ARF family G proteins and their regulators: roles in membrane transport, development and disease

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins, including the ARF-like (ARL) proteins and SAR1, regulate membrane traffic and organelle structure by recruiting cargo-sorting coat proteins, modulating membrane lipid composition, and interacting with regulators of other G proteins. New roles of ARF and ARL proteins are emerging, including novel functions at the Golgi complex and in cilia formation. Their function is under tight spatial control, which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that catalyse GTP exchange and hydrolysis, respectively. Important advances are being gained in our understanding of the functional networks that are formed not only by the GEFs and GAPs themselves but also by the inactive forms of the ARF proteins.     

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.     

普通烟草ARF基因家族序列的鉴定与表达分析

ARF(AUXIN RESPONSE FACTOR)基因含有一个B3功能域和具有转录激活或抑制活性的中心功能域,在植物发育过程中起到非常重要的作用。本研究采用生物信息学方法,根据拟南芥ARF基因序列鉴定了普通烟草基因组中的ARF基因,并对家族成员进行了序列特征、系统发生、亚细胞定位和表达模式分析。目前在普通烟草基因组中共得到50个ARF基因成员,其基因结构相对复杂,一般含有10个外显子。亚细胞定位结果表明,少数ARF蛋白定位到线粒体或叶绿体,大多数未检测到定位信号。转录组数据分析表明,ARF基因具有不同的组织表达模式,部分基因表现出组织特异性。这些研究结果为普通烟草ARF基因家族功能的深入研究奠定了基础。     

Light Promotes Protein Stability of Auxin Response Factor 7

Light is an environmental signaling, whereas Aux/IAA proteins and Auxin Response Factors (ARFs) are regulators of auxin signalling. Aux/IAA proteins are unstable, and their degradation dependents on 26S ubiquitin-proteasome and is promoted by Auxin. Auxin binds directly to a SCF-type ubiquitin-protein ligase, TIR1, facilitates the interaction between Aux/IAA proteins and TIR1, and then the degradation of Aux/IAA proteins. A few studies have reported that some ARFs are also unstable proteins, and their degradation is also mediated by 26S proteasome. In this study, by using of antibodies recognizing endogenous ARF7 proteins, we found that protein stability of ARF7 was affected by light. By expressing MYC tagged ARF activators in protoplasts, we found that degradation of ARF7 was inhibited by 26 proteasome inhibitors. In addition, at least ARF5 and ARF19 were also unstable proteins, and degradation of ARF5 via 26S proteasome was further confirmed by using stable transformed plants overexpressing ARF5 with a GUS tag.     

Localization and Characterization of the Human ADP-Ribosylation Factor 5 (ARF5) Gene

ADP-ribosylation factor 5 (ARF5) is a member of the ARF gene family. The ARF proteins stimulate thein vitroADP-ribosyltransferase activity of cholera toxin and appear to play a role in vesicular traffickingin vivo.We have mapped ARF5, one of the six known mammalian ARF genes, to a well-defined yeast artificial chromosome contig on human chromosome 7q31.3. In addition, we have isolated and sequenced an 3.2-kb genomic segment that contains the entire ARF5 coding region, revealing the complete intron–exon structure of the gene. With six coding exons and five introns, the genomic structure of ARF5 is unique among the mammalian ARF genes and provides insight about the evolutionary history of this ancient gene family.     

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.     

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.     

ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.

ADP-ribosylation factor (ARF) is an abundant and highly conserved low molecular weight GTP-binding protein that was originally identified as a key element required for the action of cholera toxin in mammalian cells, but whose physiological role is unknown. We report that ARF family proteins are highly concentrated in non-clathrin-coated transport vesicles and are coat proteins. About three copies of ARF are present on the outside of coated vesicles per alpha-COP (and thus per coatomer). ARF is highly enriched in coated vesicles as compared with parental Golgi cisternae, as shown both by biochemical and morphological methods, and ARF is removed from transport vesicles through uncoating during transport. Furthermore, ARF binds to Golgi cisternae in a GTP-dependent manner independently of coated vesicle budding. These observations strongly suggest a new role for GTP-binding proteins: ARF proteins may modulate vesicle budding and uncoating through controlled GTP hydrolysis.     

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.     

Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.     

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.     

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4

The JNK‐interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP‐binding protein ARF6. The interaction of ARF6–GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin‐1 and dynactin. Here, we report the crystal structure of ARF6–GTP bound to the JIP4‐LZII at 1.9 Å resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6–(JIP4)₂–ARF6 configuration. Comparison of the ARF6–JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site‐directed mutagenesis and surface plasmon resonance, we further show that non‐conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure‐derived model of the association of the ARF6–JIP3/JIP4 complex with membranes shows that the JIP4‐LZII coiled‐coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6‐mediated motor switch regulatory function.     

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.     

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.