Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in the yeast, Saccharomyces cerevisiae has been associated with a single nuclear gene, sst1. Strains bearing this abnormal allele lacked the capacity to accumulate trehalose during growth on glucose or galactose medium or when incubated with glucose in nonproliferating conditions. However, sst1 strains still exhibited trehalose accumulation during growth on maltose medium, provided they contained a gene for maltose fermentation (MAL gene). Introduction of a constitutive MAL ^c gene into an sst1 strain rendered the strain capable of accumulating trehalose during growth on glucose medium, but did not restore the normal capacity to convert glucose to trehalose in nonproliferating conditions. Different systems, I and II, of trehalose accumulation are proposed. System I would require the UPDG-linked synthase, whereas system II, which is normally specific for maltose, would utilize a different enzyme. It is unlikely that system II produces trehalose by trans-glucosylation, since it converted glucose to trehalose in MAL ^c sst1 strains. The results indicate that maltose specifically induces the production of the MAL gene-product, which, in turn, would stimulate the formation (or activation) of system II.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MAL ^c. Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MAL ^c strains whether or not -glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MAL ^c strains in which -glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, -methylglucosidase and a component of the trehalose accumulation system.Paper I appeared in Cell. and Molec. Biology 25: 345–354, 1979     

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain

Summary 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 -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 -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.     

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G₁ arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the K_m values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.     

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1, hxk2 grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1, hxk2 double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucoseinduced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the delection of HXK2. However, both the ggs1 and the ggs1, hk2 mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex. We discuss a possible requirement of trehalose synthesis for a metabolic balance of sugar phosphates and free inorganic phosphate during the transition from derepressed to fermentative metabolism.     

Identification of an ADPG-dependent trehalose synthase in Saccharomyces

Summary Uridine diphosphoglucose is not the sole donor for trehalose synthesis in yest cells: an ADPG-dependent trehalose synthase, has been identified in mutant strains with undetectable UDPG-dependent trehalose-6-P synthase activity. Genetic and chromatographic studies indicate that the two activities correspond to different proteins. The apparent K K_m for the nucleotide is similar for both enzymes, and Mg²⁺ is also required for both activities; however, a striking difference was observed with respect to ATP.Mg activation. This newly determined enzymatic activity in Saccharomyces clarifies previous contradictory results with mutant strains that are able to accumulate trehalose during growth yet whose UDPG-dependent trehalose synthase activity is undetectable in vitro.Abbreviations PMSF Phenyl-methyl-sulfonyl fluoride - EDTA ethileno-daminotetracetic acid - G6P glucose-6-phosphate - UDPG uridine5-diphosphoglucose - ADPG adenosine-5-diphosphoglucose - UDP uridine-5-diphosphate - ADP adenosine-5-diphosphate - PEP phosphoenol pyruvate - ATP adenosine-5-triphosphate - UTP uridine-5triphosphate - Pi inorganic phosphate - MOPS 3 (N-morpholino) propanesulfonic acid - PNPG paranitrophenylglucoside     

Insertion of non-homologous DNA sequences into a regulatory gene cause a constitutive maltase synthesis in yeast

Summary Two maltase constitutive alleles MAL1-1 ^c and MAL1-2 ^c were obtained as revertants from a defective mall-1 mutant allele not promoting maltose fermentation. Classical genetical analysis showed that the mutations were linked or allelic to the MAL1 locus. Dominance relations were established by testing -glucosidase activities in diploids containing various allele combinations.The maltose regulatory genes belonging to the MAL1, MAL1-1 ^c and MAL1-2 ^c alleles were cloned. Differences in restriction sites were found between the wild type MAL1 and the derived MAL1-constitutive alleles. The MAL1 regulatory gene was located in a 1.15 kb EcoRI fragment (Rodicio and Zimmermann 1985a, b). An EcoRI fragment of this size was found in plasmids containing the MAL1 regulatory wild type allele but was absent from plasmids carrying the constitutive alleles.The genomic organization of the MAL loci in the constitutive mutants was confirmed by Southern analysis. Various fragments containing sequences of the different MAL1 alleles were used to probe genomic digests of MAL1, MAL1-1 ^c and MAL1-2 ^c strains. The results obtained support the conclusion that the constitutive mutations had arisen by a rearrangement between the original mal1-1 mutant allele and sequences with different location in the genome.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

A regulatory mutation in yeast which affects catalase T formation and metabolism of carbohydrate reserves

Summary Mutations at the GLC1 locus in Saccharomyces cerevisiae result in a major deficiency in synthesis of catalase T, but do not affect catalase A. Three independent glc1 mutations were shown to have the same pleiotropic phenotype: catalase T deficiency, defective glycogen synthesis and defective trehalose accumulation. These three deficiencies appear to be determined by a single, nuclear gene. The possibility that glc1 mutations alter a protein kinase is considered.     

The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNAGLN (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation

Byp1-3 is an amber nonsense allele of the Sacchromyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA^GLN (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA^GLN (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA^GLN (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA^GLN (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.Communicated by K. Wolf     

Reserve carbohydrates maintain the viability of Saccharomyces cerevisiae cells during chronological aging

Glycogen and trehalose are well known to participate in many important cell functions, e.g., protection from stress factors, regulation of cell growth and division, spore formation. Since the aging is a complex process involving many aspects of cell metabolism, it was interesting to study the role of glycogen and trehalose in maintenance of viability of aging cells. We have revealed that cell aging is accompanied by an abrupt fall of glycogen and trehalose contents between the second and third weeks of aging. Simultaneously, we observed a decrease in the activity of glycolytic enzymes, phosphofructokinase and hexokinase. At the same time, the viability of aging cells abruptly declined. Although we found neither glycogen nor trehalose in the cells after the third week of aging, they remained viable for some time, apparently due to development of some compensatory metabolic pathways. In spite of this fact, complete death of the cells occurred by the eighth week of experiment, which confirmed irreplaceability of reserve carbohydrates in yeast cell metabolism. Possible reasons of the inability of aging cells to accumulate glycogen and trehalose are discussed in the work.     

Gene structure and expression of a novel Euglena gracilis chloroplast operon encoding cytochrome b6 and the β and ε subunits of the H+-ATP synthase complex

The genes encoding cytochrome b6 of the chloroplast cytochrome b6/f complex (petB) and the ATP synthase CF₁- subunit (atpB) and -subunit (atpE) were identified on the EcoD fragment of the Euglena gracilis chloroplast genome. The complete nucleotide sequence of these three genes was determined. The petB-atpB-atpE genes are cotranscribed as a tricistronic operon. This gene organization differs from that of land plants in which atpB-atpE form a discistronic operon, and petB is within the psbB-ycf8-psbH-petB-petD operon. Euglena cytochrome b6 and the -subunit of the chloroplast ATP synthase are very similar in derived amino acid sequence to the corresponding gene products from other organisms. The -subunit of the chloroplast ATP synthase complex is more divergent. In Euglena, the petB-atpB-atpE genes contain introns, including two twintrons, at eight different positions. All of the intron positions were confirmed by analysis of cDNAs. Two independent intercistronic RNA processing events and 11 splicing reactions lead to the accumulation of the mature petB, atpB and atpE monocistronic mRNAs.     

Molecular and biochemical characterization of the hexokinase from the starch-utilizing yeast Schwanniomyces occidentalis

Hexose-phosphorylating enzymes from the starch-utilizing yeast Schwanniomyces occidentalis were purified and two isoenzymes separated. The substrate pattern characterized one of these as a hexokinase phosphorylating glucose and fructose and the other as a glucokinase unable to phosphorylate fructose. The purified Schw. occidentalis hexokinase had a K_M value of 0.98 mM for glucose and 9.3 mM for fructose. The hexokinase gene was cloned by cross hybridization with a probe from the Saccharomyces cerevisiae HXK2 gene. Deletion of Schw. occidentalis hexokinase by gene replacement yielded a mutant unable to grow on fructose as sole carbon source, but still growing on glucose. Deletion mutants of Schw. occidentalis hexokinase prevented glucose repression of invertase and maltase. Growth deficiences and the defect of glucose repression of a S. cerevisiae hexokinase null mutant could be restored by heterologous expression of the Schw. occidentalis hexokinase. Moreover, the results clearly showed the existence of a separate glucokinase in Schw. occidentalis.     

Cloning and biochemical characterization of hexokinase from the methylotrophic yeast Hansenula polymorpha

We previously showed that, unlike other yeasts, Hansenula polymorpha possesses a glucokinase HPGLK1 that can mediate glucose repression in this yeast, although it cannot replace the regulatory function of hexokinase 2 in Saccharomyces cerevisiae. In the present study, the H. polymorpha hexokinase gene HPHXK1 was cloned by complementation of the glucose growth deficiency of the H. polymorpha double kinase-negative mutant A31-10 with a genomic library. The sequence of the 483-amino acid hexokinase protein deduced from the HPHXK1 gene showed the highest degree of identity (56%) with hexokinase from Schwanniomyces occidentalis, whereas the identity with hexokinase from Kluyveromyces lactis and both hexokinases from Sac. cerevisiae was 55%. The hexokinase protein was purified from crude extracts of H. polymorpha, using ion exchange chromatography and gel filtration. The K _m values of the purified enzyme for glucose, fructose and ATP were 0.26 mM, 1.1 mM and 0.32 mM, respectively. H. polymorpha hexokinase was inhibited by trehalose-6-phosphate (K _i=12 M) and ADP (K _i=1.6 mM), but not by glucose-6-phosphate. Transformation of a H. polymorpha hexokinase-negative mutant with a plasmid carrying the HPHXK1 gene restored the ability of the mutant to phosphorylate fructose and to repress the synthesis of alcohol oxidase and catalase by fructose. Therefore, hexokinase is specifically needed for the establishment of fructose repression in H. polymorpha.Communicated by S. Hohmann     

Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae

The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationaryphase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels). In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity. Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpk ^w1 BCY1 strains) or cAMP-independent (tpk1 ^w1 bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds. These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway in vivo. Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki=15 mM) and resulted unaffected by ATP in assays performed in vitro.     

New insights into a mutant of Saccharomyces cerevisiae having impaired sugar uptake and metabolism

Summary A regulatory mutant of Saccharomyces carlsbergensis unable to inactivate fructose-1,6-bisphosphatase was shown to have a normal response to the glucose signal as measured by trehalase and 6-phosphofructose-2-kinase activities. The level of fructose 2,6-bisphosphate, however, was found to be 4- to 5-fold lower than that found in the wild-type strain. A rapid and drastic depletion in ATP was confirmed. A partial revertant for growth on glucose which retained its inability to grow on fructose did not show normal levels of fructose 2,6-bisphosphate; however, ATP levels were restored. Trehalose-6-phosphate synthase activity was found in its phosphorylated, less active form. A high degree of phosphorylation at the level of enzymatic activity and of the sugar phosphorylating systems might be responsible for the impairment of control between hexose transport and metabolism, as well as for the absence of trehalose accumulation.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - PFK1 6-phosphofructose-l-kinase - FBPasel fructose-1,6-bisphosphatase - PFK2 6-phosphofructose-2-kinase - PEP phosphoenolpyruvate     

Cloning of maltase regulatory genes in Saccharomyces cerevisiae

Summary By hybridization with a putative MAL2p regulatory sequence we have identified a 19 kb long BamH1 DNA fragment to contain the MALp sequence in a MAL4 strain. A mixture of recombinant plasmids was prepared by ligation of purified 19 kb BamH1 fragments partially digested with Sau3A into the multicopy vector YEp1357. The source of DNA was a strain carrying the MAL4 locus. Yeast maltose non-fermenting strains were transformed with the plasmid mixture. A recombinant plasmid, pRM-4, containing the MAL4p regulatory gene was isolated that complements the maltose-negative phenotype. The plasmid was shown to confer the ability to synthesize maltase to recipient strains grown under inducing as well as under repressing conditions.The MAL4p regulatory sequence cloned was used as a probe in hybridization experiments to study the degrees of homology between the different MAL regulatory genes. The results showed that the sequence from MAL4 strains is strongly homologous to that of MAL3 strains whereas it shows significant differences to the ones of MAL1 and MAL2 strains.Southern analysis of the segregants of crosses between maltose-positive strains and ma10 strains allowed us to localize the maltase regulatory sequence of each MAL locus within a characteristic BamH1 fragment of genomic DNA hybridizing to the isolated sequence.     

Leucine biosynthesis in yeast

Summary Tetrad analysis indicates that -isopropylmalate synthase activity of yeast is determined by two separate genes, designated LEU4 and LEU5. LEU4 is identified as a structural gene. LEU5 either encodes another -isopropylmalate synthase activity by itself or provides some function needed for the expression of a second structural gene. The properties of mutants affecting the biosynthesis of leucine and its regulation suggest that the expression of LEU1 and LEU2 (structural genes encoding isopropylmalate isomerase and -isopropylmalate dehydrogenase, respectively) is controlled by a complex of a-isopropylmalate and a regulatory element (the LEU3 gene product). Similarities and differences between yeast and Neurospora crassa with respect to leucine biosynthesis are discussed.This is Journal Paper No. 9347 of the Agricultural Experiment Station, Purdue University