Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.     

Identification of a second cellulose synthase gene (acsAII) in Acetobacter xylinum.

A second cellulose synthase gene (acsAII) coding for a 175-kDa polypeptide that is similar in size and sequence to the acsAB gene product has been identified in Acetobacter xylinum AY201. Evidence for the presence of this gene was obtained during analysis of A. xylinum mutants in which the acsAB gene was disrupted (I.M. Saxena, K. Kudlicka, K. Okuda, and R.M. Brown, Jr., J. Bacteriol. 176:5735-5752, 1994). Although these mutants produced no detectable cellulose, they exhibited significant cellulose synthase activity in vitro. The acsAII gene was isolated by using an acsAB gene fragment as a probe. The acsAII gene coded for cellulose synthase activity as determined from sequence analysis and study of mutants in which this gene was disrupted. A mutant in which only the acsAII gene was disrupted showed no significant differences in either the in vivo cellulose production or the in vitro cellulose synthase activity compared with wild-type cells. Mutants in which both the acsAII and acsAB genes were disrupted produced no cellulose in vivo and exhibited negligible cellulose synthase activity in vitro, thus confirming that the cellulose synthase activity observed in the acsAB mutants was coded by the acsAII gene. These results establish the presence of an additional gene for cellulose synthase expressed in cells of A. xylinum, yet this gene is not required for cellulose production when cells are grown under laboratory conditions.     

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.     

Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.     

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.     

The Drosophila NR4A nuclear receptor DHR38 regulates carbohydrate metabolism and glycogen storage

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.     

Relationships between mutations affecting protein kinase and accumulation of energy reserves in Saccharomyces cerevisiae

Abstract Independently discovered mutations which alter cyclic-AMP dependent protein kinase activity in Saccharomyces cerevisiae are analysed in relation to trehalose and glycogen storage. The defective trehalose and glycogen accumulation in strains which bear the glc1 mutation results from abnormal activation of trehalase by a protein kinase which has partially lost its cAMP dependence. Cells bearing the bcy1 mutation produce an altered protein kinase due to extremely low levels of the cAMP-binding protein. This altered kinase activates trehalase, resulting in low trehalose contents in these cells. In cell-free extracts of control strains (S288C and 7Q-2D), which produce normal levels of glycogen and trehalose, the enzyme trehalase is mainly found in an inactive, cryptic form. Each of the haploid strains containing one of the mutant genes (glc1, glc4-1 and bcy1) is defective in both trehalose and glycogen accumulation and exhibits low activation ratios of trehalase by protein kinase. Genetic complementation experiments clearly establish that the bcy1 mutation involves a different gene to that altered by the glc1 mutation, since the resulting diploid behaved normally. Strain AM9-10D, previously classified as wild-type (normal for bcy1 ), is defective in the accumulation of trehalose and glycogen and exhibits almost all trehalose in the active form.     

Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.

Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells. We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen. These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase. Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis. Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen. Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase. These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell. The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase.     

Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

Relationship between the cytoplasmic activator of adenylate cyclase and glycogen metabolism in rat lung

Summary The role of cytoplasmic activator of adenylate cyclase in rat lung metabolism was investigated. Mouse adrenal tumor (MAT) cells undergo differentiation in response to choleratoxin which acts through cyclic AMP. The activator of adenylate cyclase from rat lung also produced cyclic AMP in a disrupted MAT cell preparation. However, unlike choleratoxin, it did not induce MAT cell differentiation in whole cells. These results suggest impermeability of MAT cells, and possibly other cells, to the activator. Thus, means of altering activator activity in lung cytoplasm were sought, and changes in activator activity were related to lung glycogen. Adrenalectomy (ADX) in rats led to a reduction in activator activity that was accompanied by an elevation in lung glycogen. Dexamethasone treatment of adrenalectomized rats reversed both of these effects. Streptozotocin-induced diabetes in rats elevated activator activity and lowered lung glycogen. Insulin treatment of the diabetic rats restored activator activity to the normal control values. Preweaning of rats on day 16 instead of day 22 increased activator activity on the 19th day over the controls and there was a concomitant decrease in lung glycogen. Feeding the separated pups with homogenized milk restored glycogen and activator activity to the control values. These results indicate that activator activity in rat lung cytoplasm was dependent on the circulating levels of cortisol and insulin, and that there appeared to be an inverse relationship between activator activity and glycogen level in rat lungs.     

Saccharomyces cerevisiae gene SIT4 is involved in the control of glycogen metabolism.

The gene SIT4 of S. cerevisiae, which codes for a protein structurally related to the catalytic subunit of mammalian protein phosphatase 2A, was disrupted in vitro. Analysis of glycogen synthase activity ratio in mutant haploid cells indicated that the enzyme was less active than in wild-type cells. On the contrary, glycogen phosphorylase alpha activity was much higher. The activation of glycogen synthase observed in wild-type cells after incubation with lithium ions was not detected in mutant cells. These results suggest that the product of gene SIT4, a putative protein phosphatase, could be involved in the control of glycogen metabolism in yeast cells.