Two functionally distinctive phosphopantetheinyl transferases from amoeba Dictyostelium discoideum

The life cycle of Dictyostelium discoideum is proposed to be regulated by expression of small metabolites. Genome sequencing studies have revealed a remarkable array of genes homologous to polyketide synthases (PKSs) that are known to synthesize secondary metabolites in bacteria and fungi. A crucial step in functional activation of PKSs involves their post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases). PPTases have been recently characterized from several bacteria; however, their relevance in complex life cycle of protozoa remains largely unexplored. Here we have identified and characterized two phosphopantetheinyl transferases from D. discoideum that exhibit distinct functional specificity. DiAcpS specifically modifies a stand-alone acyl carrier protein (ACP) that possesses a mitochondrial import signal. DiSfp in contrast is specific to Type I multifunctional PKS/fatty acid synthase proteins and cannot modify the stand-alone ACP. The mRNA of two PPTases can be detected during the vegetative as well as starvation-induced developmental pathway and the disruption of either of these genes results in non-viable amoebae. Our studies show that both PPTases play an important role in Dictyostelium biology and provide insight into the importance of PPTases in lower eukaryotes.     

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.     

Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum

The binding of native, 125I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 micrograms/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 micrograms/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. In kinetic experiments, actin added as monomers bound to membranes at a rate of 0.6 microgram ml-1 min-1, while pre-polymerized actin bound at a rate of 3.0 micrograms ml-1 min-1. Even in the absence of phalloidin, actin bound to membranes at concentrations well below the normal critical concentration. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. We conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins.     

The purification and characterization of Dictyostelium discoideum plasma membranes.

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5'-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome c reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

An improved procedure for the purification of plasma membranes from Dictyostelium discoideum.

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. & Weeks, G. (1977) Biochim. Biophys. Acta 464, 142-156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.     

An electrogenic proton pump in plasma membranes from the cellular slime mould Dictyostelium discoideum

Plants and fungi possess an outwardly directed plasma membrane proton pump that may regulate intracellular pH. We provide the first demonstration that amoebae of the slime mould Dictyostelium discoideum also possess a similar proton pump. It can be assayed either as an ATPase activity in highly purified plasma membranes or as a proton pump, after solubilization and reconstruction into liposomes. The pump is inhibited by vanadate, diethylstilbestrol (DES) and miconazole but not by azide or ouabain. The proton pump described here may represent the target for the action of DES and miconazole, both of which have previously been shown to induce stalk cell formation during the in vitro development of Dictyostelium.     

Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes

The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.     

The plasma membrane ATPase of Dictyostelium discoideum

During centrifugation of Dictyostelium membranes on sucrose and metrizamide gradients, an ATPase activity resistant to azide and molybdate but sensitive to diethylstilbestrol was found to copurify with the plasma membrane markers alkaline phosphatase and ¹²⁵I in cells surface-labelled by lactoperoxidase catalyzed iodination. This ATPase was enriched 50-fold in purified plasma membranes and could be separated from the mitochondrial ATPase on metrizamide gradients. The plasma membrane ATPase is very specific for ATP as substrate and Mg²⁺ as essential cofactor. Its pH optimum is 6.5 and it is inhibited by dicyclohexylcarbodiimide, diethylstilbestrol, vanadate, mercurials and Cu²⁺, but not by ouabain, molybdate, azide or oligomycin. It was not specifically affected by either monovalent cations or anions. These results suggest that the plasma membranes of Dictyostelium contain an ATPase similar to the proton-pumping ATPases recently identified in fungal and plant plasma membranes (Serrano, R. (1984) Curr. Top. Cell. Regul. 23, 87–126).     

Pertussis and cholera toxin ADP-ribosylation in Dictyostelium discoideum membranes

We report a 39 kDa substrate for cholera and pertussis toxins is present in D. discoideum membranes. This protein did not co-migrate with alpha subunits of either Gs (45 kDa and 52 kDa) or Gi (41 kDa) from control mammalian cells. The presence of GTP or its non-hydrolyzable analogs enhanced the ADP-ribosylation in response to cholera toxin, but did not significantly alter ADP-ribosylation by pertussis toxin. Divalent cations inhibited the ADP-ribosylation by both toxins. The possible association of this novel G-protein with D. discoideum adenylate cyclase may underlie some of the unique regulatory features of this enzyme. Alternatively, this G-protein may regulate one of several other cellular responses mediated by the cAMP receptor.     

Identification of concanavalin A receptors and galactose-binding proteins in purified plasma membranes of Dictyostelium discoideum

Two techniques have been modified to provide simple means for the identification of molecules which bind concanavalin A (Con A). Crossed immunoelectrophoresis was altered by replacing antibody with Con A, and receptors were identified by the precipitin arcs which they produced. Con A, tagged with fluorescein isothiocyanate, was also diffused into prefixed sodium dodecyl sulfate (SDS)-polyacrylamide gels, and additional receptors identified by fluorescence. More than 35 molecules in the plasma membranes of the cellular slime mold Dictyostelium discoideum which bind Con A were identified with these techniques. At least 12 of these diminish and 12 increase in importance as receptors during differentiation of the cells from the vegetative to the preculmination stage of development. In the course of these experiments, it was possible to confirm the presence of the galactose-binding protein discoidin, in the plasma membrane, by electrophoresing membrane proteins into an agarose gel. This lectin regains its sugar-binding activity after denaturation and electrophoresis in SDS.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Cyclic AMP is known to function as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the prespore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Abstract. Cyclic AMP is known to function as the chemo-tactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyosteliwn discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultra-microtechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the pre-spore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.     

Characterization of a glycosyl-phosphatidylinositol degrading activity in Dictyostelium discoideum membranes

Antigen 117 is a glycolipid-anchored cell surface protein implicated in cell-cell cohesion of Dictyostelium discoideum amoebae. Previous studies have demonstrated that during cell aggregation some of the protein is released from the cell surface. Here we report the characterization of the enzymatic activity involved in the 117 antigen release. The data indicate that the releasing enzyme is a phosphatidylinositol phospholipase C. The data also indicate that structural features of glycolipid anchors are conserved in a variety of organisms.     

Binding of adenosine 3':5'-monophosphate to plasma membranes of Dictyostelium discoideum amoebae

A plasma membrane preparation from Dictyostelium discoideum amoebae which contains the high affinity cAMP receptor is described. Ligand specificity and the kinetics of cAMP association and dissociation using isolated plasma membranes were similar to those of intact cells. The changes in cAMP binding activity which occur as cells proceed through their aggregation program were also reflected in the membrane preparations. However, neither the low affinity cAMP binding site nor the oscillatory cAMP binding behavior observed on intact cells was detected with the membrane preparations.