Reciprocal stabilization of transcription factor binding integrates two signaling pathways to regulate fission yeast fbp1 transcription.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Expression of Fbp2, a Newly Discovered Constituent of Memory Formation Mechanisms, Is Regulated by Astrocyte-Neuron Crosstalk.

Fbp2 (muscle isozyme of fructose 1,6-bisphosphatase) is a glyconeogenesis-regulating enzyme and a multifunctional protein indispensable for long-term potentiation (LTP) formation in the hippocampus. Here, we present evidence that expression of Fbp2 in murine hippocampal cell cultures is regulated by crosstalk between neurons and astrocytes. Co-culturing of the two cell types results in a decrease in Fbp2 expression in astrocytes, and its simultaneous increase in neurons, as compared to monocultures. These changes are regulated by paracrine signaling using extracellular vesicle (EV)-packed factors released to the culture medium. It is well accepted that astrocyte-neuron metabolic crosstalk plays a crucial role in shaping neuronal function, and recently we have suggested that Fbp2 is a hub linking neuronal signaling with redox and/or energetic state of brain during the formation of memory traces. Thus, our present results emphasize the importance of astrocyte-neuron crosstalk in the regulation of the cells' metabolism and synaptic plasticity, and bring us one step closer to a mechanistic understanding of the role of Fbp2 in these processes.

Recruitment and delivery of the fission yeast Rst2 transcription factor via a local genome structure counteracts repression by Tup1-family corepressors.

Transcription factors (TFs) determine the transcription activity of target genes and play a central role in controlling the transcription in response to various environmental stresses. Three dimensional genome structures such as local loops play a fundamental role in the regulation of transcription, although the link between such structures and the regulation of TF binding to cis-regulatory elements remains to be elucidated. Here, we show that during transcriptional activation of the fission yeast fbp1 gene, binding of Rst2 (a critical C2H2 zinc-finger TF) is mediated by a local loop structure. During fbp1 activation, Rst2 is first recruited to upstream-activating sequence 1 (UAS1), then it subsequently binds to UAS2 (a critical cis-regulatory site located approximately 600 base pairs downstream of UAS1) through a loop structure that brings UAS1 and UAS2 into spatially close proximity. Tup11/12 (the Tup-family corepressors) suppress direct binding of Rst2 to UAS2, but this suppression is counteracted by the recruitment of Rst2 at UAS1 and following delivery to UAS2 through a loop structure. These data demonstrate a previously unappreciated mechanism for the recruitment and expansion of TF-DNA interactions within a promoter mediated by local three-dimensional genome structures and for timely TF-binding via counteractive regulation by the Tup-family corepressors.

Brassinosteroid-induced CO(2) assimilation is associated with increased stability of redox-sensitive photosynthetic enzymes in the chloroplasts in cucumber plants.

Brassinosteroids (BRs) play important roles in plant growth, development, photosynthesis and stress tolerance; however, the mechanism underlying BR-enhanced photosynthesis is currently unclear. Here, we provide evidence that an increase in the BR level increased the quantum yield of PSII, activities of Rubisco activase (RCA) and fructose-1,6-bisphosphatase (FBPase), and CO(2) assimilation. BRs upregulated the transcript levels of genes and activity of enzymes involved in the ascorbate-glutathione cycle in the chloroplasts, leading to an increased ratio of reduced (GSH) to oxidized (GSSG) glutathione in the chloroplasts. An increased GSH/GSSG ratio protected RCA from proteolytic digestion and increased the stability of redox-sensitive enzymes in the chloroplasts. These results strongly suggest that BRs are capable of regulating the glutathione redox state in the chloroplasts through the activation of the ascorbate-glutathione cycle. The resulting increase in the chloroplast thiol reduction state promotes CO(2) assimilation, at least in part, by enhancing the stability and activity of redox-sensitive photosynthetic enzymes through post-translational modifications.

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1+ and fbp1+ during glucose starvation even though gld1+ and fbp1+ are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1+ was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1+ and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation.

Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure.

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP.

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

Two light-responsive elements of pea chloroplastic fructose-1,6-bisphosphatase gene involved in the red-light-specific gene expression in transgenic tobaccos.

The pea chloroplast fructose-1,6-bisphosphatase (FBPase) gene was cloned from a pea genomic library and sequenced. The gene contained three introns and four exons. Both in vitro and in vivo analyses of the promoter region of the gene were carried out simultaneously to elucidate the mechanisms of light-mediated gene expression. Two light-responsive elements were identified in gel mobility shift assays: a GT-1-like sequence for the binding of a GT-1-like factor (termed pea factor 1; PF1) and a binding site for a dark-specific factor (termed pea factor 2; PF2). The binding affinity of PF1 was higher in light-grown peas than in dark-grown peas and was affected by phosphorylation. The binding site was located at nucleotides (nt) -326 to -341. PF2 binding was dark-specific and the binding region was located upstream of the PF1-binding site (nt -492 to -412). In vivo experiments with transgenic tobacco plants suggested that the region between nt -411 and -272 contained a PF1-binding site that promoted light-mediated expression of the pea chloroplast FBPase. In contrast, the 81-bp region between nt -492 and -412, which is located further upstream than the PF1-binding site, negatively regulated light-mediated expression of FBPase. Moreover, activation of gene expression by the region (nt -411 to -272) contained a PF1-binding site that was sensitive to red-light irradiation, suggesting that the expression of the chloroplast FBPase was regulated by the phytochrome system. Interestingly, the binding region for the dark-specific factor (PF2; nt -492 to -412) not only repressed gene expression in the dark, but also acted as a light-dependent activating element of the chloroplast FBPase gene.

Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata).

Our objective is to understand the low metabolic utilization of dietary carbohydrates in fish. We compared the regulation of gluconeogenic enzymes at a molecular level in two fish species, the common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata), known to be relatively tolerant to dietary carbohydrates. After cloning of partial cDNA sequences for three key gluconeogenic enzymes (glucose-6-phosphatase (G6Pase), fructose biphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) in the two species, we analyzed gene expressions of these enzymes 6 and 24 h after feeding with (20%) or without carbohydrates. Our data show that there is at least one gluconeogenic enzyme strongly regulated (decreased expression after feeding) in the two fish species, i.e. the PEPCK for common carp and G6Pase/FBPase for gilthead seabream. In these fish species, the regulation seems to be similar to the mammals at least at the molecular level.

p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK(1)-FBPase(+) cells.

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.

Elucidating the biosynthesis of 2-carboxyarabinitol 1-phosphate through reduced expression of chloroplastic fructose 1,6-bisphosphate phosphatase and radiotracer studies with 14CO2.

2-carboxyarabinitol 1-phosphate limits photosynthetic CO2 assimilation at low light because it is a potent, naturally occurring inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase. Evidence is presented that this inhibitor is derived from chloroplastic fructose 1,6-bisphosphate. First, transgenic plants containing decreased amounts of chloroplastic fructose 1,6-bisphosphate phosphatase contained increased amounts of fructose 1,6-bisphosphate and 2-carboxyarabinitol 1-phosphate and greatly increased amounts of the putative intermediates hamamelose and 2-carboxyarabinitol, which in some cases were as abundant as sucrose. Second, French bean leaves in the light were shown to incorporate 14C from 14CO2 sequentially into fructose 1,6-bisphosphate, hamamelose bisphosphate, hamamelose monophosphate, hamamelose, and 2-carboxyarabinitol. As shown previously, 14C assimilated by photosynthesis was also incorporated into 2-carboxyarabinitol 1-phosphate during subsequent darkness.

Insulin inhibits glucocorticoid-stimulated L-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression by activation of the c-Jun N-terminal kinase pathway.

The hepatic isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K/Fru-2,6-BPase) is transcriptionally stimulated by glucocorticoids, whereas insulin blocks this stimulatory effect. Although this inhibitory effect has been extensively reported, nothing is known about the signalling pathway responsible. We have used well-characterized inhibitors for proteins involved in different signalling cascades to assess the involvement of these pathways on the transcriptional regulation of glucocorticoid-stimulated PF2K/Fru-2,6-BPase by insulin. Our results demonstrate that the phosphoinositide 3-kinase, p70/p85 ribosomal S6 kinase, extracellular signal-regulated protein kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinase pathways are not involved in the inhibitory effect of insulin on glucocorticoid-stimulated PF2K/Fru-2,6-BPase. To evaluate the implication of the MAP kinase/ERK kinase (MEK)-4-stress-activated protein kinase-c-Jun-N-terminal protein kinase ('JNK-SAPK') pathway we overexpressed the N-terminal JNK-binding domain of the JNK-interacting protein 1 ('JIP-1'), demonstrating that activation of JNK is necessary for the insulin inhibitory effect. Moreover, overexpression of MEK kinase 1 and JNK-haemagglutinin resulted in the inhibition of the glucocorticoid-stimulated PF2K/Fru-2,6-BPase. These results provide clear and specific evidence for the role of JNK in the insulin inhibition of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression. In addition, we performed experiments with a mutant of the glucocorticoid receptor in which the JNK phosphorylation target Ser-246 had been mutated to Ala. Our results demonstrate that the phosphorylation of the glucocorticoid receptor on Ser-246 is not responsible for the JNK repression of glucocorticoid-stimulated PF2K/Fru-2,6-BPase gene expression.

The xenobiotic methionine sulfoximine modulates carbohydrate anabolism and related genes expression in rodent brain.

Methionine sulfoximine is a xenobiotic amino acid derived from methionine. One of its major properties is to display a glycogenic activity in the brain. After studying this property, we investigate here a possible action of this xenobiotic on the expression of genes related to carbohydrate anabolism in the brain. Glycogen was studied by the means of electron microscopy. Astrocytes were cultured and the influence of methionine sulfoximine on carbohydrate anabolism in these cells was investigated. In vivo, methionine sulfoximine induced a large increase in glycogen accumulation. It also enhanced the glycogen accumulation in cultured astrocytes principally, when the medium was enriched in glucose. The gluconeogenic enzyme fructose-1,6-bisphosphatase may account for glycogen accumulation. Plasmids were built using antisens cDNA to permanently block the expression of fructose-1,6-bisphosphatase. An eukaryotic vector was used and the expression of fructose-1,6-bisphosphatase gene was under the control of the promoter of the glial fibrillary acidic protein. In this case, the glycogen content in cultured astrocytes largely decreased. This work shows that methionine sulfoximine enhances energy carbohydrate synthesis in the brain. Since this xenobiotic also enhances the expression of some genes related to one of the key step of glucose synthesis, it is possible that genes may be one target of methionine sulfoximine. Next investigations will study the actual effect of methionine sulfoximine in the cells.

Cloning, characterization and expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from potato.

We have isolated cDNA clones encoding the regulatory enzyme fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from a potato (Solanum tuberosum) leaf cDNA library. All clones represented transcripts of the same gene (F2KP1). Functionality of the encoded protein was verified by expression of the active enzyme in Escherichia coli. The expressed enzyme had both kinase activity which forms fructose-2,6-bisphosphate from fructose-6-phosphate and ATP, and phosphatase activity which degrade fructose-2,6-bisphosphate. The recombinant potato enzyme was radiolabelled by [2-32P]fructose-2,6-bisphosphate verifying conservation of the phosphatase catalytic mechanism which involves a phospho-protein intermediate. The deduced amino acid sequence corresponding to the catalytic core for F2KPI is homologous to the fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase isolated from animals and yeast, with conservation of amino acids involved in substrate binding and catalytic mechanisms. The sequence for F2KP1 also includes a 102 amino acids long NH2-terminal with no homology to any previously identified enzymes. This NH2 terminal may be even longer since an upstream stop codon has not yet been identified. Northern blot analysis of potato showed that the F2KP1 transcript is present in several tissues including source leaves, sink leaves and flowers, whereas the transcripts were not detectable in developing tubers. Southern blot analysis of Solanum phureja suggest there to be only one copy of the gene.

Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae.

External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms.

A regulatory factor, Fil1p, involved in derepression of the isocitrate lyase gene in Saccharomyces cerevisiae--a possible mitochondrial protein necessary for protein synthesis in mitochondria.

A mutant was isolated that failed to derepress the 5' upstream region of Candida tropicalis isocitrate lyase gene (UPR-ICL)-mediated gene expression in acetate medium, and the gene (FIL1) that complemented this mutation was isolated. The fil1 null mutant in which FIL1 is disrupted (deltafil1 strain) could not grow on acetate or ethanol, and the derepression of the isocitrate lyase encoded by ICL1 in Saccharomyces cerevisiae was also defected. The amino acid sequence of Fil1p (230 amino acids) showed similarity to ribosome recycling factors (RRFs) of prokaryotes. Compared to prokaryotic RRFs, Fil1p had an N-terminal 46-amino-acid extension which was shown to be able to function as a mitochondrial-targeting sequence. The subcellular fractionation of the deltafil1 strain showed that protein constituents of the mitochondrial fraction of the deltafil1 strain differed from those of the wild-type strain, but resembled those of chloramphenicol-treated cells or rho(o) cells. The specific activity of cytochrome c oxidase, was severely decreased in deltafil1, rho(o) and chloramphenicol-treated cells compared with wild-type cells, while enzymatic levels of mitochondrial NAD+-linked isocitrate dehydrogenase, which is encoded by nuclear DNA, were not affected. These results suggest that Fillp is necessary for protein synthesis in mitochondria of S. cerevisiae. Furthermore, cells treated with antimycin A, along with chloramphenicol-treated, rho(o), and deltafil1 cells, showed deficiency in derepression of isocitrate lyase. Northern-blot analysis showed that this can be ascribed to no increase in transcription of ICL1 and FBP1 encoding fructose 1,6-bisphosphatase. The results indicate the presence of a communication pathway between mitochondria and the nucleus which represses expression of genes encoding the key enzymes of the glyoxylate cycle and gluconeogenic pathway when there is a deficiency in the mitochondrial respiratory chain.

Effect of starvation on gene expression of regulatory enzymes of glycolysis/gluconeogenesis in genetically obese (fa/fa) Zucker rats.

OBJECTIVE: To study the mechanism that controls fructose-2,6-bisphosphate (Fru-2,6-P2) accumulation, as well as the mRNAs levels of the glycolytic/gluconeogenic regulatory enzymes in the livers of fed and starved lean (fa/-) and obese (fa/fa) Zucker rats. DESIGN: Rats were fed a standard chow or deprived of food for 24 h. SUBJECTS: Male lean (fa/-) and genetically obese (fa/fa) rats (nine weeks old). MEASUREMENTS: Fru-2,6-P2 concentration, 6-phosphofructo-2-kinase (PFK-2), glucokinase (GK), pyruvate kinase (PK) activities and the mRNA levels of GK, PFK-2, L-type pyruvate kinase, fructose-1,6-bisphosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PEPCK) were analyzed. RESULTS: PFK-2/FBPase-2 mRNA decreased during starvation in both fa/- and fa/fa animals. Although PFK-2/FBPase-2 mRNA levels were similar in fed lean and obese rats, PFK-2 concentration and activity were higher in fed obese than in fed lean animals, which might explain the high concentration of Fru-2,6-P2 observed in obese animals. During starvation, PFK-2 protein concentration decreased, correlating with the enzymatic activity and Fru-2,6-P2 levels. The activities of GK and L-pyruvate kinase (L-PK) also increased in fed obese (fa/fa) rats compared with fed lean (fa/-) animals, but decreased during starvation. The mRNA levels of glycolytic enzymes in fed obese rats were similar (PFK-2) or higher than (GK, L-PK) in fed lean animals. During starvation, they decreased in lean and obese rats with one important exception, GK mRNA remained high in obese animals. The mRNA of gluconeogenic enzymes remained constant (FBPase-1) or increased (PEPCK) during fasting. CONCLUSION: The changes observed might be explained by the hyperinsulinaemia observed in the liver of obese rats, which might lead to the stimulation of glycolysis and lipogenesis.

Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase.

By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.

Molecular characterization of a cDNA encoding chloroplastic fructose-1,6-bisphosphatase from soybean (Glycine max L.).

A full-length cDNA of soybean chloroplastic fructose-1,6-bisphosphatase was cloned and sequenced. The cDNA contained 1321 bp with 5' (26 bp) and 3' (88 bp) untranslated regions. The open reading frame of the cDNA contained 1206 bp corresponding to a polypeptide of 402 amino acids with 50 amino acid residues of a transit peptide at N-terminus that is necessary for transport into the chloroplast. A unique site relevant to the action of thioredoxin f was conserved at 221 amino acid residue. Northern blot analysis indicated that the expression of the enzyme was regulated by light illumination.

Cell volume regulates liver phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase genes.

Hypertonic-induced cell shrinkage increases glucose release in H-4-II-E rat hepatoma cells. This is paralleled by a concomitant increase in the mRNA levels of the rate-limiting enzymes of the pathway of gluconeogenesis, phosphoenolpyruvate carboxykinase (PCK) and fructose-1,6-bisphosphatase (FBP), of seven- and fivefold, respectively. In contrast, hypotonic-induced swelling of the cells results in a transient decrease in PCK and FBP mRNAs to 15% and 39% of control levels. The antagonistic effects of hyper- and hypotonicity mimic the counteracting effects of adenosine 3',5'-cyclic monophosphate (cAMP) and insulin on PCK and FBP mRNA levels. The hypertonic-induced increase in mRNA levels is due to an enhanced transcriptional rate, whereas the decrease in mRNAs caused by hypotonicity results from a decrease in transcription as well as mRNA stability. The inductive effect of hypertonicity does not require ongoing protein synthesis and acts independently of the cAMP-dependent protein kinase and protein kinase C pathways. These results suggest that cell volume changes in liver cells may play an important role in regulating hepatic glucose metabolism by altered gene expression.