Differential Membrane Association Properties and Regulation of Class I and Class II Arfs

ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.     

Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.     

Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors

The ADP ribosylation factors (Arfs) are a family of small, ubiquitously expressed and evolutionarily conserved guanosine triphosphatases that are key regulators of vesicular transport in eukaryotic cells (D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 2006;7:347-358). Although Arfs are best known for their role in the nucleation of coat protein assembly at a variety of intracellular locations, it is increasingly apparent that they are also integral components in a number of important signaling pathways that are regulated by extracellular cues. The activation of Arfs is catalyzed by a family of guanine nucleotide exchange factors (GEFs), referred to as the Sec7 family, based on homology of their catalytic domains to the yeast Arf GEF, sec7p. While there are only six mammalian Arfs, the human genome encodes 15 Sec7 family members, which can be divided into five classes based on related domain organization. Some of this diversity arises from the tissue-specific expression of certain isoforms, but all mammalian cells appear to express at least six Arf GEFs, suggesting that Arf activation is under extensive regulatory control. Here we review recent progress in our understanding of the structure, localization and biology of the different classes of Arf GEFs.     

Arf3 Is Activated Uniquely at the trans-Golgi Network by Brefeldin A-inhibited Guanine Nucleotide Exchange Factors

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20°C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20°C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.     

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of β1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.     

Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf . GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf . GTP, rather than the formation of an Arf . GDP . BFA . GBF1 complex.     

Structural perturbations in human ADP ribosylation factor-1 accompanying the binding of phosphatidylinositides

ADP ribosylation factors (Arfs) are members of a family of Ras-related GTPases that regulate a wide variety of intracellular signaling pathways, including the regulation of membrane traffic and organelle morphology. Arfs perform these functions through interactions with Arf-specific guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and effectors. Signaling phosphatidylinositides, most commonly phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) or phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)), have been shown previously to regulate the activities of a number of these regulators and effectors of Arf. The ability of Arf itself to bind these same phosphatidylinositides also has been reported previously, though without much structural detail. We investigated the ability of human Arf1.GDP (Arf1.GDP) to bind myo-inositol (1,4,5)-trisphosphate (I(1,4,5)P(3)), the soluble headgroup for PI(4,5)P(2), and a short acyl-chain soluble PI(4,5)P(2) analogue using heteronuclear single quantum coherence (HSQC)-based NMR techniques. A patch of positive electrostatic potential on the surface of Arf1.GDP is identified as being directly involved in ligand binding, but structural and stability changes extending to the N-terminal helix and nucleotide-binding site of Arf1 are also documented. The identified binding site and the resultant structural changes are discussed in terms of a possible influence of phosphatidylinositides on the binding of Arf1 to Arf1-GEF and subsequent nucleotide release.     

The LEA proteins and trehalose loving couple: a step forward in anhydrobiotic engineering

Arf family GTP-binding proteins function in the regulation of membrane-trafficking events and in the maintenance of organelle structure. They act at membrane surfaces to modify lipid composition and to recruit coat proteins for the generation of transport vesicles. Arfs associate with membranes through insertion of an N-terminal myristoyl moiety in conjunction with an adjacent amphipathic alpha-helix, which embeds in the lipid bilayer when Arf1 is GTP-bound. In this issue of the Biochemical Journal, Lundmark et al. report that myristoylated Arfs in the presence of GTP bind to and cause tubulation of liposomes, and that GTP exchange on to Arfs is more efficient in the presence of liposomes of smaller diameter (increased curvature). These findings suggest that Arf protein activation and membrane interaction may initiate membrane curvature that will be enhanced further by coat proteins during vesicle formation.     

Conformational changes in human Arf1 on nucleotide exchange and deletion of membrane-binding elements

Conformational changes associated with nucleotide exchange or truncation of the N-terminal alpha-helix of human Arf1 have been investigated by using forms of easily acquired NMR data, including residual dipolar couplings and amide proton exchange rates. ADP-ribosylation factors (Arfs) are 21-kDa GTPases that regulate aspects of membrane traffic in all eukaryotic cells. An essential component of the biological actions of Arfs is their ability to reversibly bind to membranes, a process that involves exposure of the myristoylated N-terminal amphipathic alpha-helix upon activation and GTP binding. Deletion of this helix results in a protein, termed Delta17Arf1, that has a reduced affinity for GDP and the ability to bind GTP in the absence of lipids or detergents. Previous studies, comparing crystal structures for Arf1.GDP and Delta17Arf1.GTP, identified several regions of structural variation and suggested that these be associated with nucleotide exchange rather than removal of the N-terminal helix. However, separation of conformational changes because of nucleotide binding and N-terminal truncation cannot be addressed in comparing these structures, because both the bound nucleotide and the N terminus differ. Resolving the two effects is important as any structural changes involving the N terminus may represent membrane-mediated conformational adjustments that precede GTP binding. Results from NMR experiments presented here on Arf1.GDP and Delta17Arf1.GDP in solution reveal substantial structural differences that can only be associated with N-terminal truncation.     

Overexpression of Arabidopsis AGD7 causes relocation of Golgi-localized proteins to the endoplasmic reticulum and inhibits protein trafficking in plant cells

ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bound to Arfs to GDP, which plays a pivotal role in regulating Arfs by converting the active GTP-bound forms of these proteins into their inactive GDP-bound forms. Here, we investigated the biological role of AGD7, an Arf GAP homolog, in Arabidopsis (Arabidopsis thaliana). We show that AGD7 bears a highly conserved N-terminal region and a unique C-terminal region, interacts with Arf1 both in vitro and in vivo, and stimulates Arf1 GTPase activity in a phosphatidic acid-dependent manner in vitro. In plant cells, AGD7 localized to the Golgi complex, where its overexpression was found to inhibit the Golgi localization of gamma-subunit of coat proteins and promote the relocation of Golgi proteins into the endoplasmic reticulum in both protoplasts and transgenic plants. Furthermore, overexpression of AGD7 inhibited anterograde trafficking of proteins from the endoplasmic reticulum. We propose that AGD7 functions as a GAP for Arf1 in the Golgi complex and plays a critical role in protein trafficking by controlling Arf1 activity.     

Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells. protein trafficking; immunofluorescence microscopy; Western blotting; endocytosis     

ELMOD2 is an Arl2 GTPase-activating protein that also acts on Arfs

Regulatory GTPases in the Ras superfamily employ a cycle of alternating GTP binding and hydrolysis, controlled by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), as essential features of their actions in cells. Studies of these GAPs and guanine nucleotide exchange factors have provided important insights into our understanding of GTPase signaling and biology. Within the Ras superfamily, the Arf family is composed of 30 members in mammals, including 22 Arf-like (Arl) proteins. Much less is known about the mechanisms of cell regulation by Arls than by Arfs. We report the purification from bovine testis of an Arl2 GAP and its identity as ELMOD2, a protein with no previously described function. ELMOD2 is one of six human proteins that contain an ELMO domain, and a second member, ELMOD1, was also found to have Arl2 GAP activity. Surprisingly, ELMOD2 also exhibited GAP activity against Arf proteins even though it does not contain the canonical Arf GAP sequence signature. The broader specificity of ELMOD2, as well as the previously described role for ELMO1 and ELMO2 in linking Arf6 and Rac1 signaling, suggests that ELMO family members may play a more general role in integrating signaling pathways controlled by Arls and other GTPases.     

GBF1-Arf-COPI-ArfGAP-mediated Golgi-to-ER transport involved in regulation of lipid homeostasis

Eukaryotic cells store neutral lipids and cholesteryl esters in cytoplasmic lipid droplets (LDs), which are generated from the endoplasmic reticulum (ER). Accumulating lines of evidence have indicated that Golgi-to-ER-retrograde transport mediated by COPI-coated vesicles under the control of Arf small GTPases is implicated in LD formation and utilization. However, the detailed mechanism underlying the regulation of lipid homeostasis by COPI-dependent transport has been poorly understood. Here we show that LD deposition and the cellular triacylglycerol content are significantly increased by siRNA-mediated depletion of not only β-COP (a subunit of the COPI coat complex) but also GBF1 (a guanine nucleotide exchange factor for Arfs), Arf4 and Arf5 (class II Arfs), and ArfGAP1-ArfGAP3 (GTPase-activating proteins for Arfs). Although a previous proteomic study suggested the presence of COPI subunits and Arfs on LDs, we have failed to show that components of the GBF1-Arf-COPI-ArfGAP retrograde transport machinery are directly associated with and closely apposed to LDs. Furthermore, although recent studies suggested that COPI-mediated transport and GBF1 participated in delivery of adipose triglyceride lipase (ATGL) onto the LD surface, we have found that depletion of β-COP or GBF1 does not affect association of ATGL with LDs or ATGL-mediated lipolysis. On the basis of these results, we propose other mechanisms how the GBF1-Arf-COPI-ArfGAP transport machinery is implicated in the regulation of lipid homeostasis.     

High glucose-induced membrane translocation of PKC βI is associated with Arf6 in glomerular mesangial cells

Protein kinase C (PKC)-induced changes in glomerular mesangial cell (MC) phenotypic behavior has been implicated in diabetes. The activity of diacylglycerol-sensitive PKC isoforms in MCs is altered by ambient changes in glucose, but the regulation of PKC activity and subsequent intracellular signaling events are not yet clearly defined. Small GTP-binding proteins of the ADP-ribosylation factor (Arfs) family, may regulate protein kinase membrane recruitment and hence its activity in signaling events of non-polarized cells. Members of the ARF family may coordinate membrane dynamics and other cellular functions through their interaction with PKC. We studied the activation of Arf, PKC betaI and phospholipase D (PLD) in MCs cultured under normal or high glucose conditions. MCs cultured in high glucose medium exhibited predominantly cytosolic localization of PKC betaI, Arf3 and Arf6. However, phorbol ester (PMA) stimulation of cells cultured in high glucose significantly enhanced membrane association of PKC betaI and Arf6, but not Arf3. Using [3H]choline chloride to prelabel MCs and measuring [3H]choline-containing metabolite release as PLD activity, PMA stimulated a significant increase of PLD activity under high glucose condition. Our data suggest that Arf6 plays a specific role in activation of PKC betaI and PLD under high glucose condition, and may be a significant intracellular event in the change of the mesangial cell phenotype associated with diabetic nephropathy.     

Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses

Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.     

Identification of class II ADP‐ribosylation factors as cellular factors required for hepatitis C virus replication

GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G‐proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf‐guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA‐mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.     

The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADP-ribosylation factors 1 and 6

Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.     

A novel mode of action of an ArfGAP, AMAP2/PAG3/Papa lpha, in Arf6 function

Previously we reported that AMAP2/PAG3/Papalpha/KIAA0400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, whereas it was shown to exhibit efficient GAP activities for other Arf isoforms in vitro. Here, we found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6 but not to GDP-Arf6 or other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function; it exhibits efficient catalytic GAP activity for the class I and II Arfs and yet is involved in the cellular function of the class III Arf without immediate GAP activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.