key: cord-255981-3zvwu5bd
authors: Bui, Quynh Trang; Golinelli-Cohen, Marie-Pierre; Jackson, Catherine L.
title: Large Arf1 guanine nucleotide exchange factors: evolution, domain structure, and roles in membrane trafficking and human disease
date: 2009-08-11
journal: Mol Genet Genomics
DOI: 10.1007/s00438-009-0473-3
sha: 
doc_id: 255981
cord_uid: 3zvwu5bd

The Sec7 domain ADP-ribosylation factor (Arf) guanine nucleotide exchange factors (GEFs) are found in all eukaryotes, and are involved in membrane remodeling processes throughout the cell. This review is focused on members of the GBF/Gea and BIG/Sec7 subfamilies of Arf GEFs, all of which use the class I Arf proteins (Arf1-3) as substrates, and play a fundamental role in trafficking in the endoplasmic reticulum (ER)—Golgi and endosomal membrane systems. Members of the GBF/Gea and BIG/Sec7 subfamilies are large proteins on the order of 200 kDa, and they possess multiple homology domains. Phylogenetic analyses indicate that both of these subfamilies of Arf GEFs have members in at least five out of the six eukaryotic supergroups, and hence were likely present very early in eukaryotic evolution. The homology domains of the large Arf1 GEFs play important functional roles, and are involved in interactions with numerous protein partners. The large Arf1 GEFs have been implicated in several human diseases. They are crucial host factors for the replication of several viral pathogens, including poliovirus, coxsackievirus, mouse hepatitis coronavirus, and hepatitis C virus. Mutations in the BIG2 Arf1 GEF have been linked to autosomal recessive periventricular heterotopia, a disorder of neuronal migration that leads to severe malformation of the cerebral cortex. Understanding the roles of the Arf1 GEFs in membrane dynamics is crucial to a full understanding of trafficking in the secretory and endosomal pathways, which in turn will provide essential insights into human diseases that arise from misregulation of these pathways.

The distinguishing feature of eukaryotic cells is their internal membrane organization. Membrane-bound organelles such as the endoplasmic reticulum (ER), the Golgi apparatus, and endosomes, ensure that specialized functions are carried out in the appropriate delimited environment. Each organelle possesses a characteristic lipid and protein composition, but is highly dynamic and linked to other organelles via traYcking pathways. There are two major membrane traYcking systems in eukaryotes, the secretory and the endocytic systems. The secretory pathway transports secreted and membrane proteins synthesized in the ER to their Wnal destination (plasma membrane (PM), cell exterior or intracellular organelle). Endocytosis pathways transport material from the cell exterior to intracellular organelles such as the lysosome (or vacuole in yeasts).

The Golgi apparatus is a structurally complex and highly dynamic organelle that is found at the crossroads of the secretory and endosomal membrane traYcking pathways.

The mechanisms that generate and maintain Golgi structure are not well understood. However, activation of the ADPribosylation factor 1 (Arf1) small G protein is absolutely required to maintain Golgi structure and function in eukaryotic cells, since inhibiting Arf1 activation by drugs or mutations causes complete disassembly of the Golgi apparatus and a complete block in traYcking pathways through the Golgi (Klausner et al. 1992; Jackson 2009 ). Membrane traYcking in eukaryotic cells is mediated by transport vesicles that bud from a donor compartment, then are targeted to and fuse with an acceptor compartment (Bonifacino and Glick 2004; Behnia and Munro 2005) . Arf1 is required for vesicle budding, where its activation results in recruitment of eVectors such as coat complexes to membranes. Coat complexes deform membranes and concentrate cargo into a membrane domain to form a vesicle that carries the cargo from the donor to acceptor compartment. Targeting of a vesicle to its correct acceptor compartment membrane requires tethering molecules that link the vesicle and target membranes at a distance. A series of steps ensues that result in engagement of SNARE proteins, which mediate membrane fusion (Bonifacino and Glick 2004) . The proteins involved in vesicle budding, cargo sorting, tethering, and fusion are highly conserved in evolution (Behnia and Munro 2005) .

Recent advances in genome sequencing eVorts have allowed deWnition of six major supergroups of eukaryotes (Adl et al. 2005) . Interestingly, phylogenetic studies have indicated that the Last Common Eukaryotic Ancestor (the LCEA) possessed a complex endomembrane system and that many of the major families of proteins involved in traYcking arose prior to the divergence of the eukaryotic supergroups (Jekely 2003; Dacks and Field 2007; Gurkan et al. 2007; Kloepper et al. 2007; Pereira-Leal 2008) . The LCEA probably already had small G proteins including primordial Arf and Rab proteins, coat complexes, membrane tethering complexes, and SNAREs (Gurkan et al. 2007; Koumandou et al. 2007; Kloepper et al. 2008; Pereira-Leal 2008; Dacks et al. 2009 ).

There are nine subfamilies of Sec7 domain Arf guanine nucleotide exchange factors (GEFs) in eukaryotes (Cox et al. 2004) . In this review, we will focus on two of these subfamilies, the GBF/Gea and BIG/Sec7 GEFs. Other wellstudied subfamilies of Arf GEFs include the ARNO/cytohesin, SYT1, SYT2, EFA6, and BRAG subfamilies, which are in general smaller proteins, and which function primarily in endosomal-PM traYcking pathways Casanova 2007; Gillingham and Munro 2007) . Of the nine subfamilies of Arf GEFs, only those of the GBF/Gea, BIG/Sec7, ARNO/cytohesin, and RalF subfamilies have signiWcant activity on the class I Arfs, Arf1-Arf3. RalF is the founding member of a family of bacterial Arf1 GEFs, Wrst found in Legionella pneumophila and Rickettsia prowazekii (Nagai et al. 2002; Amor et al. 2005 ).

These proteins do not arise from the bacteria themselves, but were incorporated into these bacterial genomes through horizontal transfer from their eukaryotic host (Nagai et al. 2002 ). We will not discuss this special subfamily of Arf1 GEFs in this review. Among the eukaryotic Arf1 GEFs, there has been some controversy as to whether ARNO/ cytohesin GEFs actually use class I Arfs as substrates in vivo, as they localize primarily to the endosomal-PM system where Arf6 functions. However, a key paper from the Donaldson laboratory has provided new insights into this issue through identiWcation of a GTPase cascade involving both Arf1 and Arf6 in cells (Cohen et al. 2007) . Cohen et al. demonstrated that interaction of Arf6-GTP with the PH domain of ARNO is essential for its recruitment to the PM, where it then activates Arf1. Structural and biochemical analyses showing release of an autoinhibitory interaction within ARNO by Arf6-GTP provide further molecular insight into this regulatory cascade (DiNitto et al. 2007) . Hence, although the ARNO/cytohesin proteins are bona Wde Arf1 GEFs, we will focus this review on the GBF/Gea and BIG/Sec7 Arf1 GEFs, which function in Golgi and endosomal membrane traYcking pathways.

The GBF/Gea and BIG/Sec7 subfamilies are related, with members sharing sequence similarity principally in Wve homology domains (Mouratou et al. 2005) . The majority of these Arf1 GEFs are high molecular weight proteins, on the order of 200 kDa, and for this reason they have been referred to as the large Arf GEFs. The GBF/Gea and BIG/ Sec7 Arf1 GEFs function in internal membrane systems such as the Golgi apparatus, the trans-Golgi network (TGN) and endosomal pathways (Zhao et al. 2002; Park et al. 2005; Casanova 2007; Gillingham and Munro 2007) .

Although the GBF/Gea and BIG/Sec7 Arf1 GEFs share a common domain structure, they clearly form two distinct subfamilies (Cox et al. 2004; Mouratou et al. 2005) . Members of each subfamily have diVerent localizations and functions, with GBF/Gea GEFs in general functioning in the ER-early Golgi membrane system and BIG/Sec7 GEFs functioning in the late-Golgi and endosomal membrane systems. Mammalian GBF1 localizes primarily to the intermediate compartment between the ER and the Golgi (ERGIC) and to cis-Golgi membranes (Kawamoto et al. 2002; Zhao et al. 2002; , and yeast Gea1p and Gea2p localize to the cis-Golgi (Chantalat et al. 2003) . In plants, all the Arf GEFs fall into the GBF/Gea and BIG/Sec7 subfamilies, so the situation is more complicated. Like its yeast and mammalian counterparts, the A. thaliana Arf1 GEF GNL1 localizes to and functions at the cis-Golgi, but another member of the GBF/Gea subfamily, GNOM, localizes to and functions in the endosomal membrane system, although it still retains a capacity to function in the early Golgi (Richter et al. 2007 ). To compensate for the lack of endosomal-PM Arf GEFs, plants have thus diversi-Wed the functions of the GBF/Gea subfamily members, and possibly those of the BIG/Sec7 subfamily as well. In mammals and yeast, the BIG/Sec7 proteins localize to and function at the trans-Golgi, the TGN and endosomes (FranzusoV et al. 1991; Shinotsuka et al. 2002a; Shinotsuka et al. 2002b; Zhao et al. 2002; Charych et al. 2004; .

In this review, we will discuss the evolution of these Arf1 GEFs, and present an analysis of conserved domains that are common to both subfamilies as well as those that are speciWc to either the GBF/Gea or BIG/Sec7 proteins. We will describe the interacting partners of these Arf1 GEFs, indicating which of the homology regions of these proteins are involved in each interaction. Finally, we will describe studies that have implicated the Arf1 GEFs in human disease, including recent studies describing speciWc inhibitors of members of the GBF/Gea subfamily of Arf1 GEFs.

The number of complete genome sequences has increased dramatically in the past few years, and now are available in public databanks for a wide variety of both prokaryotes and eukaryotes. Analyses of these genome sequences have indicated that there are six major supergroups of eukaryotes that diverged from the LCEA (Adl et al. 2005) . These supergroups are the Excavata, the Chromalveolata, the Archaeplastidia, the Opisthokonta, the Amoebozoa, and the Rhizaria. Fungi, including the well-studied model system Saccharomyces cerevisiae and mammals, are part of the same supergroup, the Opisthokonta. Complete genome sequences from members of Wve of the six eukaryotic supergroups are available, with the Rhizaria being the one exception. We searched the complete genome sequences of representative members of these Wve supergroups for members of the two subfamilies of large Arf1 GEFs, and found members of both the GBF/Gea and the BIG/Sec7 proteins (Tables 1, 2). Humans have two BIG/Sec7 proteins, called BIG1 and BIG2, and only one GBF/Gea subfamily member, GBF1. Arabidopsis, which like other plant species of the Archaeplastida, have only GBF/Gea and BIG/Sec7 Arf1 GEF subfamilies and no others, have Wve BIGs and three GBFs. Paramecium tetraurelia, a protozoan that belongs to the Chromalveolata supergroup, has nine BIG/Sec7 members, but no GBF/Gea sequences. The model organism Saccharomyces cerevisiae has one member of the BIG/Sec7 subfamily, Sec7p, and two members of the GBF/Gea subfamily, Gea1p and Gea2p (likely due to a recent genome duplication event). The human pathogen Cryptosporidium parvum, a member of the Apicomplexa, has no BIG/Sec7 proteins, and only one GBF/Gea sequence. The C. parvum GBF protein shares a high level of homology with plant GBF proteins such as the Arabidopsis thaliana GNOM protein. The Apicomplexa are characterized by the apicoplast, an organelle which is thought to be the remnant of an endosymbiotic algal cell (Abrahamsen et al. 2004; Huang et al. 2004) . It is likely that the C. parvum GBF gene, like other plant-like genes in this organism, was transferred from the primordial endosymbiosed algal cell to the nucleus of the Apicomplexan ancestor that engulfed it (Huang et al. 2004 ). The closely related Cryptosporidium hominis species has a very divergent Arf GEF that appears neverthe-less to be a member of the GBF/Gea subfamily (Xu et al. 2004) .

Phylogenetic analysis of the BIG/Sec7 and GBF/Gea subfamilies of Arf GEFs using members from Wve eukaryotic supergroups indicates a clear separation of these two subfamilies (Fig. 1) . The entire Arf GEF sequences were used for the phylogenetic analysis, not just the Sec7 domains as in previous studies (Cox et al. 2004; Mouratou et al. 2005) . Using three diVerent programs, the same tree topology was obtained, with high conWdence levels for a separation of the BIG/Sec7 and GBF/Gea sequences into two diVerent clades (Fig. 1) . Hence the presence of two subfamilies of large Arf GEFs is ancient, and it is likely that both GBF/Gea and BIG/Sec7 proteins existed within the LCEA before the separation of the diVerent eukaryotic lineages. The nine protozoan Paramecium tetraurelia Arf GEFs, including the Wve denoted GGG1-5, clearly fall into the BIG/Sec7 subfamily (Fig. 1 ).

As described previously, the large Arf1 GEFs have Wve major sequence homology domains that are common to members of both the GBF/Gea and the BIG/Sec7 subfamilies (Mouratou et al. 2005) . These domains are the DCB and the HUS domains upstream of the catalytic Sec7 domain, and HDS1, HDS2, and HDS3 that lie downstream of the Sec7 domain (Fig. 2) . This analysis was based on sequence data from organisms found primarily within two eukaryotic supergroups. When representative members of Wve of the six eukaryotic supergroups are analyzed, the Wve common homology domains are present in the Arf1 GEFs of most of the organisms examined, in both GBF/Gea and BIG/Sec7 subfamily members (Figs. 3, 4, 5, 6, 7, 8) . However, HDS2 and HDS3 are the least conserved domains, particularly for the GBF/Gea proteins, and we were unable to Wnd evidence for these domains in the GBF protein of the Chromalveolata supergroup member C. parvum. Because the GBF/Gea and the BIG/Sec7 proteins have distinct localizations and functions, it is pertinent to ask the question of whether there exist homology regions speciWc to each of the two subfamilies of large Arf1 GEFs. Examination of homology regions both upstream and downstream of the Sec7 domain indicated that among the representatives of all Wve eukaryotic supergroups, only one domain, speciWc to the BIG/ Sec7 subfamily, lies outside of the common domains ( Fig. 10 ). All other regions of high sequence conservation speciWc to either the GBF/Gea or the BIG/Sec7 subfamilies that were present among members of all the eukaryotic supergroups we examined were within the common homology regions DCB, HUS, HDS1, HDS2, and HDS3. Hence it appears that there is a highly conserved function and/or structure of the large Arf1 GEFs that is preserved in both the subfamilies, and speciWc functions are superposed on this common structural organization. Overall, there is a higher level of sequence homology between the BIG/Sec7 subfamily members than among GBF/Gea sequences.

Outside of the catalytic Sec7 domain, which is the most highly conserved domain of the Arf1 GEFs, the N-terminal domains DCB and HUS have the next highest levels of homology, particularly among GBF/Gea family members. As described previously (Ramaen et al. 2007 ), the DCB domain was redeWned to include residues upstream of the originally deWned domain (Mouratou et al. 2005) (Fig. 3) . The most highly conserved region outside of the catalytic Sec7 domain is the "HUS box," a region of seven amino The sequences used for the analysis are shown in Tables 1 and 2. Branches of the tree are color coded to indicate the eukaryotic supergroup that each sequence belongs to: Opisthokonta (red), Archaeplastidia (green), Amoebozoa (blue), Chromalveolata (violet), and Excavata (beige) 0.2

acids near the end of the HUS domain ( Fig. 4) (Mouratou et al. 2005; Park et al. 2005) . Including a wide range of the eukaryotic sequences conWrms the high level of homology of this region, which has the consensus sequence Y/F N Y/F D C D/E/N (where stands for hydrophobic). The highly conserved aspartic acid residue within this motif is invariant in all but one of the sequences examined (Fig. 4) . In vivo, this residue plays an important role in traYcking and in GBF/Gea function in both mammalian cells and in yeast (Park et al. 2005; Ramaen et al. 2007; Deng et al. 2009 ). On either side of this motif, there are residues conserved within each subfamily of the Arf1 GEFs, speciWc either for GBF/Gea or BIG/Sec7 proteins (Fig. 4) .

In the catalytic Sec7 domain, the loop between helices 6 and 7 in yeast, plant, and mammalian large Arf1 GEFs has the invariant sequence FRLPGE, where the Wnal residue is the glutamic acid Wnger that is essential for exchange activity (Goldberg 1998; Beraud-Dufour et al. 1999) . When members of other eukaryotic families are included, only residues F and GE of this motif are invariant (Fig. 5) . SigniWcantly, the catalytic glutamic acid residue is invariant even in this wide range of eukaryotic organisms. Within the Sec7 domain itself, there are several residues that are highly conserved within each subfamily (Fig. 5) . Again, there are more conserved residues among BIG/Sec7 subfamily members than among GBF/Gea sequences.

In the C-terminal homology regions HDS1, HDS2, and HDS3, the BIG/Sec7 sequences had a signiWcantly higher level of homology than the GBF/Gea sequences (Figs. 6, 7, 8) . Both HDS1 and HDS2 are more highly conserved among the BIGs than among the GBFs (Figs. 6, 7) , with the second half of HDS2 showing a particularly high number of conserved residues among BIG/Sec7 proteins of diverse eukaryotic origin (Fig. 7) . Indeed, the NCBI conserved domain database has identiWed a portion of the BIG/Sec7 HDS2 domain as highly conserved and named it DUF1981. In contrast, there are few if any residues in the second half of HDS2 that are conserved among all the GBF/Gea proteins (Fig. 7) .

The HDS3 domain was described previously, but our analysis has indicated that this domain is 50-80 amino acids longer than that proposed by Mouratou et al. (2005) , with the additional homology region located at the C-terminal end of the originally deWned HDS3 domain (Fig. 8) .

Including representatives of the Wve eukaryotic super families indicates that there is only weak homology within this domain (Fig. 8) , but when representative members of only fungi, plants, and mammals are included, a signiWcant level of homology is found throughout the HDS3 domain among members of both BIG/Sec7 (not shown) and GBF/Gea subfamilies (Fig. 9 ).

There is one homology region speciWc to a subfamily of the large Arf1 GEFs, which we call HDS4, and which is present only among BIG/Sec7 proteins (Fig. 10) . No equivalent homology region can be found in the GBF/Gea proteins. We found HDS4 sequences in eukaryotic supergroups other than the Opisthokonta (animals, fungi) and Archaeplastida (plants, algae), but with less homology. Both Tetrahymena thermophila and Paramecium tetraurelia BIG sequences have a region homologous to amino acids 1,675-1,740 of the human BIG1 HDS4 domain (Fig. 2 , data not shown). However, we did not Wnd evidence for an HDS4 domain in the Amoebozoa or Excavata supergroup members Dictyostelium discoideum and Trichomonas vaginalis, respectively.

The highly conserved sequence homology domains of the GBF/Gea and the BIG/Sec7 Arf1 GEFs suggest that they have important, conserved functions in evolution, and are likely to be involved in protein-protein interactions. Indeed, studies from a number of laboratories have Fig. 10 is conserved in the Chromalveolata members that we analyzed (amino acid residues 1,600-1,665 of the Tetrahymena thermophila BIG protein, corresponding to amino acids 1,675-1,740 of human BIG1). The orange box within the HUS domain represents the highly conserved HUS box, sequence Y/F N Y/F D C D/E/N ( : hydrophobic) Fig. 3 Conserved residues within the N-terminal region of the BIG/ Sec7 and GBF/Gea subfamilies of Arf GEFs containing the DCB domain. Multiple sequence alignment showing conserved residues speciWc to the BIG/Sec7 subfamily (pink), speciWc to the GBF/Gea subfamily (blue), or both subfamilies (yellow). Invariant residues in all the sequences are shown in red. The most highly conserved portion of this region contains the DCB domain. Secondary structure prediction of alpha-helical regions is shown above alignment in pink for BIG/Sec7 sequences, and below alignment in blue for GBF/Gea sequences. Deletions in sequences are indicated by red Xs, and correspond to: S18-G217, S319-P336 in SEC7_Sacc, R17-M82, S186-P202 in SEC7_Klul, P106-G109 in BIG_Dicd, V120-H154 in BIG_Ostt, V89-N116 in BIG_Chlr, and K101-D113 in BIG_Tett. Protein sequence alignments were created using Clustal W 1.83 (Thompson et al. 1994) and T-coVee (Notredame et al. 2000) with default parameters. The multiple alignments were manually adjusted and edited using BioEdit version 7.0.8 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The GBF/Gea and BIG/Sec7 alignments (done separately) were imported into BioEdit, then these were manually corrected to correspond to the combined alignment. Aligned sequences were displayed with ESPript (Gouet et al. 1999 ) using the BLOSUM62 matrix with a similarity global score of 0.15 and a diVerence score between conserved groups of 0.5. Secondary structure predictions on multiple alignments were performed at the Pôle Bioinformatique Lyonnais (http://pbil.univ-lyon1.fr/) and the consensus of three diVerent programs (the PHD, Predator, and GOR IV) is indicated. Green box indicates AKAP domain of BIG2 (Table 3) . It is important to point out that many of these interacting partners have been identiWed by yeast two-hybrid screens or by co-immunoprecipitation of proteins from cells, which demonstrate a physical associa-tion, but not a direct protein-protein interaction. Further studies are required to determine whether these interactions are direct or via a bridging protein. Many of the interactions described in Table 3 have been previously reviewed (Cox et al. 2004; ; Mouratou et al. Table 3 . For BIG1, these include the exocyst subunit Exo70 (Xu et al. 2005) , the Myosin IXb molecular motor (Saeki et al. 2005) , and the BIG1 protein itself (Ramaen et al. 2007 ). The interactions with Exo70 and Myosin IXb suggest a role for BIG1 in targeting secretory vesicles to the PM. The BIG1 DCB domain has been demonstrated directly to form a dimer through puriWcation of this domain from E. coli and gel Wltration analysis (Ramaen et al. 2007) . This data conWrmed the initial observations for the Arabidopsis thaliana GNOM Arf1 GEF that the DCB domain is involved in dimerization (Grebe et al. 2000) .

For mammalian GBF1, two proteins have been shown to interact directly, the small G protein Rab1 (Monetta et al. 2007 ) and the COPI coat subunit gamma-COP (Deng et al. 2009 ). Rab1 plays an essential role in traYcking between the ER and cis-Golgi (Behnia and Munro 2005) . Like the Arf small G proteins, Rabs cycle between GDP-and GTPbound forms, and target eVector proteins to membranes when in their active GTP-bound conformation. Rab proteins are primarily involved in membrane fusion, with invariant residues (red). Secondary structure prediction of alpha-helical regions is shown above alignment in pink Islam et al. (2007 Islam et al. ( , 2008 roles in regulation of tethering complexes and the pairing of SNAREs which directly mediate fusion (Behnia and Munro 2005) . GBF1 was shown to be an eVector for Rab1 in vitro, and active Rab1 in cells was shown to increase the association of both GBF1 and COPI to membranes (Monetta et al. 2007 ). Interestingly, another Rab1 eVector, the tethering protein p115, has also been shown to interact with GBF1 (Garcia-Mata and Sztul 2003). p115 not only acts to tether membranes in ER-Golgi traYcking, but also interacts directly with and promotes pairing of the ER-Golgi SNARE proteins, which are required for membrane fusion (Short et al. 2005) . A precise molecular explanation for the Rab1-GBF1-p115 series of interactions has not been demonstrated, but suggests a role for GBF1 in coordination of vesicle budding and fusion processes. The interaction between GBF1 and the gamma-COP subunit of the COPI coat complex is conserved from yeast to humans. This interaction is a direct protein-protein interaction in both systems (Deng et al. 2009 ; MPG-C and CLJ, unpublished data). Studies from several laboratories have indicated that GBF1 is involved speciWcally in recruitment of COPI to early Golgi membranes. Knockdown of GBF1 by RNAi inhibits COPI association with membranes with-out aVecting other coats such as AP-1 (Ishizaki et al. 2008; Manolea et al. 2008; Deng et al. 2009 ). In contrast, knockdown of the BIG1 and BIG2 proteins prevents AP-1 association with TGN membranes, without aVecting COPI (Ishizaki et al. 2008; Manolea et al. 2008) . A model explaining this speciWcity has been proposed, whereby COPI interacts with the Arf1 GEF GBF1 (Gea1p and Gea2p in yeast) prior to nucleotide exchange on Arf1, so that the eVector COPI is in place at the time Arf1-GTP is formed (Deng et al. 2009 ). This model oVers an explanation for the observation that a single Arf protein, Arf1, can recruit at least Wve diVerent coat complexes, including COPI, AP1/clathrin, GGA/clathrin, AP3, and AP4, to diVerent sites within cells (Bonifacino and Lippincott-Schwartz 2003) . Interestingly, all these coat complexes share sequence homology and are structurally similar (Bonifacino and Lippincott-Schwartz 2003; McMahon and Mills 2004) , and function in a speciWc traYcking pathway (Bonifacino and Lippincott-Schwartz 2003; McMahon and Mills 2004) . This is also true of the large Arf1 GEFs, whose function is required to recruit these coat complexes to membranes. One of the regions of gamma-COP that interacts with the Arf1 GEFs is the appendage domain (Deng et al. 2009 ), a region that is structurally conserved in all Wve coats recruited to membranes by Arf1-GTP (McMahon and Mills 2004) . These results suggest the possibility that the diVerent Arf1 GEFs within these two subfamilies regulate coat assembly via a common mechanism involving GEF-coat interactions. An interaction between GBF1 and GGA coat complexes has been proposed, although it has not been determined if this interaction is direct (Lefrancois and McCormick 2007) (Table 3) . GBF1 functions and localizes to the cis-Golgi, whereas the GGA proteins function and localize primarily to the trans-Golgi and TGN (Hirst et al. 2001; Puertollano et al. 2001; Mattera et al. 2003; Nakayama and Wakatsuki 2003; Puertollano et al. 2003) . Severe perturbations of the early Golgi caused by interfering with GBF1 function also aVect late-Golgi functions, where the GGA proteins act. Hence, the results of this study are likely due to a more indirect mechanism than by direct interaction of GBF1 and the GGAs.

An important role for the Arf1 GEFs in cell signaling has emerged with the identiWcation of BIG1 and BIG2 as A-kinase anchoring proteins or AKAPs. AKAPs are PKA regulatory subunit binding proteins that act as scaVolds to compartmentalize signaling cascades. This function is accomplished through formation of multi-protein complexes that spatially restrict the PKA-cAMP signaling cascade at speciWc membrane sites within cells. BIG1 and BIG2 both contain AKAP domains that mediate binding of the regulatory subunit RII of protein kinase A (PKA) (Figs. 2, 3) , and BIG1 accumulates in nuclei upon increase in cAMP levels in cells (Li et al. 2003; Padilla et al. 2004; Citterio et al. 2006) . Both BIG1 and BIG2 immunoprecipitated from HepG2 cells can be phosphorylated by recombinant PKA, and this results in a decrease in GEF activity (Kuroda et al. 2007) . One PKA phosphorylation site in BIG1 has been identiWed, at the C-terminus of the Sec7 domain (Li et al. 2003) (Fig. 4) . Furthermore, endogenous protein phosphatase 1-(PP1-) was found in a complex with BIG1 or BIG2 in microsomal fractions, indicating that a phosphatase of this signaling pathway is physically associated with BIG1 and BIG2 GEFs (Kuroda et al. 2007) . A recent study from this group has shown that another regulatory enzyme in this cascade, phosphodiesterase PDE3A, is functionally associated with BIG1 and BIG2 (Puxeddu et al. 2009 ). PDE enzymes break down cAMP to terminate cAMP signaling, reversing its eVects on BIG1 and BIG2. Puxeddu et al. showed that PDE3A (but not PDE4) co-immunoprecipitates with both BIG1 and BIG2, and that knockdown of PDE3A led to a partial redistribution of BIG1 and BIG2 from Golgi membranes to cytosol. In addition to the cAMP-PKA regulatory proteins themselves, an AKAP-binding protein AMY-1 has also been found to associate with the RII -binding (AKAP) domains of both BIG1 and BIG2 (Ishizaki et al. 2006) . In cells, AMY-1 TGN localization is abrogated in cells depleted of BIG2, but not of BIG1, indicating that BIG2 is the physiological partner of AMY-1 at the TGN (Ishizaki et al. 2006) .

What are the functional consequences of BIG1 and BIG2 involvement as AKAPs in cAMP-PKA signaling? For BIG1, phosphorylation by PKA results in its redistribution from the Golgi and cytoplasm to the nucleus (Citterio et al. 2006 ). The precise function of BIG1 in the nucleus is not known, but it involves interactions with nuclear proteins including nucleolin, Wbrillarin and RNA-binding protein La, as well as the small nuclear RNA snoU3 (Padilla et al. 2008) .

The type I tumor necrosis factor receptor (TNFR1) is one of two receptors for TNF that mediates its eVects on inXammation, apoptosis, and the innate immune response. TNFR1 can be released from cells by incorporation into exosome-like vesicles which are shed from the surface of cells (Hawari et al. 2004) . BIG2 has been shown to play a role in regulating this process by two mechanisms, one involving its GEF activity, the other through its AKAP domains. Activation of Arf1 and Arf3 by BIG2 is required for extracellular release of TNFR1 in exosome-like vesicles, and involves a physical association between BIG2 and TNRF1 (Islam et al. 2007 ). In addition, cAMP-dependent protein kinase A signaling induces TNFR1 exosomelike vesicle release through binding of PKA regulatory subunit RII to AKAP domains of BIG2 (Islam et al. 2008 ).

Mutations in the BIG2 Arf1 GEF have been linked to autosomal recessive periventricular heterotopia (ARPH), a disorder of neuronal migration that leads to severe malformation of the cerebral cortex (microcephaly) and severe developmental delay (Sheen et al. 2004) . Two BIG2 disease alleles have been identiWed, including a frameshift mutation that results in truncation of the majority of the protein (Sheen et al. 2004 ). The disease symptoms are a result of the failure of a speciWc class of neurons to migrate from their point of origin in the lateral ventricular proliferative zone to the cerebral cortex (Sheen et al. 2004; Ferland et al. 2009 ). This defect arises from a defect in vesicular traYcking that alters the adhesion properties of these neurons (Ferland et al. 2009 ). Interestingly, mutations in other traYcking proteins can give rise to periventricular heterotopia in humans or in model systems, indicating that traYcking defects are likely the root cause of this disease (Ferland et al. 2009 ). Hence, the Arf1 GEFs not only serve essential roles in fundamental cell biological processes, but also play an important role in human development and neuronal function (Sheen et al. 2004) .

The Arf1 GEFs have been shown to act as host factors for pathogens mediating human disease. Replication of several viruses has been shown to require GBF1, including poliovirus, coxsackievirus, and coronavirus. Poliovirus and coxsackievirus are enteroviruses belonging to the Picornaviridae family of positive strand non-enveloped RNA viruses. Despite the fact that enteroviruses are not enclosed by a membrane, their replication never-the-less depends completely on host cell membranes. Upon infection, enteroviruses cause a massive reorganization of the intracellular membranes of their host, including ER and Golgi membranes (Bienz et al. 1983; Salonen et al. 2005) . Replication of poliovirus and coxsackievirus is completely inhibited by BFA, and it has been shown that GBF1 is the major target of this drug in poliovirus replication (Belov et al. 2007a; Belov et al. 2008) . The enteroviral 3A protein has also been shown to block secretion in host cells, which inhibits the innate immune response of the host (Doedens et al. 1997; Wessels et al. 2006) . This block in secretion also occurs via GBF1, through physical association of 3A with GBF1 which inhibits its function on Golgi membranes in the host cell (Wessels et al. 2006; Wessels et al. 2007 ). Hence, polio and coxsackieviruses subvert the host membrane by blocking GBF1 activity on host cell Golgi membranes, reorganizing these membranes, then activating GBF1 function on Arf1 for viral replication on these virally induced membrane structures.

Although a major role for GBF1 in enteroviral replication has been demonstrated, there is evidence that the BIG1 and the BIG2 Arf1 GEFs are also involved. Interestingly, diVerent viral proteins have been shown to recruit diVerent coats to virally reorganized membranes. In an in vitro system, it has been shown that the 3A protein recruits GBF1 to membranes (Belov et al. 2008) , whereas the viral 3CD protein speciWcally recruits BIG1 and BIG2 (Belov et al. 2007b) . Furthermore, 3A recruitment of GBF1 results in speciWc binding of COPI to membranes (Belov et al. 2008) , whereas, 3CD recruits the GGA3 coat to membranes (Belov et al. 2007b) . These observations support an intimate coupling between recruitment of speciWc GEFs with speciWc coats to membranes, and provide another line of evidence supporting a connection between GBF1 and COPI, and also a connection between BIG1/BIG2 and the late-Golgi coat GGA3.

In addition to polio and coxsackieviruses, the RNA replication of a number of positive-strand viruses is BFA-sensitive (Gazina et al. 2002; Molina et al. 2007; Tai et al. 2009 ). Among these is Hepatitis C virus (HCV), which has been shown recently to depend also on the Arf1 eVectors COPI and phosphatidylinositol-4-kinase for its replication (Tai et al. 2009 ). These results strongly suggest that GBF1 is a required host factor for HCV replication. BFA also inhibits replication of other families of viruses, including the enveloped positive-strand RNA coronaviruses (Verheije et al. 2008) . For one member of the coronaviruses, mouse hepatitis coronavirus, GBF1 and Arf1 have been demonstrated to be essential host factors for its RNA replication (Verheije et al. 2008) .

Given the important roles of the Arf1 GEFs of the GBF/ Gea and BIG/Sec7 subfamilies in human disease, development of drugs that speciWcally target these proteins is of medical interest. As mentioned above, BFA is a natural substance that is a potent and speciWc inhibitor of members of both of these subfamilies. Recently, a chemical (Golgicide A) which speciWcally inhibits GBF1, but not BIG1 or BIG2, has been described (Saenz et al. 2009 ). Golgicide A was identiWed in a screen for inhibitors of the cytotoxicity of Shiga toxin, a protein produced by the pathogenic bacterium Shigella dysenteriae (Saenz et al. 2009 ). This discovery, in addition to providing an important inhibitor of GBF1, demonstrates a crucial role for this Arf1 GEF as a host factor in S. dysenteriae infection.

Complete genome sequence of the apicomplexan, Cryptosporidium parvum

The new higher level classiWcation of eukaryotes with emphasis on the taxonomy of protists

The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site

Large ARF guanine nucleotide exchange factors in membrane traYcking

Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

Organelle identity and the signposts for membrane traYc

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

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

A critical role of a cellular membrane traYc protein in poliovirus RNA replication

Dual interaction of ADP ribosylation factor 1 with Sec7 domain and with lipid membranes during catalysis of guanine nucleotide exchange

Intracellular distribution of poliovirus proteins and the induction of virus-speciWc cytoplasmic structures

The mechanisms of vesicle budding and fusion

Coat proteins: shaping membrane transport

Regulation of Arf activation: the sec7 family of Guanine nucleotide exchange factors

A novel Golgi membrane protein is a partner of the ARF exchange factors Gea1p and Gea2p

The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular traYcking, interacts with the beta subunits of the GABA receptors

EVect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei

Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains

Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers

Evolution of the eukaryotic membrane-traYcking system: origin, tempo and mode

Evolution of speciWcity in the eukaryotic endomembrane system

Sec7p directs the transitions required for yeast Golgi biogenesis

A COPI coat subunit interacts directly with an early-Golgi localized Arf exchange factor

Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors

Inhibition of endoplasmic reticulum-to-Golgi traYc by poliovirus protein 3A: genetic and ultrastructural analysis

Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia

Localization of components involved in protein transport and processing through the yeast Golgi apparatus

The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange factor

ADP-ribosylation factor/COPI-dependent events at the endoplasmic reticulum-Golgi interface are regulated by the guanine nucleotide exchange factor GBF1

DiVerential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

The small G proteins of the Arf family and their regulators

Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching

ESPript: analysis of multiple sequence alignments in PostScript

A conserved domain of the Arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding

An evolutionary perspective on eukaryotic membrane traYcking

Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors

GGAs: roles of the diVerent domains and comparison with AP-1 and clathrin

A Wrst glimpse into the pattern and scale of gene transfer in Apicomplexa

AMY-1 (associate of Myc-1) localization to the trans-Golgi network through interacting with BIG2, a guanine-nucleotide exchange factor for ADP-ribosylation factors

Redundant roles of BIG2 and BIG1, guanine-nucleotide exchange factors for ADP-ribosylation factors in membrane traYc between the trans-Golgi network and endosomes

The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles

cAMP-dependent protein kinase A (PKA) signaling induces TNFR1 exosome-like vesicle release via anchoring of PKA regulatory subunit RIIbeta to BIG2

Mechanisms of transport through the Golgi complex

Small GTPases and the evolution of the eukaryotic cell

GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors, is localized to the cis-Golgi and involved in membrane association of the COPI coat

Brefeldin A: insights into the control of membrane traYc and organelle structure

An elaborate classiWcation of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system

SNAREing the basis of multicellularity: consequences of protein family expansion during evolution

Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins

Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) and BIG2 activity via PKA and protein phosphatase 1 gamma

The Arf GEF GBF1 is required for GGA recruitment to Golgi membranes

Protein kinase A-anchoring (AKAP) domains in brefeldin Ainhibited guanine nucleotide-exchange protein 2 (BIG2)

Distinct functions for Arf guanine nucleotide exchange factors at the Golgi complex: GBF1 and BIGs are required for assembly and maintenance of the Golgi stack and trans-Golgi network, respectively

Divalent interaction of the GGAs with the Rabaptin-5-Rabex-5 complex

COP and clathrin-coated vesicle budding: diVerent pathways, common approaches

AMP-activated protein kinase phosphorylates Golgi-speciWc brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus

DiVerential inhibition of cellular and Sindbis virus translation by brefeldin A

Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes

The structure and function of GGAs, the traYc controllers at the TGN sorting crossroads

T-CoVee: A novel method for fast and accurate multiple sequence alignment

Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): eVects of FK506

Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors

Association of guanine nucleotide-exchange protein BIG1 in HepG2 cell nuclei with nucleolin, U3 snoRNA, and Wbrillarin

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes

The Ypt/Rab family and the evolution of traYcking in fungi

Functional analysis of ADP-ribosylation factor (ARF) guanine nucleotide exchange factors Gea1p and Gea2p in yeast

The GGAs promote ARF-dependent recruitment of clathrin to the TGN

Morphology and dynamics of clathrin/ GGA1-coated carriers budding from the trans-Golgi network

Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and eVect on ARF1 activity

Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors

Functional diversiWcation of closely related ARF-GEFs in protein secretion and recycling

BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity

Golgicide A reveals essential roles for GBF1 in Golgi assembly and function

Viral RNA replication in association with cellular membranes

Mutations in ARFGEF2 implicate vesicle traYcking in neural progenitor proliferation and migration in the human cerebral cortex

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1

Interaction of brefeldin A-inhibited guanine nucleotideexchange protein (BIG) 1 and kinesin motor protein KIF21A

Guanine nucleotide-exchange factors for Arf GTPases: their diverse functions in membrane traYc

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor, speciWcally aVects membrane traYcking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation

Golgins and GTPases, giving identity and structure to the Golgi apparatus

A functional genomic screen identiWes cellular cofactors of hepatitis C virus replication

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speciWc gap penalties and weight matrix choice

Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation

A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1

Molecular determinants of the interaction between coxsackievirus protein 3A and guanine nucleotide exchange factor GBF1

The genome of Cryptosporidium hominis

Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70

Localization of large ADPribosylation factor-guanine nucleotide exchange factors to diVerent Golgi compartments: evidence for distinct functions in protein traYc

Acknowledgments This study was supported by a Chaire d'Excellence from the Agence Nationale de la Recherche, France. We thank Jacqueline CherWls and Valerie Biou for assistance with bioinformatics programs and for critical reading of the manuscript.