Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K_m values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS. © 1996 Wiley-Liss, Inc.     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

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 hydrolyzed 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.     

Isolation and characterization of mutations affecting UDPG pyrophosphorylase activity in Dictyostelium discoideum

A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in vivo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed.     

Isolation and characterization of a component of the surface sheath of Dictyostelium discoideum

Aggregates of Dictyostelium discoideum are surrounded by a surface sheath which functions to maintain polarity and integrity during development. We have isolated and partially characterized a component of the surface sheath. It is composed of 60% cellulose, 15% protein, 3% heteropolysaccharide (heteropolymer), 5% lipid, and 1% sulfate when isolated from migrating slugs. The sheath, isolated from aggregates prior to tip formation, has less protein, a different heteropolymer, and cellulose of a lower crystallinity than the sheath of migrating slugs. The increase in crystallinity of the cellulose during development may be important in determining the strength of the surface sheath.     

The hydrophobic character of the membrane-bound phosphodiesterase from Dictyostelium discoideum

A phosphodiesterase activity is shown to copurify with the plasma membrane fraction prepared by the two-phase partition method. The enrichment in phosphodiesterase parallels that of alkaline phosphatase, which is thought to be a typical membranous enzyme. Up to 66% of the phosphodiesterase activity can be solubilized by a treatment with 0.2% Triton X-100. Higher doses were ineffective in solubilizing more activity. Analysis by native gel electrophoresis showed that an activity extracted by 2 M NaCl migrated at the same position as 'soluble' phosphodiesterase of cytosolic or extracellular origin. In contrast, the Triton-solubilized enzyme had an apparently higher molecular weight. When subjected to charge shift electrophoresis on agarose gels in the presence of an ionic detergent, the Triton-solubilized phosphodiesterase displayed a hydrophobic character. This behaviour contrasts with that of 'soluble' phosphodiesterases, the electrophoretic mobility of which is unaffected by the presence of an anionic detergent. The hydrophobic character of the membranous enzyme was lost after gentle hydrolysis by papain.     

Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Isolation of germination mutants of Dictyostelium discoideum

A simple method to separate spores from amoebae of Dictyostelium discoideum has been devized and used to isolate spore germination mutants. A subclass of these mutants is temperature sensitive for germination and growth.     

The small nuclear RNAs of the cellular slime mold Dictyostelium discoideum. Isolation and characterization

Three species of small nuclear RNA from the lower eucaryote Dictyostelium discoideum have been isolated and characterized with regard to size, cellular abundance, modified nucleotide content, and 5'-end structures. Previous studies had shown that the nuclei of mammalian cells contain a number of discrete low molecular weight, nonribosomal, nontransfer RNA molecules known as small nuclear RNAs. The mammalian small nuclear RNAs range in size from approximately 100 to 250 nucleotides and are quite abundant, in some cases approaching ribosomal RNA in number of copies/cell. Some of these molecules have an unusual cap structure at their 5'-ends similar to that found on eucaryotic messenger RNAs, and a number contain a characteristic set of internal modifications as well. Our results indicate that the small nuclear RNAs of Dictyostelium resemble their counterparts in higher eucaryotic cells structurally, but are present in significantly fewer copies/cell. The implications of these findings for small nuclear RNA function are discussed.     

Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum

To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 218 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group II, fgdC in group III, and fgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to cAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.     

Isolation and characterization of a 30,000-dalton calcium-sensitive actin cross-linking protein from Dictyostelium discoideum

A calcium-sensitive actin-binding protein having a subunit molecular mass of 30,000 daltons (30K protein) has been isolated from Dictyostelium discoideum. Structural, immunological, and functional analyses demonstrated that the 30K protein was distinct from other actin-binding proteins of D. discoideum. A native molecular mass of 31,700 daltons was determined by equilibrium sedimentation, indicating that the protein is monomeric. The Stokes radius was 30 A. The frictional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape. The 30K protein induced an increase in the viscosity of a solution of F-actin. Bundles of actin filaments were observed in negatively stained mixtures of actin and the 30K protein. Both the formation of filament bundles and the increases in viscosity of actin induced by the 30K protein were observed in the presence of 1 X 10(-8) M but not 2 X 10(-6) M calcium. Variation of the pH from 6.6 to 7.8 had no effect on the activity of the 30K protein. Calcium induced neither a large change in quaternary structure of the 30K protein nor a restriction of the lengths of actin filaments by the 30K protein. The apparent affinity of the 30K protein for actin was decreased in the presence of calcium. Reversible cross-linking of actin filaments by the 30K protein may contribute to regulation of the consistency and contractility of cytoplasm in D. discoideum.