Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis.
(9/1111)
The crystal structure of the complex of ARF1 GTPase bound to GDP and the catalytic domain of ARF GTPase-activating protein (ARFGAP) has been determined at 1.95 A resolution. The ARFGAP molecule binds to switch 2 and helix alpha3 to orient ARF1 residues for catalysis, but it supplies neither arginine nor other amino acid side chains to the GTPase active site. In the complex, the effector-binding region appears to be unobstructed, suggesting that ARFGAP could stimulate GTP hydrolysis while ARF1 maintains an interaction with its effector, the coatomer complex of COPI-coated vesicles. Biochemical experiments show that coatomer directly participates in the GTPase reaction, accelerating GTP hydrolysis a further 1000-fold in an ARFGAP-dependent manner. Thus, a tripartite complex controls the GTP hydrolysis reaction triggering disassembly of COPI vesicle coats. (+info)
Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain.
(10/1111)
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. (+info)
Expression and distribution of adenosine diphosphate-ribosylation factors in the rat kidney.
(11/1111)
BACKGROUND: Adenosine diphosphate (ADP)-ribosylation factors (ARFs) are small guanosine triphosphatases involved in membrane traffic regulation. Aiming to explore the possible involvement of ARF1 and ARF6 in the reabsorptive properties of the nephron, we evaluated their distribution along the different renal epithelial segments. METHODS: ARFs were detected by immunofluorescence and immunogold cytochemistry on renal sections, using specific anti-ARF antibodies. RESULTS: ARF1 was detected in proximal and distal tubules, thick ascending limbs of Henle's loops, and cortical and medullary collecting ducts. By immunofluorescence, labeling was mostly localized to the cell cytoplasm, particularly in Golgi areas. By electron microscopy, the Golgi apparatus and the endosomal compartment of proximal and distal tubular cells were labeled. ARF6 immunofluorescence was observed in brush border membranes and the cytoplasm of proximal convoluted tubular cells, whereas it was restricted to the apical border of proximal straight tubules. ARF6 immunogold labeling was detected over microvilli and endocytic compartments of proximal tubular cells. CONCLUSIONS: This study demonstrates the following: (a) the heterogeneous distributions of ARF1 and ARF6 along the nephron, (b) the existence of cytosolic and membrane-bound forms for both ARFs, and (c) their association with microvilli and endocytic compartments, suggesting an active participation in renal reabsorption. (+info)
Purification and cloning of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors.
(12/1111)
Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport. (+info)
Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1.
(13/1111)
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. (+info)
ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes.
(14/1111)
The assembly of clathrin-coated vesicles on Golgi membranes is initiated by the GTP-binding protein ADP ribosylation factor (ARF), which generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex. Once bound, the AP-1 recruits clathrin triskelia, which polymerize to form the coat. We have found that ARF.GTP also recruits AP-1 and clathrin onto protein-free liposomes. The efficiency of this process is modulated by the composition of the liposomes, with phosphatidylserine being the most stimulatory phospholipid. There is also a requirement for cytosolic factor(s) other than ARF. Thin-section electron microscopy shows the presence of clathrin-coated buds and vesicles that resemble those formed in vivo. These results indicate that AP-1-containing clathrin-coated vesicles can form in the absence of integral membrane proteins. Thus, ARF.GTP, appropriate lipids, and cytosolic factor(s) are the minimal components necessary for AP-1 clathrin-coat assembly. (+info)
GTP-dependent binding of ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23.
(15/1111)
A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs. (+info)
Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
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Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL. (+info)