ArfGAP1 function in COPI mediated membrane traffic: currently debated models and comparison to other coat-binding ArfGAPs

The ArfGAPs are a family of proteins containing an ArfGAP catalytic domain that induces the hydrolysis of GTP bound to the small guanine nucleotide binding-protein ADP-ribosylation factor (Arf). Functional models for Arfs, which are regulators of membrane traffic, are based on the idea that guanine nucleotide-binding proteins function as switches: Arf with GTP bound is active and binds to effector proteins; the conversion of GTP to GDP inactivates Arf. The cellular activities of ArfGAPs have been examined primarily as regulatory proteins that inactivate Arf; however, Arf function in membrane traffic does not strictly adhere to the concept of a simple switch, adding complexity to models explaining the role of ArfGAPs. Here, we review the literature addressing the function Arf and ArfGAP1 in COPI mediated transport, focusing on two critical and integrated functions of membrane traffic, cargo sorting and vesicle coat polymerization. We briefly discuss other ArfGAPs that may have similar function in Arf-dependent membrane traffic outside the ER-Golgi.     

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.     

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.     

The structural GDP/GTP cycle of human Arf6

The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPγS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.     

Control of cell signaling by Arf GTPases and their regulators: Focus on links to cancer and other GTPase families

The ADP-ribosylation factors (Arfs) comprise a family of regulatory GTP binding proteins. The Arfs regulate membrane trafficking and cytoskeleton remodeling, processes critical for eukaryotes and which have been the focus of most studies on Arfs. A more limited literature describes a role in signaling and in integrating several signaling pathways to bring about specific cell behaviors. Here, we will highlight work describing function of Arf1, Arf6 and several effectors and regulators of Arfs in signaling.     

Munc18 interacting proteins: ADP-ribosylation factor-dependent coat proteins that regulate the traffic of beta-Alzheimer's precursor protein

Coat proteins cycle between soluble and membrane-bound locations at the time of vesicle biogenesis and act to regulate the assembly of the vesicle coat that determines the specificity in cargo selection and the destination of the vesicle. A transmembrane cargo protein, an Arf GTPase, and a coat protein (e.g. COPs, APs, or GGAs) are minimal components required for budding of vesicles. Munc18 interacting proteins (MINTs) are a family of three proteins implicated in the localization of receptors to the plasma membrane. We show that MINTs bind Arfs directly, co-localize with Arf and the Alzheimer's precursor protein (beta-APP) to regions of the Golgi/trans-Golgi network, and can co-immunoprecipitate clathrin. We demonstrate that MINTs bind Arfs through a region of the PTB domain and the PDZ2 domain, and Arf-MINT interaction is necessary for the increased cellular levels of beta-APP produced by MINT overexpression. Knockdown (small interference RNA) experiments implicate beta-APP as a transmembrane cargo protein that works together with MINTs. We propose that MINTs are a family of Arf-dependent, vesicle-coat proteins that can regulate the traffic of beta-APP.     

Ancient Complexity,Opisthokont Plasticity,and Discovery of the 11th Subfamily of Arf GAP Proteins

The organelle paralogy hypothesis is one model for the acquisition of nonendosymbiotic organelles, generated from molecular evolutionary analyses of proteins encoding specificity in the membrane traffic system. GTPase activating proteins (GAPs) for the ADP‐ribosylation factor (Arfs) GTPases are additional regulators of the kinetics and fidelity of membrane traffic. Here we describe molecular evolutionary analyses of the Arf GAP protein family. Of the 10 subfamilies previously defined in humans, we find that 5 were likely present in the last eukaryotic common ancestor. Of the 3 most recently derived subfamilies, 1 was likely present in the ancestor of opisthokonts (animals and fungi) and apusomonads (flagellates classified as the sister lineage to opisthokonts), while 2 arose in the holozoan lineage. We also propose to have identified a novel ancient subfamily (ArfGAPC2), present in diverse eukaryotes but which is lost frequently, including in the opisthokonts. Surprisingly few ancient domains accompanying the ArfGAP domain were identified, in marked contrast to the extensively decorated human Arf GAPs. Phylogenetic analyses of the subfamilies reveal patterns of single and multiple gene duplications specific to the Holozoa, to some degree mirroring evolution of Arf GAP targets, the Arfs. Conservation, and lack thereof, of various residues in the ArfGAP structure provide contextualization of previously identified functional amino acids and their application to Arf GAP biology in general. Overall, our results yield insights into current Arf GAP biology, reveal complexity in the ancient eukaryotic ancestor and integrate the Arf GAP family into a proposed mechanism for the evolution of nonendosymbiotic organelles.     

Modifications to the C‐Terminus of Arf1 Alter Cell Functions and Protein Interactions

Arf family proteins are ≈21‐kDa GTP‐binding proteins that are critical regulators of membrane traffic and the actin cytoskeleton. Studies examining the complex signaling pathways underlying Arf action have relied on recombinant proteins comprised of Arf fused to epitope tags or proteins, such as glutathione S‐transferase or green fluorescent protein, for both cell‐based mammalian cell studies and bacterially expressed recombinant proteins for biochemical assays. However, the effects of such protein fusions on the biochemical properties relevant to the cellular function have been only incompletely studied at best. Here, we have characterized the effect of C‐terminal tagging of Arf1 on (i) function in Saccharomyces cerevisiae, (ii) in vitro nucleotide exchange and (iii) interaction with guanine nucleotide exchange factors and GTPase‐activating proteins. We found that the tagged Arfs were substantially impaired or altered in each assay, compared with the wild‐type protein, and these changes are certain to alter actions in cells. We discuss the results related to the interpretation of experiments using these reagents and we propose that authors and editors consistently adopt a few simple rules for describing and discussing results obtained with Arf family members that can be readily applied to other proteins.     

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.     

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.     

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.     

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.     

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.     

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.     

Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.     

Arf GAPs as regulators of the actin cytoskeleton

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

Arf GAPs as regulators of the actin cytoskeleton.

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

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.     

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.     

Substrate specificities and activities of AZAP family Arf GAPs in vivo

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401-413, 2004), are characterized by the presence of at least one NH(2)-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599-610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH(2)-terminal sterile-alpha motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH(2)-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.