Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.     

Acquisition process of differentiation competence in Dictyostelium discoideum

It was previously shown [K. Okamoto, J. Gen. Microbiol. 127, 301 (1981)] that Dictyostelium discoideum cells dissociated from early aggregates, but not aggregation competent cells obtained in a suspension culture, undergo prespore differentiation, when transferred into a medium containing glucose, albumin, and cAMP. Therefore, the former, but not the latter, is considered to have been acquired "differentiation competence." In the present work, the requirements for cells to acquire the differentiation competence are investigated with D. discoideum NC4 strain. On solid substratum, the incubation above a threshold density is absolutely required for this process, while cell aggregation itself is not essential. In suspension cultures, the competence is acquired only under hypertonic conditions. Inhibition of protein synthesis or depletion of cAMP does not affect the acquisition process of the competence. The requirement of hypertonic treatment was also investigated with several other D. discoideum strains.     

Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum

In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.     

Maturation of asparagine-linked oligosaccharides in Dictyostelium discoideum analyzed with modification-specific probes

Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Codon preference in Dictyostelium discoideum.

Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.     

Phosphotyrosine-containing proteins in Dictyostelium discoideum.

Phosphotyrosine-containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205-220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [gamma-32P]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205-220 kDa bands.     

A Dictyostelium discoideum mutant exhibiting calcium-dependent, high-level detergent resistance.

We report the isolation of a mutant of the cellular slime mold Dictyostelium discoideum that is highly resistant to the lethal action of the nonionic detergent Triton X-100. The resistance is completely dependent on the presence of divalent cations, of which Ca2+ is the most effective.     

Effect of intracellular carbohydrates on heat resistance of Dictyostelium discoideum spores.

The effect of intracellular trehalose and glycogen on the survival of spores of Dictyostelium discoideum ATCC 25697 after exposure to supraoptimal temperatures was examined. Cells metabolically perturbed by incubation in glucose and inorganic phosphate have intracellular trehalose and glycogen concentrations fivefold and twofold higher, respectively, than those of the controls. These cells were more resistant to the lethal effects of wet heat (45 degrees to 55 degrees C) than were control cells. The presence of 40 mM trehalose in the buffer during heat stress increased the survival of nonperturbed cells to approximately the level of the perturbed cells. No protection was observed when cells were heated in the presence of exogenous glycogen. Glucose or disaccharides other than trehalose when present during heat stress, had no effect on heat resistance. Nonperturbed cells preincubated in 40 mM trehalose and washed before heat stress were more resistant to killing than were controls. Cells perturbed with inorganic phosphate, which has been shown to increase trehalose concentrations but decrease glycogen concentrations, were also more resistant to the lethal effects of wet heat than were controls. The data suggest that trehalose has an effect on the wet-heat resistance of D. discoideum. Some possible mechanisms are suggested.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

Acetylated and nonacetylated actins in Dictyostelium discoideum.

We have carried out a two-dimensional gel analysis of the actin system of Dictyostelium discoideum. Our results show that on the basis of isoelectric focusing, there is a single major [35S]methionine-labeled species which corresponds both to the actin purified by Uyemura et al. (Uyemura, D., Brown, S.S., and Spudich, J.A. (1978) J. Biol. Chem. 253, 9088-9095) and to the Coomassie Blue staining species seen in whole cell lysates of the organism. We also detect a minor labeled actin species, x, which has no corresponding Coomassie Blue staining counterpart. This species turns over much more rapidly than the major actin and has one more positive charge. It is not labeled with [3H]acetate, whereas the major actin is. When D. discoideum RNA is added to a mRNA-dependent rabbit reticulocyte lysate protein translation system, only one major actin is seen, and this species corresponds to the major actin observed in vivo. If endogenous acetyl coenzyme A is removed from the translation system, a second major actin appears corresponding in position to x. These results indicate that in D. discoideum, there is present a single major actin species in addition to a small amount of a rapidly turning over actin which is a nonacetylated form of the major actin. Additional experiments examining these actins through the developmental cycle of the organism show no consistent differences with the results obtained using vegetative cells.     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

Differentiation without mitosis in Dictyostelium discoideum.

Dictyoselium discoideum Ax-2 amoebae incubated in the presence of the microtubule inhibitor nocodazole, irreversibly lost their ability to multiply. Nocodazole-treated cells remained viable and RNA and protein synthesis continued for at least 48 h. When nocodazole-treated amoebae were allowed to develop on Millipore filters or on agar slides they differentiated with some delay when compared with controls. These results show that mitosis, naturally present during the developmental cycle of Dictyostelium discoideum Ax-2, is not indispensible for differentiation.     

Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

Endogenous biotinylated proteins in Dictyostelium discoideum.

Biotin-dependent enzymes are involved in carboxylation, decarboxylation and transcarboxylation reactions. Here, we have used sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting followed by probing with avidin to identify biotin-containing polypeptides in Dictyostelium discoideum. Twenty biotinyl polypeptides were visualized, with a 23 kDa protein appearing transiently. Based upon the molecular mobility of the biotinyl polypeptides, D. discoideum may contain the biotin-dependent enzymes acetyl CoA carboxylase, proprionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase.     

Structure and function of beta-blucosidases in Dictyostelium discoideum.

There are two isozymes of beta-glucosidase in developing cells of Dictyostelium discoideum. A procedure for screening large numbers of clones for beta-glucosidase activity was utilized to obtain mutations which directly affect the activity. We recovered seven strains which lack both isozymes and four strains with residual activity in which enzymatic and physical properties of both isozymes are altered. Beta-Glucosidase appears to act as a block to selfing in macrocyst formation as shown by the fact that ssite mating type to form macrocyst-like structures. Immunological evidence utilizing antisera prepared against purified beta-glucosidase-1 demonstrates that most of the glycosidases in Dictyostelium discoideum share a common antigenic determinant which appears to be added post-translationally. The two isozymes of beta-glucosidase share common protein subunits but the antigenic determinant is either lacking or masked in beta-glucosidase-2. This may account for some of the enzymatic and physical differences between the two isozymes.