Rescue of a Dictyostelium discoideum mutant defective in cyclic nucleotide phosphodiesterase

One of the developmentally induced gene products that is essential for chemotaxis of Dictyostelium amoebae is a cyclic nucleotide phosphodiesterase. The enzyme can be secreted or exist in a membrane bound form. This enzyme is missing in the mutant HPX235 which, as a consequence, does not aggregate unless exogenous cAMP phosphodiesterase is supplied. We have introduced multiple copies of the cloned phosphodiesterase gene into mutant amoebae and restored aggregation. The formation of anatomically correct fruiting bodies, which does not occur when exogenous enzyme is added, is also restored by transformation with the gene. The construct that we have used gives rise only to secreted phosphodiesterase and therefore the membrane bound form of the enzyme is not absolutely required for normal aggregation and morphogenesis.     

Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictyostelium discoideum

Cyclic 3',5'-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20-100 ng could be assayed in a reaction coupled to 5'-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 microM cAMP, cAMP-PD activity in aggregates ranged from 20-40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5-10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40-60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5-10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50-80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.     

A developmentally regulated cysteine proteinase in Dictyostelium discoideum.

We have determined the sequence of a Dictyostelium mRNA encoding a protein with a high degree of homology to plant and animal cysteine proteinases. The degree of homology is highest in the region of the cysteine residue which is transiently acylated during peptide hydrolysis but all other residues known to be important in catalysis are also conserved. We have named this protein cysteine proteinase 1. There is a hydrophobic signal peptide of 18 amino acids and an additional 99 amino acids at the N terminus, which are not present in other cysteine proteases and which may be cleaved off during processing of the enzyme. There is a single copy of the gene in the Dictyostelium genome. The cysteine proteinase 1 mRNA is absent from growing cells and from cells isolated during the first 6 h of development but it constitutes approximately 1% of cellular mRNA by 10-12 h of development. During the development of Dictyostelium a major fraction of cellular protein is degraded to provide amino acids and a source of energy. Cysteine proteinase 1 may play a role in this auto-digestion.     

The cyclic nucleotide phosphodiesterase inhibitory protein of Dictyostelium discoideum. Purification and characterization

We have purified the glycoprotein inhibitor of the extracellular cyclic nucleotide phosphodiesterase of Dictyostelium discoideum to apparent homogeneity. The inhibitor has a molecular weight of 47,000 measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interaction of the inhibitor and the cyclic nucleotide phosphodiesterase occurs with 1:1 stoichiometry and with a dissociation constant of about 10(-10) M. Periodate oxidation of the inhibitor or of the enzyme destroys concanavalin A binding ability but does not affect the formation of the enzyme-inhibitor complex. Inhibitor is not produced by cells during logarithmic growth but appears in quantity during stationary phase and after transfer from growth medium to phosphate buffer.     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the single-phosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pdsA gene and four fgd genes affect the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.     

Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination

mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.     

Disaggregation-induced changes of developmentally regulated proteins in Dictyostelium discoideum

By means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1 mM cAMP or 250 micrograms/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact.     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

Identification and characterization of developmentally regulated mRNP proteins of Dictyostelium discoideum

The isolation of poly(A)+ polysomal and nonpolysomal RNPs by oligo(dT)-cellulose chromatography has led to the identification of more than 20 polypeptides that bind to the poly(A)+ mRNA in growing Dictyostelium cells. Most of these polypeptides were identified in experiments using short-wave UV light (254 nm) to crosslink specifically bound proteins to the RNA. Digestion of the RNPs with ribonucleases A and T1 prior to their application to oligo(dT)-cellulose permitted the isolation of the 3' poly(A)-protein complexes. In polysomal RNPs, two major polypeptides, with molecular weights of 31,000 (p31) and 31,500 (p31.5), are bound to poly(A). These proteins can also be purified from cytoplasmic extracts by affinity chromatography on poly(A)-Sepharose. Partial proteolytic digestion of p31 and p31.5 indicates that they are closely related. The UV-crosslinking experiments established that p31 and p31.5 bind to the non-poly(A) segments of mRNA as well. In nonpolysomal RNPs, p31 and a polypeptide with a molecular weight of 29,500 (p29.5) are the major species associated with poly(A). Partial proteolytic digestion of p29.5 indicates that it is closely related to p31 and p31.5. Only small amounts of p29.5 were observed in the polysomal poly(A)-protein complexes. Early in Dictyostelium development, when cellular translation activity is sharply reduced, most of the p29.5, p31 and p31.5 present is selectively degraded. These observations are consistent with a translational role for these proteins.     

The wacA gene of Dictyostelium discoideum is a developmentally regulated member of the MIP family

We isolated a cDNA from Dictyostelium discoideum that encodes a 30 kDa protein with significant similarity to members of the major intrinsic protein (MIP) family of membrane transporters. The most closely related protein in the public data bases is an aquaporin from Cicadella viridis which shows 34% identity. The cDNA was used to isolate and characterize genomic fragments carrying the Dictyostelium gene which we named wacA. Genomic probes were used to recognize wacA mRNA isolated at various stages of development. The results showed that the gene is developmentally regulated such that the mRNA first appears at 12 h of development and is retained throughout the remainder of development. In situ hybridization of whole mounts prepared at 15 h of development showed that wacA mRNA accumulates exclusively in prespore cells and is absent from prestalk cells. Although wacA expression is prespore specific, disruption of the gene by homologous recombination did not result in observable alterations in the formation of spores or their resistance to osmotic challenges.     

Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.