Interspecies relationships among ADP-ribosylation factors (ARFs): Evidence of evolutionary pressure to maintain individual identities

ADP-ribosylation factors (ARFs) are ～20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.     

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.     

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.     

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.     

ADP-ribosylation factors: a family of ∼20-kDa guanine nucleotide-binding proteins that activate cholera toxin

ADP-ribosylation factors (ARFs) comprise a family of 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G_s, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.Abbreviations ARF ADP-ribosylation factor - sARF I and sARF II soluble ADP-ribosylation factors purified from bovine brain - mARF purified membrane-associated ARF - hARF human ARF - bARF bovine ARF - yARF yeast ARF - ARF bacterially-expressed recombinant ARF - gARF Giardia ARF - dARF Drosophila ARF - G protein guanine nucleotide-binding protein - G_s G protein responsible for stimulation of adenylyl cyclase - GTPS guanosine-5-O-(3-thio-triphosphate) - CIAI cholera toxin A1 subunit - DMPC dimyristoylphosphatidylcholine - SDS sodium dodecyl sulfate     

A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis

Although auxin response factors (ARFs) are the first well-characterized proteins that bind to the auxin response elements, elucidation of the roles of each ARF gene in auxin responses and plant development has been challenging. Here we show that ARF19 and ARF7 not only participate in auxin signaling, but also play a critical role in ethylene responses in Arabidopsis (Arabidopsis thaliana) roots, indicating that the ARFs serve as a cross talk point between the two hormones. Both arf19 and arf7 mutants isolated from our forward genetic screens are auxin resistant and the arf19arf7 double mutant had stronger auxin resistance than the single mutants and displayed phenotypes not seen in the single mutants. Furthermore, we show that a genomic fragment of ARF19 not only complements arf19, but also rescues arf7. We conclude that ARF19 complements ARF7 at the protein level and that the ARF7 target sequences are also recognized by ARF19. Therefore, it is the differences in expression level/pattern and not the differences in protein sequences between the two ARFs that determines the relative contribution of the two ARFs in auxin signaling and plant development. In addition to being auxin resistant, arf19 has also ethylene-insensitive roots and ARF19 expression is induced by ethylene treatment. This work provides a sensitive genetic screen for uncovering auxin-resistant mutants including the described arf mutants. This study also provides a likely mechanism for coordination and integration of hormonal signals to regulate plant growth and development.     

ADP-Ribosylation Factors Do Not Activate Yeast Phospholipase Ds but Are Required for Sporulation

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.     

GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.     

Small GTP-binding proteins in vesicular transport

Recent recognition of the abundance of small GTP-binding proteins in eukaryotic cells has sparked off a search for the possible function of these proteins. Evidence is accumulating that SAR1, ARF, SEC4 and YPT1 in yeast and the rab and arf family in mammalian cells play a central role in the regulation of vesicle transport and organelle function.     

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes.

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.     

Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins

A new member, hARF4, of the ADP-ribosylation factor (ARF) family, a subset of the superfamily of regulatory GTP-binding proteins, has been cloned from a cDNA expression library. Two other human ARF cDNA sequences, designated human ARF1 and ARF3, have been reported previously and are 96% identical in amino acid sequence. A human ARF1 cDNA, significantly longer than previously described clones, was obtained, by cross-species hybridization using a bovine ARF1 cDNA probe. Bovine ARF1p and human ARF1p are 100% identical while each is only 80% identical to hARF4p. Thus, hARF4p is the most divergent of the mammalian ARF proteins identified. Northern blot analysis revealed the expression of at least three different ARF messages in human placenta and adrenal carcinoma cells. Both hARF1 and hARF4 encode GTP-binding proteins with predicted molecular masses of 20,000-21,000 Da. Biochemical analysis of the purified recombinant proteins revealed a high degree of conservation of nucleotide binding properties and in vitro ARF activities. ARF is an essential gene in the yeast, Saccharomyces cerevisiae, and is encoded by two genes. Expression of either hARF1p or hARF4p in yeast was found to rescue the lethal double mutant, arf1-arf2-, thus demonstrating the functional conservation of ARF functions between yeast and man. The combination of in vivo and in vitro assays for ARF function provides a specific and unambiguous means of determining bona fide ARF proteins from divergent species from among the rapidly increasing number of structurally related, small molecular weight GTP-binding proteins.     

Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins

Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c. 20 kDa, termed ADP-ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.     

Interaction of the PDZ domain of human PICK1 with class I ADP-ribosylation factors

We have cloned the cDNA encoding human PICK1 (protein interacting with C kinase 1), a PDZ domain-containing protein of 415 amino acids, and also identified the Drosophila homologue by search of the databank. Northern blot analysis shows a single mRNA of about 2.0 kb ubiquitously expressed in human tissues. Although PICK1 proteins harbor a region homologous to arfaptin1 and arfaptin2, two proteins that bind to the ARF (ADP-ribosylation factor), this region of PICK1 does not interact with ARFs in the yeast two-hybrid system. On the other hand, the PDZ domain of PICK1 is capable of interacting with constitutively active, GTP-bound forms of ARF1 and ARF3, but neither with those of ARF5/6 nor with the GDP-bound ARFs. The PICK1-ARF interaction is abrogated by introduction of mutations in the PDZ domain or by deletion of the extreme C-terminus of ARF1. Thus, PICK1 specifically interacts with ARF1/3 in the GTP-bound state, suggesting that PICK1 participates in ARF1/3-mediated cellular processes.     

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.     

Specificity, promiscuity and localization of ARF protein interactions with NCS-1 and phosphatidylinositol-4 kinase-III beta

ADP-ribosylation factor (ARF) proteins are involved in multiple intracellular vesicular transport pathways. Most studies have focused on the functions of ARF1 or ARF6 and little is known about the remaining ARF isoforms. Although the mammalian ARF proteins share a high degree of sequence identity, recent evidence has indicated that they may control distinct trafficking steps within cells. A unanswered issue is the degree of specificity of ARF family members for different interacting proteins. To investigate potential functional differences between the human ARF proteins, we have examined the localization of all human ARF isoforms and their interactions with two ARF1 binding proteins, neuronal calcium sensor-1 (NCS-1) and phosphatidylinositol-4 kinase-IIIbeta (PI4Kbeta). Use of a fluorescent protein fragment complementation method showed direct interactions between ARFs 1, 3, 5 and 6 with NCS-1 but at different intracellular locations in live HeLa cells. Photobleaching experiments indicated that complementation did not detect dynamic changes in protein interactions over short-time scales. A more specific interaction between ARFs 1/3 and PI4Kbeta was observed. Consistent with these latter findings ARF1 but not ARF5 or 6 enhanced the stimulatory effect of PI4Kbeta on regulated exocytosis, suggesting a specific role for class-I ARFs in the regulation of PI4Kbeta.