Trehalose enzymes as models for signal transduction in Saccharomyces.
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Disaccharides↗
Biomedical subjects
A D Panek
Publications and source records attributed to A D Panek.
Fructose 2,6-bisphosphate and trehalose metabolism in Saccharomyces cerevisiae.
1. A regulatory mutant of Saccharomyces (fdp) unable to activate fructose 1,6-bisphosphatase presented a normal response to the glucose and fructose signals as measured by trehalase activation, indicating that the inability of the strain to grow on these sugars is caused by a defect located beyond membrane interactions. 2. In vivo experiments with a mutant strain bearing a phosphoglucoisomerase gene (pgil-delta) deletion showed that activation of trehalase and deactivation of the tehalose-6-phosphate synthase complex occurred to the same extent whether glucose or fructose was used as signal. 3. These results suggest that fructose-2,6-bisphosphate is not involved in the interconversion of forms of the enzymes of trehalose metabolism. Furthermore, when fructose-2,6-bisphosphate was assayed on trehalose synthesizing activity using cell-free extracts and partially purified preparations of the complex, no effect was observed. 4. We conclude that regulation by cAMP fulfills the requirements for control of trehalose levels in Saccharomyces.
Cyclic AMP↗
Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces. I. Interconversion of forms by phosphorylation.
Trehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.
Genes↗
Catabolite inactivation of trehalose synthesis during growth of yeast on maltose.
1. The effects of catabolite inactivation upon the trehalose pathway linked to maltose utilization were investigated in Saccharomyces cerevisiae. Mutant strains devoid of UDPG-trehalose synthase activity were used in this study. 2. Trehalose accumulation was also susceptible to catabolite inactivation as has been reported for the carrier protein, one of the components of the maltose system. Reversibility was only achieved when incubation with glucose did not exceed 5 min and was dependent upon protein synthesis.
Culture Media↗
Partial purification and characterization of the interconvertible forms of trehalase from Saccharomyces cerevisiae.
Cryptic trehalase from Saccharomyces cerevisiae was purified about 3000-fold. The recovery of 970% of the original "activity" indicated the removal of an inhibitor of the enzyme. Active trehalase, obtained through phosphorylation of cryptic trehalase by cAMP-dependent protein kinase, was isolated by chromatography on DEAE-cellulose. A major phosphorylated protein, with an apparent Mr of 86,000, was detected after SDS-polyacrylamide gel electrophoresis. This protein band correlated exactly with the elution profile of trehalase activity and 32Pi incorporation into the enzyme on DEAE-cellulose chromatography. Partially purified active trehalase showed absolute specificity towards trehalose with an apparent Km of 4.79 X 10(-3) M. Both forms of the enzyme showed an apparent molecular weight of 160,000, by gel filtration. Centrifugation on a glycerol density gradient indicated multiple forms of trehalase-c, with Mr of 320,000, 160,000, and 80,000. After activation of each of these forms by protein kinase, a single form of trehalase-a was observed, with a Mr of 160,000. Trehalase-c appears to be a totally inactive form of the enzyme. The only mechanism of activation seems to be phosphorylation by cAMP-dependent protein kinase. When the protein kinase concentration was varied, at a fixed trehalase-c concentration, a sigmoidal activation plot was obtained. This result suggests the occurrence of multiple forms of cryptic trehalase.
Cyclic AMP↗
Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain.
A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5-20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic alpha-glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and alpha-glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.
Cloning, Molecular↗
Further evidence for the alternative pathway of trehalose synthesis linked to maltose utilization in Saccharomyces.
Yeast strains bearing a deficiency in trehalose-6-phosphate synthase activity are unable to accumulate trehalose on any carbon source unless they contain one of the MAL genes. If the gene is inducible then synthesis of trehalose occurs specifically during growth on maltose: when the MAL gene is constitutive then trehalose accumulation can also be seen when cells are grown on glucose. Different systems for trehalose synthesis were suggested: one of them would require the UDPG-linked trehalose synthase whereas the second would utilize an alternative pathway. We proposed a mechanism by which the gene-product of a MAL gene would serve as a common positive regulator for the expression of the genes coding for maltose permease, alpha-glucosidase and some component of the trehalose accumulation system. In order to elucidate this novel pathway a strain lacking UDPG-linked trehalose synthase activity and harboring a defect in maltose uptake was constructed. Excessive maltose uptake resulted in accumulation of intracellular maltose, and twice as much trehalose as in a control strain. Partial inhibition of hexokinase by xylose affected the ratio between internal maltose and trehalose and significantly reduced glycogen synthesis. Sodium fluoride also blocked glycogen synthesis but allowed for trehalose accumulation. Moreover, a mutant which lacks hexokinase I and II was unable to accumulate trehalose when grown on glucose in spite of the presence of a constitutive MAL2 gene. These results suggest that trehalose synthesis would require G-6-P formation derived from maltose. Such a deviation would allow for slowing down the glycolytic flux which, in turn, would favour efficient maltose utilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Disaccharides↗
Investigation of the relationship between sst1 and fdp mutations in yeast and their effect on trehalose synthesis.
A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in Saccharomyces has been associated with a single nuclear gene SST1 (M.S. Operti et al., Current Genetics, 5: 69-76, 1982). The sst1 phenotype was first observed in a pleiotropic fdp mutant which does not grow on mannose, sucrose, glucose or fructose, and in its partial spontaneous revertant (Q6R2) which acquired the capacity to grow on glucose but retained the mutant phenotype with respect to fructose. The sst1 mutants described in this paper are genetically derived from this partial revertant, a fact which suggested a link between sst1 and fdp mutations. All sst1 strains tested showed reduced growth on fructose in 24 h, which also characterizes the fdp mutation. Linkage studies revealed that the SST1 gene maps very close to the FDP gene. Furthermore, another mutant (cif) described as similar to fdp also lacked trehalose-6-phosphate synthase activity. Complementation tests between cif and fdp mutants indicated that the genes are allelic. These results suggest that SST1 and FDP are allelic and that the sst1 mutation might not be associated with the structural gene for trehalose-6-phosphate synthase but corresponds to one of the effects of the pleiotropic fdp mutation.
Alleles↗
Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system.
Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation. Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity. This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation. Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP. Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from [gamma-32P]ATP into protamine. In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells. Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2. Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP. Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction. Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells. This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form. The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase.
Adenosine Triphosphate↗
In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.
To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase.
Culture Media↗
Regulation of porphyrin biosynthesis in yeast. Level of delta-aminolevulinic acid in porphyrin mutants of Saccharomyces cerevisiae.
Saccharomyces cerevisiae mutants bearing mutations at the cyc4 locus are partially deficient in cytochrome synthesis. Although the mutation is not in the structural gene for delta-aminolevulinic acid (Alv) synthase, the mutants are deficient in Alv synthesis in vivo as indicated by abnormally low intracellular Alv concentrations. The cyc4 mutation causes cells to grow very slowly in minimal glucose medium, but not in yeast extract-peptone-glucose medium. A simple nutritional defect caused by the cyc4 mutation is not involved because cytochrome deficiency is enhanced by growing cyc4 cells in yeast extract-peptone medium. A regulatory role for CYC4 is indicated. Evidence for negative feed-back control of Alv synthase by heme is provided by the observation of enhanced intracellular Alv accumulation in yeast mutants partially deficient in decarboxylation of uroporphyrinogen and coproporphyrinogen, respectively.
5-Aminolevulinate Synthetase↗
Regulation of energy metabolism in yeast. Inheritance of a pleiotropic mutation causing defects in metabolism of energy reserves, ethanol utilization and formation of cytochrome a.a3.
The recessive, nuclear gene mutation glc1, which causes glycogen deficiency in Saccharomyces cerevisiae, is highly pleiotropic. Studies of the inheritance of glc1 revealed two classes of phenotypic characteristics: I. Traits invariably associated with the mutant gene and II. Traits whose expressions require the presence of glc1 and one or more additional genes. Class I traits include glycogen deficiency and the loss of capacity to accumulate trehalose in nonproliferating conditions. Traits in the second class include a decreased rate of growth on ethanol medium, a deficiency in cytochrome a.a3 and an enhanced accumulation of pigment, probably a metalloporphyrin. Constructed strains containing both glc1 and the constitutive maltose fermentation gene MAL4c can accumulate trehalose but not glycogen during growth on glucose. However, accumulated trehalose is degraded when cells are exposed to nonproliferating conditions. It is proposed that the glc1 mutation affects a regulatory system, probably involving a protein kinase and/or protein phosphatase, which regulates glycogen synthase and trehalase. Independent regulation of trehalose synthesis by a system controlled by MAL4c is indicated.
Cytochromes↗
Permeabilization of yeast for in situ determination of alpha-glucosidase.
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Cell Membrane Permeability↗
Biochemical genetics of trehalose metabolism in Saccharomyces cerevisiae.
Different strains of Saccharomyces cerevisiae exhibit alternative patterns of trehalose accumulation during growth on a glucose medium. The active pattern is designated as TAC(+) phenotype and the alternative, low-activity pattern as tac(-) phenotype. The tac(-) phenotype is expressed only during growth, since tac(-) strains actively accumulate trehalose during incubation in a glucose medium lacking a nitrogen source. The tac(-) phenotype appears to be determined by a single, recessive gene tac1. The quantitative expression of the dominant, alternative allele TAC1 is subject to wide variation. A highly active pattern of trehalose accumulation requires TAC1 and an amplification factor, TAM, which consists of one or more dominant gene(s). TAM does not appear to alter significantly the expression of tac1. Highly amplified TAC(+) strains may contain a labile factor not present in a TAC(+) strain which accumulates intermediate levels of trehalose.
Cells, Cultured↗
Relationship between succinate excretion and cytochrome levels in Saccharomyces cerevisiae.
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Cytochromes↗
Isoenzymes of transaldolase in strains of Candida utilis and effect of different growth conditions.
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Candida↗
Modulation of cytochrome biosynthesis in yeast by antimetabolite action of levulinic acid.
Levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was used to inhibit cytochrome biosynthesis in growing yeast cells. In Saccharomyces cerevisiae the antimetabolite acts by inhibiting delta-aminolevulinic acid dehydratase in vivo, causing an accumulation of intracellular delta-aminolevulinic acid and simultaneous decreases in all classes of mitochondrial cytochromes. Changes in cellular cytochrome content with increasing levulinic acid concentration suggested the existence of different regulatory patterns in S. cerevisiae and Candida utilis. In C. utilis, cytochrome a.a3 formation is very resistant to the antimetabolite action of levulinic acid. In this aerobic yeast, cytochrome c+c1 is the most sensitive to levulinic acid, and cytochrome b exhibits intermediate sensitivity.
Candida↗
Mouse kidney trehalase: purification and properties.
Trehalase (E.C.3.2.1.28) was isolated from mouse kidney and purified to homogeneity. The enzyme was solubilized with n-octanol and activated by freezing and thawing before precipitation with ammonium sulfate. A 1700-fold purification was achieved by chromatography on DEAE-cellulose, SP-Sephadex, followed by gel filtration on Sephadex G-200. Only a single form of enzyme activity was shown throughout the fractionation as confirmed by gel electrophoresis of the final preparation. The enzyme was specific for trehalose and its estimated molecular weight by filtration on Sephadex G-200 was 73,000. The Km for trehalose was shown to be 2.67 x 10(-3)M and the optimum pH was in the range of 5.5-5.6. We have also shown that the optimum temperature of the enzyme is 60 degrees C, but in the absence of substrate, thermal inactivation occurred at 55 degrees C.
Animals↗