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R A Kahn

Publications and source records attributed to R A Kahn.

At least 19 recordsLinked to original sources
PubMed1996

Human ARF4 expression rescues sec7 mutant yeast cells.

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.

ADP-Ribosylation Factor 1↗
PubMed1995

ADP-ribosylation factor translocation correlates with potentiation of GTP gamma S-stimulated phospholipase D activity in membrane fractions of HL-60 cells.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP gamma S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP gamma S-stimulated PLD activity in the membrane fractions of these cells. Inactive phorbol esters induced no such potentiation. Both fMLP and active phorbol esters induced a 2-3-fold increase in GTP gamma S-stimulated PLD in HL-60 membranes. Membranes derived from stimulated HL-60 cells contained 60-70% more ARF as compared with membranes derived from control cells. Membrane contents of ARF were assessed by Western blotting with the anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation. Therefore, ARF translocation correlates with the potentiation of the GTP gamma S-stimulated PLD activity. The effect on PLD activity and ARF membrane content achieved through fMLP stimulation was greatly enhanced by prior treatment of the cells with cytochalasin B. Membranes derived from control and fMLP-stimulated cells were assayed for PLD activity in the presence of exogenous ARF and a 50-kDa fraction known to contain elements implicated in PLD activation. The ability of ARF and the 50-kDa fraction to enhance GTP gamma S-sensitive PLD activity was significantly reduced when the membranes were derived from fMLP-stimulated cells. The data indicate that, in addition to ARF, elements of the 50-kDa PLD-inducing factors were likely already translocated to the membranes upon stimulation. We propose that ARF, upon stimulation with agonists such as fMLP or phorbol esters, is translocated to the membrane and in concert with other protein components of the 50-kDa fraction enhances PLD activity.

ADP-Ribosylation Factors↗
PubMed1995

Partial purification and characterization of Arf-sensitive phospholipase D from porcine brain.

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s20,w of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.

ADP-Ribosylation Factors↗
PubMed1995

The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch.

ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.

ADP-Ribosylation Factor 1↗
PubMed1995

Mutational analysis of Saccharomyces cerevisiae ARF1.

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

ADP-Ribosylation Factor 1↗
PubMed1995

Arf proteins: the membrane traffic police?

Cofactor for cholera toxin; activator of phospholipase D; regulator of coat-protein assembly; inhibitor of membrane traffic; ability to cause expansion of the endoplasmic reticulum and vesiculation of the Golgi; sensitivity to membrane phospholipids--each of these activities has been attributed to Arf proteins. Can a single molecular mechanism link them all?

ADP-Ribosylation Factors↗
PubMed1995

Nitric oxide modulates dopamine release during global temporary cerebral ischemia.

Dopamine (DA) is released in large quantities from the striatum during cerebral ischemia. Along with excitatory neurotransmitters, DA plays a role in cellular neuronal ischemic injury. In this study we examined the role of nitric oxide (NO) in the ischemia-induced release of DA. A microdialysis probe was stereotactically placed into the corpus striatum of 16 Sprague-Dawley rats for DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) level determinations. After probe stabilization, the animals received either NG-nitro-L-arginine-methyl ester (L-NAME), a NO synthase inhibitor, or vehicle through the microdialysis probe. Temporary global forebrain ischemia was induced using bilateral carotid artery ligature tightening and controlled hemorrhagic hypotension for 15 min. L-NAME administration caused a reduction in ischemic estimated extraneuronal DA concentration by 60% (P < 0.005) compared with control. There was an increase in both DOPAC and HVA concentrations during the recovery period compared to baseline values in the control group (P < 0.05). L-NAME also caused a reduction in HVA concentration compared to vehicle administration during the latter part of recovery (P < 0.05). These data support the concept that ischemic dopamine release may be mediated by NO. This NO-modulated DA release may contribute to the previously reported deleterious neurotoxic effects of NO during ischemia.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1994

Structure of the human ADP-ribosylation factor 1 complexed with GDP.

ADP-ribosylation factors (ARFs) are essential and ubiquitous in eukaryotes, being involved in vesicular transport and functioning as an activator of phospholipase D (refs 1, 2) and cholera toxin. The functions of ARF proteins in membrane traffic and organelle integrity are intimately tied to its reversible association with membranes and specific interactions with membrane phospholipids. One common feature of these functions is their regulation by the binding and hydrolysis of GTP. Here we report the three-dimensional structure of full-length human ARF1 (M(r) 21,000) in its GDP-bound non-myristoylated form. The presence of a unique amino-terminal alpha-helix and loop, together with differences in Mg2+ ligation and the existence of a non-crystallographic dimer, set this structure apart from other GTP-binding proteins. These features provide a structural basis for the GTP-dependent modulation of membrane affinity, the lack of intrinsic GTPase activity, and the nature of effector binding surfaces.

ADP-Ribosylation Factor 1↗
PubMed1994

The amino terminus of ADP-ribosylation factor (ARF) 1 is essential for interaction with Gs and ARF GTPase-activating protein.

The role of the amino terminus in the actions of ADP-ribosylation factor 1 (ARF1) was examined by comparing wild type ARF1, a 13-residue NH2-terminal deletion mutant ([delta 13]ARF1), and a 17-residue NH2-terminal deletion mutant ([delta 17]ARF1). The amino-terminal 13 residues of ARF1 are required for cofactor activity in the ADP-ribosylation by cholera toxin when Gs is the substrate. This is in marked contrast to the finding that cofactor activity is the same for wild type and [delta 13]ARF1 when agmatine is substrate (Hong, J.-X., Haun, R. S., Tsai, S.-C., Moss, J., and Vaughan, M. (1994) J. Biol. Chem. 269, 9743-9745). These data support the conclusion that ARF1 interacts with both cholera toxin and Gs and that the amino terminus of ARF1 is required specifically for binding Gs. Surprisingly, this result also clearly revealed that the two principal assays for ARF activity, cofactor activity for cholera toxin using either Gs or agmatine as substrates, used for over 10 years in different laboratories, can yield quite different results. While both NH2-terminal deletion mutants failed to support the ADP-ribosylation of Gs by cholera toxin, [delta 13]ARF1, but not [delta 17]ARF1, inhibited the activity of the wild type protein. The GTPase activity of [delta 13]ARF1 was activated to a small extent by ARF GTPase-activating protein (GAP), whereas that of [delta 17]ARF1 was unaffected. We conclude that residues 14-17 are involved in the interaction of ARF with both cholera toxin and ARF GAP. The co-purifying nucleotides, nucleotide exchange kinetics, and dependence of exchange on phospholipids for the mutant proteins were all different from the wild type ARF1 proteins. The importance of monitoring the nucleotide binding to ARF proteins under the conditions used in the ARF assay and expressing ARF activities as specific activities, normalized to GTP binding sites, particularly when comparisons between different proteins or preparations are made, is discussed.

ADP-Ribosylation Factor 1↗
PubMed1994

Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. Evidence for specific interaction with phosphatidylinositol 4,5-bisphosphate.

ADP-ribosylation factors (ARFs) have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in yeast and mammalian cells. More specific interactions have also been described for ARF proteins with an ARF-specific GTP-ase-activating protein and as activators of phospholipase D activity. These protein interactions have defined requirements for phosphatidylinositol 4,5-bisphosphate (PIP2). Direct interactions between ARF1 and PIP2 or other phospholipids were tested by examining effects on guanine nucleotide binding kinetics. PIP2 (400 microM) increased the rate of GDP dissociation > 100-fold. Several other acid phospholipids had more modest effects (4-7-fold) on GDP dissociation rates, while other phospholipids had no effect. PIP2 also had the greatest effect on the rate of binding of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), increasing it almost 100-fold at early time points. However, at later times (> 5 min), PIP2 caused a paradoxical loss of nucleotide binding to ARF1. PIP2 was found to stabilize the nucleotide-free form of ARF1 as subsequent dilution of PIP2 allowed ARF1 to bind GTP gamma S to high stoichiometry. The demonstration of direct interaction between ARF1 and PIP2 provides the basis for a model in which PIP2 acts as a cofactor in some of the interactions between ARF1 and other proteins.

ADP-Ribosylation Factor 1↗
PubMed1994

ADP-ribosylation factor (ARF)-like 3, a new member of the ARF family of GTP-binding proteins cloned from human and rat tissues.

The human and rat homologues of a new member of the ADP-ribosylation factor (ARF) family of 21-kDa GTP-binding proteins, termed Arl3, were identified as an expressed sequence tag (human) and as a product of polymerase chain reaction amplification using degenerate probes derived from conserved sequences in members of the ARF family (rat). Alignments of the full-length open reading frames of the human and rat homologues revealed the encoded proteins to be over 97% identical to each other and 43% identical to human ARF1. Northern blots of mRNA from seven human tissues and four rat tissues revealed the presence of a ubiquitous band of about 1 kilobase in length that hybridized with the corresponding Arl3 probes. A number of human tumor cell lines expressed Arl3, as determined by immunoblotting with an Arl-specific antibody, raised against a peptide derived from the human Arl3 sequence. The level of Arl3 expressed in these cell lines was on the order of 0.01% of total cell protein. Purified recombinant human Arl3 was shown to bind guanine nucleotides but lacks ARF activity and intrinsic or ARF GTPase-activating protein-stimulated GTPase activity. In contrast to ARF proteins, the Arl3 protein has reduced dependence on phospholipids and magnesium for guanine nucleotide exchange. Thus, Arl3 is a ubiquitously expressed GTP-binding protein in the ARF family with distinctive biochemical properties consistent with its having unique, but unknown, role(s) in cell physiology.

ADP-Ribosylation Factor 1↗
PubMed1994

GTP hydrolysis by ADP-ribosylation factor is dependent on both an ADP-ribosylation factor GTPase-activating protein and acid phospholipids.

ADP-ribosylation factor (ARF) is a 21-kDa GTP binding protein that regulates eukaryotic membrane traffic. Both the binding and hydrolysis of GTP by ARF have been shown to be necessary for this function. However, purified mammalian ARF lacks intrinsic GTPase activity (< 0.0015 min-1). We document the presence, in bovine brain extracts, of a protein with the predicted properties for an ARF GTPase-activating protein (ARF GAP). This activity was highly dependent on phospholipids. An acid phospholipid fraction from bovine brain (containing primarily phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidylserine) had no effect on intrinsic GTPase activity of purified ARF but increased the ARF GAP activity of bovine brain homogenates about 8-fold. This dependence on acid phospholipids was retained after > 100-fold purification of ARF GAP, making it, likely, an inherent property of this reaction. PIP2 alone stimulated ARF GAP activity up to 30-fold with a half-maximal effect at 100-300 microM but had no effect on the GTPase rate of ARF alone. Phosphatidylinositol 4-phosphate was also active but had only 50% of the maximal effect and twice the EC50 of PIP2. Phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and diacylglycerol either alone or in the presence of ARF GAP do not stimulate ARF GTPase activity. ARF proteins have been identified recently as regulators of phospholipase D. The product of the phospholipase D reaction, phosphatidic acid, stimulated ARF GAP approximately 5-fold and reduced the PIP2 concentration needed for GAP stimulation about 6-fold. The substrate of phospholipase D, phosphatidylcholine, inhibited ARF GAP activity, but this inhibition seen with phosphatidylcholine was partially reversed by phosphatidic acid. A feedback loop for the coordinate regulation of phospholipase D and ARF activities is proposed.

ADP-Ribosylation Factors↗
PubMed1994

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.

ADP-Ribosylation Factor 1↗
PubMed1994

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.

ADP-Ribosylation Factors↗