin labels a distinct endosomal structure in neurons and that ARNO and ARF6 mediate neurite extension through the regulation of this compartment.     

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIgamma restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIgamma with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.     

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.     

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

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

ARF6 in the nervous system

Actin cytoskeleton dynamics and membrane trafficking are tightly connected and are among the most important driving forces of neuronal development, basic synaptic transmission events, and synaptic plasticity. One group of proteins involved in coordination of these two processes is the family of ADP ribosylation factors (ARFs) regulating actin dynamics, lipid modification and membrane trafficking. ARF6 is the only member of the ARF family that can simultaneously regulate actin cytoskeleton changes and membrane exchange between plasma membrane and endocytic compartments. The presence of ARF6 and its guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in the brain, as well as its capability to regulate several aspects of neuronal development and synaptic plasticity, has been recently demonstrated. The main purpose of this review is to present the current knowledge about how ARF6 can influence morphological processes crucial for proper formation of the neuronal circuits in the brain, including dendrite and axon differentiation, development of dendritic arbor complexity and dendritic spine formation. Potential effects of ARF6 on synaptic events resulting from its ability to control exo- and endocytosis will be also discussed.     

ARF6 controls post-endocytic recycling through its downstream exocyst complex effector

The small guanosine triphosphate (GTP)-binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP-bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.     

Regulation of neuroendocrine exocytosis by the ARF6 GTPase-activating protein GIT1

Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release.     

A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1.     

Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6

Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.     

Centaurin-alpha1 is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent GTPase-activating protein for ARF6 that is involved in actin cytoskeleton organization

The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6GDP. Centaurin-alpha1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we show that centaurin-alpha1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-alpha1 also inhibited cortical actin formation in a PIP3-dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-alpha1. An artificially plasma membrane-targeted centaurin-alpha1 bypasses the requirement of PIP3 for its involvement in ARF6 inactivation, suggesting that PIP3 is required for recruitment of centaurin-alpha1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-alpha1 negatively regulates ARF6 activity by functioning as an in vivo PIP3-dependent ARF6 GAP.     

Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope- tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.     

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.     

Localization and regulation of phospholipase D2 by ARF6

Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.