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The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development. Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1%. A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.

The Dictyostelium ERK2 protein is transiently activated when cells are treated with the chemotactic agents cAMP or folic acid. Activating phosphorylation is markedly inhibited in strains overexpressing the constitutively activated RasG protein. This is in marked contrast to mammalian cells where the highly related mitogen-activated protein kinases (MAPKs) are stimulated by Ras activation.

4 páginas, 3 figuras., It has been found that the inhibition of Dictyostelium discoideum fructose-1,6-bisphosphatase by fructose 2,6-P2 greatly diminished when the pH was raised to the range 8.5–9.5, which resulted in a marked decrease of the affinity for the inhibitor with no change in the Km for the substrate. This provides evidence for the involvement of an allosteric site for fructose 2,6-P2. Moreover, the fact that excess substrate inhibition also decreased at the pH values for minimal fructose 2,6-P2 inhibition, and was essentially abolished in the presence of fructose 2,6-P2, strongly suggests that this inhibition takes place by binding of fructose 1,6-P2 as a weak analogue of the physiological effector fructose 2,6-P2., This work was supported by grants from the CAICYT and FIS.

The two smallest polypeptide components of D. discoideum cytochrome c oxidase, whose alternative expression depends on oxygen concentration [Schiavo G. and Bisson R. (1989) J. Biol. Chem. 264, 7129-7134], have been partially sequenced. They show 45$ homology and are isoforms of the same subunit, which must be encoded on two different genes.

In crude cell lysates of the cellular slime mould Dictyostelium discoideum we identified a protein kinase C (PKC)-like enzyme activity. This activity, measured as phosphorylation of a synthetic EGF-receptor-derived peptide [(1987) J. Biol. Chem. 262, 772–777], was regulated by Ca2+, phosphatidylserine (PS), 1,2-dioleoyl-rac-glycerol (DG) and the phorbol ester PMA. PS and DG stimulated the enzyme in a synergistic manner. The stimulation by these lipids was, in contrast to what has been found for ‘classical’ mammalian PKC, not dependent on Ca2+. The D. discoideum enzyme was strongly stimulated by nanomolar concentrations of PMA, and inhibited by PKC-inhibitor staurosporine.

The nucleoside queuosine (Q) is found in the first position of the anticodon of tRNATy r, tRNA His, tRNAAsp and tRNA Asn of eubacteria and eukaryotes except yeast [1]. In eukaryotes queuine is inserted into the tRNAs in exchange for guanine by the enzyme queuine: guanine tRNAtransglycosylase [2,3]. Experiments with germ-free mice, fed on queuine-free diet, showed that mice do not synthesize queuine de novo [4,5]. The modification queuine in the Q-family of tRNA was found to be incomplete in tumours from various sources and to change in cells and tissues undergoing differentiation or ageing [6-10]. The role of queuine in these processes is unknown. Here, we show that Dictyostelium discoideum can be applied as a model system to evaluate the biological significance of queuine. When myxamoebae of the axenic strain AX-2 are grown in a well-defined synthetic medium supplemented with yeast extract but without peptone their tRNAs comprise guanine in place of queuine. The addition of queuine to the medium, at 10 -7 M, causes an almost complete modification of the corresponding tRNAs. Analysis of tRNA from the wildtype strain, NC-4, that needs E. coli B/r for growth indicates that bacteria supply D. discoideum with queuine.

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH i and those in glycolytic rate are not causally related events.

Cell cycle phase at the onset of development in Dictyostelium influences cell fate. Cells in the G2 phase, which tend to become spores, show a more rapid induction of expression of the cell surface receptor involved in the chemotaxis. We show that differential induction of developmental expression is restricted to some transcripts, including those encoding proteins required for chemotaxis, and thus is not due to general transcriptional repression during mitosis. We also show that cells showing rapid induction of one such gene are preferentially located at the centre of early aggregates. These results are consistent with cells derived from G2 phase being at the centre of early aggregates because selective differences in gene regulation render them more efficient at aggregation.

A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium discoideum. This transferase requires divalent cations and is inhibited by N-ethylmaleimide and detergent treatment. Apparent calculated Km values for GDP-Fuc and lacto-N-biose I are 1.27 microM and 2.80 mM, respectively. The activity is quantitatively recovered in the supernatant after centrifugation at 100000 x g for 1 h. The reaction product, as determined by gel permeation chromatography, sensitivity to fucosidases, and analysis of partially methylated derivatives, is Fucalpha1-2Galbeta1-3GlcNAc (H type 1 trisaccharide).

The F-actin crosslinking molecule α-actinin from the slime mould Dictyostelium discoideum carries two characteristics EF-hand structures at the C-terminus. The calcium-binding loops contain all necessary liganding oxygens and most likely form the structural basis for the calcium sensitivity of strictly calcium-regulated non-muscle α-actinins. Furthermore, the sequence exhibits at the N-terminal site of the molecule a high degree of homology to chicken fibroblast α-actinin. This stretch of amino acids appears to have remained essentially constant during evolution and might represent the actin-binding site. The findings have led us to propose a model for the inhibitory action of Ca 2+ on non-muscle α-actinins.

Phosphatidylinositol kinase was examined in Dictyostelium discoideum since this organism offers molecular and genetic advantages to study the role of phosphatidylinositol metabolism during cell growth and development. D. discoideum homogenates phosphorylated phosphatidylinositol to form phosphatidylinositol 4-phosphate in a reaction which was found to be linear with time and cell concentration. Optimal activity was obtained in the presence of 1 mM MgCl 2 and pH 7.6 and has an apparent K m for ATP of about 250 μM. Changes in phosphatidylinositol kinase were examined during D. discoideum development. Activity increased about 2-fold, 4 h after removal of the food source, to decline to almost no activity at late aggregation. During slug formation the activity increased about 15-fold and remained constant during further development. These results suggest a role for D. discoideum phosphatidylinositol kinase during development.

Starving Dictyostelium discoideum cells monitor the local density of other starving cells by simultaneously secreting and sensing CMF. CMF regulates signal transduction through the chemoattractant cAMP receptor, cAR1. cAR1 activates a heterotrimeric G protein by stimulating Gα2 to release GDP and bind GTP. We show here that the rate of cAMP-stimulated GTP hydrolysis in membranes from cells exposed to CMF is roughly 4 times slower than in membranes from untreated cells, even though the rate of GTP binding is the same. This hydrolysis is abolished in cells lacking Gα2. Our data thus suggest that CMF regulates cAMP signal transduction in part by prolonging the lifetime of the Gα2-GTP complex. © 1997 Federation of European Biochemical Societies.

A tail fragment of Dictyostelium discoideum myosin has been cloned and expressed as a fusion protein with the N-terminal region of MS-2 polymerase. The cloned fragment was phosphorylated with myosin heavy chain kinase II from aggregation-competent D.discoideum cells that specifically phosphorylate threonine residues on the myosin tail. Phosphopeptide maps showed the same site specificity of phosphorylation with the fusion protein as a substrate as with native myosin. An improved assay for the kinase was developed in which the fusion protein is precipitated with a monoclonal antibody that inhibits polymerization of the myosin tails without preventing their phosphorylation. Sites of phosphorylation were tentatively localized to a sequence in the C-terminal region of the heavy chain where four threonine residues are found.

In a wide range of cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pH i ). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pH i . About 1.5 min after stimulation, pH i starts increasing from pH i ∼7.45 to pH i ∼7.60, as is revealed independently by two different pH null-point methods. The rise in pH i is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pH i is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum .

Chemotaxis is very important during the whole life cycle of the cellular slime molds. In the vegetative stage the amoebae have to find their bacterial food in the soil which they inhabit. At this time, the amoebae are chemotactic to folic acid and pterin [ 1,2], both of which are excreted by bacteria; therefore it seems probable that this mechanism is used to find food [l]. When the food source is exhausted the amoebae aggregate to form a multicellular slug which then differentiates into a fruiting body. Different species of slime mold use different compounds as the chemoattractant for this aggregation. The best studied system is Dictyostelium discoideum which utilizes pulsatile signals of CAMP [3]; however, chemoattractants from other species have been partially purified [4,5]. of the change during chemotactic stimulation [14,15] which suggests that it may play an important role in the transduction of the signal.

The effect of phorbol 12-myristate 13-acetate upon the light-scattering response to cAMP of a D. discoideum cell suspension was investigated. It was found that the first spike of the cAMP-mediated light-scattering change (peaking at about 15–20 s after stimulation) was inhibited by the phorbol ester. This effect was concentration dependent with an half-maximum value for the inhibition of 4 nM. The inhibition was found to be maximal after a 10–20 min incubation time. The phorbol ester was shown to affect the dose-response relationship between the cAMP concentration and the relative amplitude of the light-scattering change, more by decreasing the number of cAMP receptors than by decreasing their apparent affinity for cAMP.

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolysed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.