Martha Vaughan

Molecules in the ARF Orbit

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins, members of the Ras GTPase superfamily that were initially recognized and purified because of their ability to stimulate the ADP-ribosyltransferase activity of the cholera toxin A subunit (CTA). We now know that they are critical components of several different vesicular trafficking pathways in all eukaryotic cells and activators of specific phospholipase Ds (PLDs) (reviewed in Refs. 1–3). ARF interacts with many proteins and other molecules that regulate its state of activation or are involved in its intracellular function (Fig. 1). As with other members of the Ras superfamily, ARF with GDP bound is inactive. Replacement of bound GDP with GTP produces active ARF-GTP, which can associate with membranes. ARF-GTP is the form that activates CTA and PLD. Both forms are important in vesicular transport, which requires that the ARF molecule cycle between active and inactive states. Like the many other GTPbinding proteins or GTPases that are molecular switches for the selection, amplification, timing, and delivery of signals from diverse sources, ARF functions via differences in conformation that depend on whether GTP or GDP is bound. Vectorial signaling results from the necessary sequence of GTP binding, hydrolysis of bound GTP, and release of the GDP product (Fig. 2). Under physiological conditions, release of GDP from ARF, the prerequisite for GTP binding, is very slow and is markedly accelerated by guanine nucleotide-exchange proteins or GEPs, several of which are now known. Hydrolysis of bound GTP to yield ARF-GDP, i.e. the inactivation or “turn-off” reaction, is similarly very slow (undetectable) in the absence of specific GTPase-activating proteins or GAPs. During the last 3 years, a large part of the new information related to ARF structure and function concerns these two major types of ARF regulatory proteins, which are the subjects of this review. In addition, the relatively small ;180-amino acid ARF proteins interact with numerous other molecules (not all simultaneously). These include CTA, PLD, and guanylyl nucleotide, of course, as well as PIP2 (4), coatomers (5), arfaptin (6), G protein bg subunits (7, 8), and Gas (7), about which information is much more limited (Fig. 1). Self-association of ARF in functional dimers or tetramers has also been suggested (5), and the crystal structure of a dimer is published (9). Criteria for designation as an ARF have been the ability to activate cholera toxin and to rescue mutant Saccharomyces cerevisiae bearing the lethal double deletion of ARF1 and ARF2 genes. ARF-like proteins (ARLs), which are structurally very similar to ARFs, were initially believed not to possess these ARF activities. It is now known, however, that under suitable assay conditions, at least some ARLs can exhibit ARF activity (10), and there is perhaps a continuum of ARF-ARL function. Neither of the two criteria may, in fact, reflect accurately the properties that are required for physiological ARF function in vesicular trafficking or PLD activation. The mutant yeast rescue assay seems ambiguous, because diverse ARFs from many species can be effective when overexpressed, although yeast ARF3, which resembles most closely mammalian ARF6, is ineffective (11). In the assay of CTA (or PLD or guanine nucleotide binding), dramatic effects of specific phospholipids and detergents as well as concentrations of Mg and salt on the activities of individual ARFs are well known and make it difficult to draw valid inferences about the functional relevance of many in vitro observations. Mammalian ARFs are divided into three classes based on size, amino acid sequence, gene structure, and phylogenetic analysis; ARF1, ARF2, and ARF3 are in class I, ARF4 and ARF5 are in class II, and ARF6 is in class III (3). Non-mammalian class I, II, and III ARFs have also been found (3). A role for class I ARFs 1 and 3 in ER to Golgi and intra-Golgi transport is well established (1, 2). ARF6 has been implicated in a pathway involving plasma membrane and a tubulovesicular compartment that is distinct from previously characterized endosomes (12, 13). Vesicular transport has been extensively studied in the Golgi and ER to Golgi pathways (1, 2). The mechanisms, including the molecules and their functions, are likely very similar in other pathways. Formation of a transport vesicle begins when activated ARF with GTP bound associates with the cytoplasmic surface of a donor membrane. Just how the initiation site is identified remains unknown. Activated ARF interacts with a coat protein, one of seven in the coatomer complex. Recruitment of multiple ARF molecules followed by coatomers causes membrane deformation and budding. Bilayer fusion at the base of a bud induced by fatty acyl-CoA results in vesicle release. Roles for PLD in both vesicle formation (14) and fusion (15) have been suggested. Removal of the coat, which is necessary for vesicle fusion at the target membrane, requires inactivation of ARF by hydrolysis of bound GTP to GDP. This description is necessarily greatly oversimplified. There is no consideration of the many other molecules, protein and lipid, in each transport vesicle that have structural, metabolic, or signaling functions. Emr and Malhotra (57) have edited an excellent series of reviews on several aspects of vesicle-mediated transport. Some are cited individually here. * This minireview will be reprinted in the 1998 Minireview Compendium, which will be available in December, 1998. This is the third article of five in the “Small GTPases Minireview Series.” ‡ To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, 10 Center Dr. MSC 1434, National Institutes of Health, Bethesda, MD 208921434. Tel.: 301-496-4554; Fax: 301-402-1610; E-mail: vaughanm@ gwgate.nhlbi.nih.gov. 1 The abbreviations used are: ARF, ADP-ribosylation factor; ARL, ARFlike protein; CTA, cholera toxin A subunit; PLD, phospholipase D; ER, endoplasmic reticulum; PIP2, phosphatidylinositol 4,5-bisphosphate; GEP, guanine nucleotide-exchange protein; GAP, GTPase-activating protein; PH, pleckstrin homology; ARNO, ARF nucleotide-binding site opener; BFA, brefeldin A; PC, phosphatidylcholine; GST, glutathione S-transferase; GTPgS, guanosine 59-O-(3-thiotriphosphate); GDPbS, guanosine 59-O-(2thiodiphosphate); GRP1, general receptor for phosphoinositides. FIG. 1. Molecules in the ARF orbit. ARF interacts with three general types of molecules. Regulatory proteins GEP and GAP (Fig. 2) are discussed in the text. Arfaptins (6) are ;44-kDa proteins identified in a yeast twohybrid screen of a HL-60 cDNA library using dominant active ARF3 (Q71L) as bait. The recombinant GST-arfaptin fusion protein bound activated recombinant human ARFs 1, 3, 5, and 6 to an extent decreasing in that order. The possibility that the complex of activated ARF and arfaptin is a functional entity remains to be explored. Small non-protein molecules like guanylyl nucleotides and PIP2 with specific binding sites have major effects on ARF conformation and, therefore, activity. PLD and coatomer are two major effectors with which ARF interacts. The functional significance of its demonstrated interaction with G protein bg subunits (7, 8) is unknown. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 34, Issue of August 21, pp. 21431–21434, 1998 Printed in U.S.A.

Lipids of alveolar macrophages, polymorphonuclear leukocytes, and their phagocytic vesicles

Phagocytic vesicles were isolated from rabbit alveolar macrophages and guinea pig polymorphonuclear leukocytes that had ingested emulsified paraffin oil. Phospholipids and their fatty acids were determined in whole cells and in the phagocytic vesicle and pellet fractions separated from them. The cholesterol-to-phospholipid ratios in the vesicle fractions were distinctly higher than those of the respective whole cells or pellet fractions. The vesicle fractions also had higher phospholipid-to-protein ratios than did the whole cells. The phospholipids of the phagocytic vesicle fraction from macrophages contained relatively more sphingomyelin, lyso-(bis)phosphatidic acid, and phosphatidylserine and less lecithin, phosphatidylethanolamine, and phosphatidylinositol than did the whole cells or pellet fractions. The phospholipids of phagocytic vesicles from polymorphonuclear leukocytes contained significantly more phosphatidylinositol than did the pellet fractions. Lyso(bis)phosphatidic acid, which constituted 15% of the phospholipid in rabbit alveolar macrophages and 25% of that in their phagocytic vesicles, contained almost 60% oleic acid and 20% linoleic acid. This lipid was not detected in rabbit peritoneal macrophages or in rat alveolar macrophages.The polyunsaturated fatty acids of leukocyte phospholipids were chiefly linoleic, whereas in macrophages arachidonic accounted for almost 20% of the total fatty acids. The macrophages produced malondialdehyde when ingesting polystyrene beads or emulsified paraffin oil, from which it was inferred that peroxidation of endogenous lipid can occur during phagocytosis. Polymorphonuclear leukocytes in which less than 3% of phospholipid fatty acids were arachidonic did not produce malondialdehyde during phagocytosis of these inert particles, but did when ingesting an emulsion containing linolenate, thus providing evidence for peroxidation of ingested lipid. Isolated phagocytic vesicles from alveolar macrophages contained lipid peroxides and generated malondialdehyde when incubated with ADP, FeCl(3), and NADH.

Quantitative Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

Effects of the Prostaglandins on Hormone-induced Mobilization of Free Fatty Acids

About 30 years ago, Goldblatt (1, 2) and von Euler (3, 4) showed that human and ovine seminal plasma contain an acidic lipid (prostaglandin) with vasodepressor activity and smooth muscle-stimulating activity. The early studies have been summarized by von Euler (5, 6). In 1960 Berg-strom and Sjdvall isolated from sheep vesicular glands a highly potent crystalline material, prosta-glandin E1 (PGE1) (7).1 The structure of this compound (Figure 1) was established as 2

Effects of prostaglandin E opposing those of catecholamines on blood pressure and on triglyceride breakdown in adipose tissue

The effect of prostaglandin E1 on release of glycerol from adipose tissue in the presence of lipolytic hormones (epinephrine norepinephrine adrenocorticotropic hormone thyroid-stimulating hormone and glucagon) was determined in vitro by incubating fat pads for 1 hour at 37 degrees in 3 ml of Krebs bicarbonate medium containing bovine serum albumin (30 mg/m1) and hormone in pairs 1 with PGE1 and 1 without. The effects of PGE1 in the absence of added hormones were variable. But data from 16 pairs of PGE hormone-exposed tissues indicated a significant effect of PGE1: -.3 + or -.06 mcmol of glycerol/gm of tissue perhour: on the other hand mean gycerol release for control tissues without hormone was .9 mcmol/gm/hour. The effect of epinephrine (or norepinephrine) PGE1 and a combination of the 2 on mean arterial pressure on dogs was also tested. Injection of catecholamine alone elevated arterial pressure; simultaneous injection of PGE1 reduced the observed pressor response significantly (P < .001). Injection of PGE1 alone caused a profound drop in pressure with a gradual return to normal over about 15 minutes.

Purification and Cloning of a Brefeldin A-inhibited Guanine Nucleotide-exchange Protein for ADP-ribosylation Factors

Activation of ADP-ribosylation factors (ARFs), ∼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: ∼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 ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼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 ∼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 ∼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 andBIG2 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 ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼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.

Isolation and Properties of Phagocytic Vesicles from Polymorphonuclear Leukocytes

A method for the isolation of intact phagocytic vesicles from guinea pig peritoneal-exudate granulocytes and human peripheral-blood leukocytes is presented. After leukocytes ingested the particles of a stable emulsion of paraffin oil, the uningested emulsion was washed away and the cells were homogenized. The homogenate was placed in the middle of a three-step discontinuous sucrose gradient and centrifuged for 1 hr at 100,000 g. The phagocytic vesicles, containing the low density paraffin-oil particles, were simultaneously washed and collected by floatation, while the other organelles, chiefly granules, sedimented through the lower wash layer, and the particle-free supernatant remained in the middle of the gradient. Emulsion particles stained with Oil Red O were employed to assay the rate of phagocytosis and to mark the location of the particles in subcellular fractions. The dye was extracted from washed cells or cell fractions with dioxane and colorimetrically quantified. The purity of phagocytic vesicles obtained by this method was assessed by electron microscopy, chemical analysis, and assay of enzyme composition. Granule-associated enzymes, acid phosphatase, alkaline phosphatase, beta-glucuronidase, and peroxidase were present in the phagocytic vesicles and originated from the granules. Cyanide-resistant NADH (reduced form of diphosphopyridine nucleotide) oxidase was also found. Enzymes associated with the vesicles exhibited latency to Triton X-100. Uptake of particles and the transfer of total protein and phospholipid into phagocytic vesicles occurred simultaneously Accumulation of acid and alkaline phosphatase in the vesicles continued until phagocytosis ceased. Peroxidase, NADH oxidase, and beta-glucuronidase activities in the phagocytic vesicles, on the other hand, were maximal by 30 min and increased little thereafter even when phagocytosis was still going on.

Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize

Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane–related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 μg·g−1 fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 μg·mL−1, kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function.

ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize

Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 μg g−1 fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles β-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze β-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.