Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources.

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.     

Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae

Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+. The phosphatase is strongly inhibited by heparin and fluoride. L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM. The molecular mass of the native phosphatase was found to be 180,000 Da. Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each. Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7. Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic [32P] phosphate was demonstrated.     

Kinetic differences between two interconvertible forms of fructose-1,6-bisphosphatase from Saccharomyces cerevisiae

Newly synthesized, [³⁵S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, 11β-hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [³⁵S]methionine-labeled 11β-hydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.     

Ubiquitous presence of gluconeogenic regulatory enzyme, fructose-1,6-bisphosphatase, within layers of rat retina

To shed some light on gluconeogenesis in mammalian retina, we have focused on fructose-1,6-bisphosphatase (FBPase), a regulatory enzyme of the process. The abundance of the enzyme within the layers of the rat retina suggests that, in mammals in contrast to amphibia, gluconeogenesis is not restricted to one specific cell of the retina. We propose that FBPase, in addition to its gluconeogenic role, participates in the protection of the retina against reactive oxygen species. Additionally, the nuclear localization of FBPase and of its binding partner, aldolase, in the retinal cells expressing the proliferation marker Ki-67 indicates that these two gluconeogenic enzymes are involved in non-enzymatic nuclear processes.     

Liver peptide stabilizing factor protects phosphofructokinase against inactivation by fructose-1,6-bisphosphatase.

Rabbit liver fructose-1,6-bisphosphatase (FDPase) can reversibly inactivate both rabbit muscle and rat liver phosphofructokinases (PFK) under appropriate conditions. The peptide factor which stabilizes rat liver PFK-L₂ against thermal inactivation has now been found to protect both PFKs from inactivation by FDPase. Assay at high ATP (ca. 3 mM) is necessary to demonstrate these reversible changes. In addition, the activation of FDPase by liver cytosol, by oleate plus cytosol, or by oleate plus muscle PFK is lowere about 50% in the presence of peptide factor. These observations suggest an active participation of the peptide factor in regulation of liver glycolysis and gluconeogenesis.     

Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.     

Purification and regulation of fructose-1,6-bisphosphatase from Bacillus licheniformis.

The fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the spore-forming bacterium Bacillus licheniformis was purified approximately 800-fold (with a 20% yield of activity) by a procedure that included ammonium sulfate precipitation, precipitation by MnCl2, and gamma-alumina gel absorption. Catalysis by this enzyme in vitro was specific for fructose 1,6-bisphosphate (Km of approximately 20 muM) and proceeded optimally at pH 8.0 to 8.5. Fructose-1,6-bisphosphatase was found to be rapidly inactivated by incubation in the presence of AMP or in the absence of Mn2+. The AMP inactivation was prevented by adding P-enolpyruvate to the incubation mixture. The enzyme was slowly inactivated when incubated in the presence of stabilizing concentrations of Mn2+ (5 mM) at protein concentrations of less than 8 mg of protein per ml. An additional system is produced during sporulation which specifically inactivates fructose bisphosphatase in vitro. This system, which is distinctly different from the AMP inactivating system, can be blocked by P-enolpyruvate. This fructose bisphosphatase, like fructose bisphosphatases from other sources, was strongly inhibited by AMP, exhibiting a Ki of approximately 5 muM. This inhibition, however, could be completely overcome by P-enolpyruvate. P-enolpyruvate was also found to be an activator of the enzyme and exhibited a Km of approximately 2 muM. This activation was prevented in a competitive manner by AMP, exhibiting a Ki of approximately 5 muM. No other effector of fructose bisphosphatase was identified in an extensive search. The specific activity of fructose bisphosphatase in crude extracts was found to be independent of the stage of the life cycle of the bacterium or of the nature of the carbon-energy source supporting growth. Immunoprecipitation studies indicate that no new species of fructose biphosphatase is produced during gluconeogenic growth or sporulation. The enzyme extracted from cells under a variety of physiological conditions exhibited a molecular weight of about 5 times 10-5 as determined by sucrose density centrifugation. Therefore, it is proposed that a single constitutively synthesized fructose bisphosphatase is present in B. licheniformis. Measurements of the intracellular level of fructose 1,6-bisphosphate indicate that the variation in the level of substrate throughout growth (1 mM) and sporulation (0.3 mM) does not regulate the in vivo activity of this enzyme, since the Km of the enzyme for fructose 1,6-bisphosphate is approximately 10-fold lower than the lowest in vivo concentration of substrate. P-enolpyruvate is proposed as the major regulator of fructose bisphosphatase activity in vivo.     

Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.     

Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.     

The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K_m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.     

Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site

In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of [32P] orthophosphate. Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography. Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939-3943). The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11. We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests. Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit. However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures. An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures. The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.     

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisphosphatase resembles other regulatory enzymes involved in photosynthetic CO2 assimilation in its activation by chloroplast metabolites.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.