Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.     

Developmental changes in soluble proteins during the early development of Dictyostelium discoideum

Changes in the pattern of soluble proteins that accumulatedat the growth phase, interphase and late-aggregation phase ofthe cellular slime mold Dictyostelium discoideum were studiedby two-dimensional polyacrylamide gel electrophoresis. Amongthe 300 proteins detected during the early development, themost soluble do not change during the growth and aggregationphases, but about 90 proteins show changes in their relativeintensities on staining. During the transition from growth tothe interphase, the predominant changes were the disappearanceof 16 spots, the decrease in 30 spots, the appearance of 13new spots, and the increase in 14 spots. In contrast, from theinterphase to the late-aggregation phase, there were remarkableprogressive increases in 13 spots, an overall increase in 6spots, a decrease in 16 spots, the appearance of 8 new spotsand the disappearance of 4 spots. (Received July 13, 1979; )     

The effect of altered glycosylation on the characteristics of lysosomal trehalase in Dictyostelium discoideum

The trehalase I of Dictyostelium discoideum exhibits characteristics of a typical lysosomal enzyme. The enzyme is glycosylated and carries a number of negatively charged components which cause it to be a very acidic protein. Strain M31, bears a recessive mutation mod A which alters the post-translational modification of several lysosomal enzymes including trehalase. A direct consequence of this mutation is a reduction of the negatively charged components on lysosomal enzymes. This reduction in negativity is observed in the altered chromatographic and electrophoretic behaviour of M31 trehalase.Trehalase I is synthesized during spore germination. Tunicamycin prevents the formation of recoverable trehalase from germinating spores but does not interfere with the germination process. These results indicate that the trehalase I synthesized during spore germination is not required for the successful completion of spore germination. Minor modification in the glycosylation, as seen in strain M31, does not affect the enzymatic activity. However, when glycosylation is greatly reduced by tunicamycin the enzyme is inactive.     

Developmental commitment in Dictyostelium discoideum

Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.     

Developmental decisions in Dictyostelium discoideum

Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system. Key signaling pathways identified as regulating cell fate decisions are likely to be conserved with metazoa and are providing insight into differentiation decisions under circumstances where considerable cell movement takes place during development.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Modifications of lysosomal enzymes in Dictyostelium discoideum

This paper has two purposes. The first is to review the past studies on the structure, biosynthesis, and immunological properties of a class of glycoproteins, the lysosomal enzymes, in Dictyostelium discoideum. The second purpose is to present new data on the analysis of mutant strains altered in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides, and on the characterization of new carbohydrate antigenic determinants found on multiple proteins in Dictyostelium. We will also show how a combination of genetic, biochemical and immunochemical approaches have been used to unravel a portion of the glycosylation pathway in Dictyostelium.The long-term goal of these studies is to use Dictyostelium discoideum as a model system to understand the functions of a variety of glycoconjugates in a multicellular organism. The existence of a large number of mutant strains which are altered in a variety of cellular functions, development and the posttranslational modification of multiple proteins, offers a great opportunity to explore this area.     

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.     

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.     

Actin-associated proteins in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is becoming the premier system for the explication of the biochemical and cellular events that occur during motile processes. Proteins associated with the actin cytoskeleton, in particular, appear to play key roles in cellular responses to many external stimuli. This review summarizes our present understanding of the actin-associated proteins in Dictyostelium, including their in vitro activities and their structural and/or functional analogues in mammalian cells.     

Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum

In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.     

The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum

We have examined the relationship of N-linked oligosaccharide structures to the proper targeting and proteolytic processing of two lysosomal enzymes, alpha-mannosidase and beta-glucosidase, in the slime mold Dictyostelium discoideum. Two different mutant strains, HL241 and HL243, each synthesize the same nonglucosylated, truncated, lipid-linked oligosaccharide precursor, Man6GlcNAc2. [3H]Mannose-labeled N-linked oligosaccharides were studied following their release from immunoprecipitated alpha-mannosidase and beta-glucosidase by digestion with peptide:N-glycosidase F. The oligosaccharides from both mutants resembled each other, but they were smaller and contained fewer anionic groups than those from the wild-type. The oligosaccharides from the mutants strains were reduced in sulfate and Man-6-P content, and all Man-6-P was in the form of acid-stable phosphodiesters. Pulse-chase radiolabeling experiments using [35S] methionine indicated that the precursor forms of both enzymes were smaller than wild-type, and that this difference was due solely to differences in N-linked oligosaccharides. The precursor forms of the enzymes were not over-secreted, but appeared to be proteolytically processed into mature forms at approximately 50% the rate of wild-type. This is mainly due to their prolonged retention in the rough endoplasmic reticulum, but, ultimately, both enzymes were properly targeted to lysosomes. These studies indicate that a reduction in the amount of sulfation, phosphorylation or size of the N-linked oligosaccharides in these mutants is not critical for the proteolytic processing and targeting of the lysosomal enzymes, but that these changes may influence their rate of exit from the rough endoplasmic reticulum.     

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.     

Developmental regulation of cell-type-enriched mRNAs in Dictyostelium discoideum

We describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during development of Dictyostelium discoideum and two new mRNAs that are expressed in a non-cell-type-specific manner. None of the prespore-enriched mRNAs are detectable in Dictyostelium cells until 13-15 h of development but then they increase dramatically and peak at 18-22 h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk-enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.     

Hypertonicity-induced phosphorylation of proteins in Dictyostelium discoideum

Abstract By exposing the cells of Dictyostelium discoideum to a high concentration (120 mM) of KCl, several species of proteins (188 kilodalton (kDa), 95 kDa, and 71 kDa) are specifically phosphory-lated. This phosphorylation is induced irrespective of the time of starvation of cells by KCl, but not by cAMP, and inhibited by cycloheximide. The ³²P-labeled phosphoryl groups of 95- and 71-kDa proteins disappear by chasing during the subsequent differentiation step in a liquid shake culture. The majority of the 188- and 95-kDa proteins exist in the plasma membrane fraction.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

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.     

Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.