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R A Kahn

Publications and source records attributed to R A Kahn.

At least 91 records · Page 5Linked to original sources
PubMed1992

Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport.

We have used an intra-Golgi transport assay to identify GTP-binding proteins involved in regulation of protein traffic. Two soluble proteins of 20 kd were purified by their ability to mediate GTP gamma S-dependent inhibition of transport. These GTP-dependent Golgi binding factors, or GGBFs, exhibit a 3-fold difference in activity and are differentiated by their hydrophobicity, isoelectric points, and apparent size. Removal of 80% of GGBFs from cytosol abolishes GTP gamma S sensitivity but does not affect inhibition by aluminum fluoride. We demonstrate that GGBFs are members of the ADP-ribosylation factor (ARF) family. Recombinant ARF1 exhibits GGBF activity and myristoylation is required. The distinct biochemical properties of GGBFs indicate that members of the ARF family may have related but distinct functions in intracellular transport.

ADP-Ribosylation Factor 1↗
PubMed1992

The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport.

Deletion of the amino-terminal 17 residues from human ADP-ribosylation factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind guanine nucleotides with high affinity. Unlike the wild type, the binding of guanine nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.

ADP-Ribosylation Factor 1↗
PubMed1992

Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion.

We have used an in vitro endosome fusion assay, recombinant ARF, synthetic peptides, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to study the role of ARF during endocytosis. Previous work has shown that GTP gamma S stimulates in vitro endosome fusion in dilute cytosol (less than 0.5 mg/ml) but inhibits fusion in concentrated cytosol (greater than 1.0 mg/ml). Two peptides corresponding to the NH2-terminal 16 amino acids of human ARF1 and ARF4 blocked GTP gamma S stimulation of fusion in dilute cytosol and reversed GTP gamma S inhibition of fusion in concentrated cytosol. The addition of recombinant human ARF1 to endosomes in dilute or concentrated cytosol resulted in GTP gamma S-dependent inhibition of fusion. Only the myristoylated form of ARF inhibited fusion. The NH2-terminal ARF1 peptide reversed inhibition by recombinant ARF1. Preincubation experiments showed that endosomes could form an ARF-resistant intermediate during the fusion process. Western blot analysis revealed clathrin-coated vesicles extracted with detergent retained ARF. The results suggest that ARF is involved in both the stimulatory and inhibitory effects of GTP gamma S in dilute and concentrated cytosol, respectively. Furthermore, myristoylation, the NH2-terminal domain, and binding to GTP appear to be critical for ARF activity during an early prefusion step required for endocytosis.

ADP-Ribosylation Factor 1↗
PubMed1992

ADP-ribosylation factor is required for vesicular trafficking between the endoplasmic reticulum and the cis-Golgi compartment.

We describe the potential role of ADP-ribosylation factor (ARF) in vesicular trafficking using an in vitro assay that efficiently reconstitutes transport between the endoplasmic reticulum (ER) and the cis-Golgi compartment in mammalian semi-intact cells, a population of cells in which the plasma membrane is physically perforated to reveal intact ER and Golgi compartments. We demonstrate that peptides identical to the amino-terminal domain of ARF, which inhibit ARF cofactor activity in cholera toxin-catalyzed ADP-ribosylation of G alpha S (Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046), inhibit transport of the vesicular stomatitis virus G protein between the ER and cis-Golgi compartment. Inhibition of transport was rapid (t1/2 = 30-60 s) and irreversible. Half-maximal inhibition was observed at concentrations of 15 and 22 microM with peptides identical to the amino-terminal domain of the human ARF4 (hARF4) protein and the human ARF1 protein, respectively. Kinetic analysis of vesicular stomatitis virus G protein transport suggested that the hARF4 peptide inhibits a late vesicle fusion step. In addition, incubation of semi-intact cells in the presence of the myristoylated form human ARF1 (hARF1myr) protein, but not the nonmyristoylated form of ARF1, inhibited transport. In contrast to peptide, the hARF1myr blocked an early transport step, similar to that observed with guanosine 5'-3-O-(thio)triphosphate. These results suggest that ARF and components facilitating ARF function play an important role in the cyclical fission and fusion of transport vesicles mediating ER to Golgi trafficking.

ADP-Ribosylation Factor 1↗
PubMed1991

Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein.

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.

ADP-Ribosylation Factors↗
PubMed1991

Activation of a small GTP-binding protein by nucleoside diphosphate kinase.

Genes that encode nucleoside diphosphate kinases (NDKs) have been implicated as regulators of mammalian tumor metastasis and development in Drosophila melanogaster. However, the cellular pathways through which NDKs function are not known. One potential mechanism of regulation is phosphorylation of guanosine diphosphate (GDP) bound to regulatory guanosine triphosphate (GTP) binding proteins. NDK-catalyzed phosphorylation of bound GDP was investigated for the adenosine diphosphate ribosylation factor (ARF), a 21-kilodalton GTP-binding protein that functions in the protein secretion pathway. Bovine liver NDK, recombinant human NDK, and the protein product of the mouse gene nm23-1, which suppresses the metastatic potential of certain tumor cells, used ARF-GDP as a substrate, thereby allowing rapid and efficient production of activated ARF (ARF-GTP) in the absence of nucleotide exchange. These data are consistent with the proposed function of NDK as an activator of a small GTP-binding protein and provide a mechanism of activation for a regulatory GTP-binding protein that is independent of nucleotide exchange.

Animals↗
PubMed1991

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.

ADP-Ribosylation Factors↗
PubMed1991

Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins.

Effects of aluminum, magnesium, and fluoride (AMF) on members of both the trimeric G protein and smaller (20-25 kDa) monomeric GTP-binding protein families were examined. The dissociation of GDP from G proteins was blocked by AMF but was unchanged with the addition of AMF to any of six of the monomeric GTP-binding proteins. Biochemical activities and properties of one of the smaller GTP-binding proteins, ADP-ribosylation factor, were also found to be unaffected by AMF. It is concluded that the ability of AMF to activate the trimeric G proteins is not shared by the smaller GTP-binding proteins and thus should prove to be a useful discriminator between cellular activities regulated by these two families of regulatory proteins.

Adenosine Diphosphate Ribose↗
PubMed1991

Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues.

Low-molecular-mass GTP-binding proteins of the ras family were analysed by [32P]GTP binding after PAGE and transfer to nitrocellulose membranes. By this technique, several GTP-binding proteins in the 20-30 kDa range were detected in both cytosolic and microsomal fractions of RINm5F cells. One of these, displaying an apparent molecular mass of about 20 kDa and a pI of 6.7, was mainly cytosolic and was shown to be the ADP-ribosylation factor (ARF) by using specific antibodies. When permeabilized RINm5F cells were incubated with the stable GTP analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) the amount of ARF increased in a fraction containing both Golgi and plasma-membrane markers, but not in the fraction containing secretory granules, mitochondria and lysosomes. GTP, GDP and its beta-thio analogue did not enhance ARF binding to membranes, smg25/rab3 and rho, as well as all the other small GTP-binding proteins detected by the [32P]GTP binding, did not redistribute under these conditions. As GTP[S] stimulates insulin secretion in these cells, we also examined the relationship between ARF translocation and insulin secretion. Both phenomena were elicited by GTP[S] with an EC50 (median effective concentration) of about 10 microM. p[NH]ppG was equipotent with GTP[S] in inducing insulin secretion (EC50 about 10 microM), but higher concentrations (about 500 microns) were required to achieve the same maximal ARF redistribution. These results suggest that: (1) ARF is subject to cycling between a membrane-associated and a free/loosely attached form, determined by the species of bound guanine nucleotide; (2) ARF alone does not seem to regulate exocytosis in insulin-secreting cells.

ADP-Ribosylation Factors↗
PubMed1991

The arflike gene encodes an essential GTP-binding protein in Drosophila.

We have identified a Drosophila gene (arflike, arl) encoding a protein that is structurally related (approximately 55% identity) to the ADP-ribosylation factors (ARFs) of yeast and mammals. Biochemical analyses of purified recombinant arl-encoded protein revealed properties similar to the ARF proteins, including the ability to bind and hydrolyze GTP. Clear functional differences between arl and ARF proteins, including a complete lack of ARF activity, suggest that arl is not a functional homolog of ARF. A recessive lethal arl mutation was recovered, demonstrating that the arl locus is an essential gene. We conclude that the arl locus encodes an essential member of the ARF subfamily of small GTP-binding proteins in Drosophila.

ADP-Ribosylation Factors↗
PubMed1991

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.

ADP-Ribosylation Factor 1↗
PubMed1991

Purification of a synthetic myristylated peptide by counter-current chromatography.

A preparative purification of myristyl-Gly-Asn-Ile-Phe-Ala-Asn-Leu-Phe-Lys-Gly-Leu-Phe-Gly-Lys-Lys-Glu -NH2 was accomplished using the multi-coil counter-current chromatograph. A partition coefficient was determined in the n-butanol-acetic acid-water (4:1:5) system. Chromatographic runs were made in this system and one modified with ethyl acetate. The peptide material showed anomalous elution behavior due to its surfactant properties. It was found that by loading the sample exclusively in the stationary phase, satisfactory retention of the compound occurred. Finally, conditions utilizing the upper phase as the mobile phase successfully separated the impurities.

Amino Acid Sequence↗
PubMed1990

ADP-ribosylation factor is functionally and physically associated with the Golgi complex.

ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.

ADP-Ribosylation Factor 1↗
PubMed1990

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.

ADP-Ribosylation Factor 1↗
PubMed1990

Neurological presentation of neuro-Behçet's syndrome: clinical categories.

In an attempt to categorize the diverse neurological manifestations of Behçet's syndrome, a group of 10 patients was prospectively studied; the diagnosis of Behçet's syndrome was not known in any of them prior to their neurological presentation. A reasonably clear distinction can be made to divide the manifestations into three general categories with some overlap. Category I includes patients with increased intracranial pressure with or without cerebral venous sinus thrombosis. Category II are those presenting with a stroke. Category III are those patients who present with spinal cord involvement and CSF pleocytosis. Brainstem involvement was noted independently of the group. In general, patients with Neuro-Behçet's syndrome showed little ocular involvement. The course of the illness both in morbidity and mortality tended to be better in category I, and mortality was only seen in category II patients. In populations susceptible to the disease, the clinical categorisation proposed may alert the clinician to the possibility of neuro-Behçet's syndrome whenever patients with pseudotumour cerebri, dural sinus thrombosis, stroke in the young, unexplained myelitis or possible multiple sclerosis are encountered.

Behcet Syndrome↗