Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.

Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.     

Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers

The eukaryotic family of ADP-ribosylation factor (Arf) GTPases plays a key role in the regulation of protein trafficking, and guanine-nucleotide exchange is crucial for Arf function. Exchange is stimulated by members of another family of proteins characterized by a 200-amino acid Sec7 domain, which alone is sufficient to catalyze exchange on Arf. Here, we analyzed the phylogeny of Sec7-domain-containing proteins in seven model organisms, representing fungi, plants, and animals. The phylogenetic tree has seven main groups, of which two include members from all seven model systems. Three groups are specific for animals, whereas two are specific for fungi. Based on this grouping, we propose a phylogenetically consistent set of names for members of the Sec7-domain family. Each group, except for one, contains proteins with known Arf exchange activity, implying that all members of this family have this activity. Contrary to the current convention, the sensitivity of Arf exchange activity to the inhibitor brefeldin A probably cannot be predicted by group membership. Multiple alignment reveals group-specific domains outside the Sec7 domain and a set of highly conserved amino acids within it. Determination of the importance of these conserved elements in Arf exchange activity and other cellular functions is now possible.     

Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism

ARF GTPases are activated by guanine nucleotide exchange factors (GEFs) of the Sec7 family that promote the exchange of GDP for GTP. Brefeldin A (BFA) is a fungal metabolite that binds to the ARF1*GDP*Sec7 complex and blocks GEF activity at an early stage of the reaction, prior to guanine nucleotide release. The crystal structure of the ARF1*GDP*Sec7*BFA complex shows that BFA binds at the protein-protein interface to inhibit conformational changes in ARF1 required for Sec7 to dislodge the GDP molecule. Based on a comparative analysis of the inhibited complex, nucleotide-free ARF1*Sec7 and ARF1*GDP, we suggest that, in addition to forcing nucleotide release, the ARF1-Sec7 binding energy is used to open a cavity on ARF1 to facilitate the rearrangement of hydrophobic core residues between the GDP and GTP conformations. Thus, the Sec7 domain may act as a dual catalyst, facilitating both nucleotide release and conformational switching on ARF proteins.     

Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain

We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p. Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro. We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.     

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ～200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.     

Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that ate regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (~180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of ~200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6.     

Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6

ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.     

Distinct regulations of ARF1 by two Aplysia Sec7 isoforms

Sec7 protein is a guanine nucleotide exchange factor in the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Aplysia Sec7 proteins (ApSec7s) play many roles in neurite outgrowth and synaptic facilitation in Aplysia neurons. However, the binding property of Aplysia ARF1 by ApSec7 isoforms has not been examined. In this study, we found that the cloned Aplysia ARF1 (ApARF1) protein only localized to the Golgi complex when it was expressed alone in HEK293T cells; however, if ApARF1 was co-expressed with plasma membrane-targeted ApSec7, it localized to both the plasma membrane and the Golgi complex via association with the Sec7 domain of ApSec7. Moreover, in HEK293T cells expressing both ApARF1 and another Sec7 isoform, ApSec7(VPKIS), the pleckstrin homology domain of ApSec7(VPKIS) associated with ApARF1, resulting in its localization to the Golgi complex. Overall, we propose a model in which ApSec7(VPKIS) activates ApARF1 in the Golgi complex, while ApSec7 recruits ApARF1 to the plasma membrane where it activates ApARF1/6 downstream signaling.     

ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.     

Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.     

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at 相似文献   

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

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

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.     

Mechanism of domain closure of Sec7 domains and role in BFA sensitivity

Activation of small G proteins of the Arf family is initiated by guanine nucleotide exchange factors whose catalytic Sec7 domain stimulates the dissociation of the tightly bound GDP nucleotide. The exchange reaction involves distinct sequential steps that can be trapped by the noncompetitive inhibitor brefeldin A, by mutation of an invariant catalytic glutamate, or by removal of guanine nucleotides. Arf-GDP retains most characteristics of its GDP-bound form at the initial low-affinity Arf-GDP-Sec7 step. It then undergoes large conformational changes toward its GTP-bound form at the next step, and eventually dissociates GDP to form a nucleotide-free high-affinity Arf-Sec7 complex at the last step. Thus, Arf proteins evolve through different conformations that must be accommodated by Sec7 domains in the course of the reaction. Here the contribution of the flexibility of Sec7 domains to the exchange reaction was investigated with the crystal structure of the unbound Sec7 domain of yeast Gea2. Comparison with Gea2 in complex with nucleotide-free Arf1 Delta 17 [Goldberg, J. (1998) Cell 95, 237-248] reveals that Arf induces closure of the two subdomains that form the sides of its active site. Several residues that determine sensitivity to brefeldin A are involved in interdomain and local movements, pointing to the importance of the flexibility of Sec7 domains for the inhibition mechanism. Altogether, this suggests a model for the initial steps of the exchange reaction where Arf docks onto the C-terminal domain of the Sec7 domain before closure of the N-terminal domain positions the catalytic glutamate to complete the reaction.     

A Sec7-related protein in Paramecium.

We have cloned and sequenced a SEC7-related gene in Paramecium tetraurelia that contains an open reading frame for 1135 amino acids encoding a 133 kDa protein, PSec7. Sec7, first identified in vesicular trafficking mutants in yeast, and its phylogenetic homologues function as guanine-nucleotide exchange factors for small G-proteins such as ARF (ADP-ribosylation factor). The deduced amino acid sequence in PSec7 for the motifs that form the ARF binding site are more than 70% identical to yeast Sec7 and similarly identical to ARNO, the human ARF exchange factor, with correct positioning of the critical glutamic acid residue within the motif region. Overall, the identity of PSec7 to yeast Sec7 is 32%. The deduced amino acid sequence also has five sequences that resemble IQ motifs, EF hand binding domains found in all myosins, and two pleckstrin homology domains. Similar sequences are present in yeast Sec7 and other Sec7-related molecules. A protein kinase A phosphorylation site may also be present. Southern blots suggest that a single gene encodes PSec7. Northern blots show that the message encoding PSec7 is induced on deciliation, followed by ciliogenesis, which suggests a role for PSec7 in cilia such as transport or targeting of ciliary membrane components.     

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.     

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.     

The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP

Cytohesin is a guanine nucleotide exchange factor that regulates members of the ADP-ribosylation factor (ARF) family of small GTPases. All of the members of the cytohesin family (including ARNO, ARNO3, and the newly characterized cytohesin-4) have a similar domain distribution consisting of a Sec7 homology domain, a pleckstrin homology domain, and an N-terminal coiled coil. In this study, we attempt to identify proteins that interact specifically with the coiled coil motif of cytohesin. Yeast two-hybrid screening of a B cell library using the cytohesin N terminus as bait, identified CASP, a scaffolding protein of previously unknown function, as a binding partner. CASP contains an internal coiled coil motif that is required for cytohesin binding both in vitro and in COS-1 cells. The specificity of the coiled coil of CASP is not restricted to cytohesin, however, because it is also capable of interacting with other members of the cytohesin/ARNO family, ARNO and ARNO3. In immunofluorescence experiments, CASP localizes to perinuclear tubulovesicular structures that are in close proximity to the Golgi. These structures remain relatively undisturbed when the cells are treated with brefeldin A. In epidermal growth factor-stimulated COS-1 cells overexpressing cytohesin and CASP, cytohesin recruits CASP to membrane ruffles, revealing a functional interaction between the two proteins. These observations collectively suggest that CASP is a scaffolding protein that facilitates the function of at least one member of the cytohesin/ARNO family in response to specific cellular stimuli.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.     

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

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