FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2056952404> ?p ?o ?g. }

• W2056952404 endingPage "22501" @default.
• W2056952404 startingPage "22495" @default.
• W2056952404 abstract "The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the β-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the α-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at ∼2 mm. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase. The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the β-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the α-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at ∼2 mm. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase. Glycogen is a storage form of glucose in a wide variety of organisms and cell types. In the yeast Saccharomyces cerevisiae, glycogen accumulation is normally initiated in response to nutrient limitation, prior to entry into stationary phase (1François J. Blazquez M.A. Ariño J. Gancedo C. Yeast Sugar Metabolism.in: Zimmermann F.K. Technomics Publishing Co. Inc., Lancaster, PA1997Google Scholar). Glycogen biosynthesis requires a self-glucosylating initiator protein, glycogenin, glycogen synthase for chain elongation, and branching enzyme to introduce the α-1,6-glycosidic branch points (2Skurat A. Roach P.J. LeRoith D. Olefsky J.M. Taylor S.I. Diabetes Mellitus: A Fundamental and Clinical Text. Lippincott-Raven Publishers, Philadelphia, PA1996: 213-222Google Scholar). Over the past few years, the yeast genes encoding enzymes responsible for glycogen biosynthesis have been identified, including two glycogen synthase genes, GSY1 and GSY2 (3Farkas I. Hardy T.A. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1990; 265: 20879-20886Abstract Full Text PDF PubMed Google Scholar, 4Farkas I. Hardy T.A. Goebl M.G. Roach P.J. J. Biol. Chem. 1991; 266: 15602-15607Abstract Full Text PDF PubMed Google Scholar).GSY2 encodes the major nutritionally regulated form of glycogen synthase (4Farkas I. Hardy T.A. Goebl M.G. Roach P.J. J. Biol. Chem. 1991; 266: 15602-15607Abstract Full Text PDF PubMed Google Scholar). One control of GSY2 is by its increased expression at the approach of stationary phase (4Farkas I. Hardy T.A. Goebl M.G. Roach P.J. J. Biol. Chem. 1991; 266: 15602-15607Abstract Full Text PDF PubMed Google Scholar). In addition, this protein is regulated by covalent phosphorylation, which decreases its activity when measured in the absence of glucose-6-P (5Rothman-Denes L.B. Cabib E. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 967-974Crossref PubMed Scopus (83) Google Scholar, 6Huang K-P. Cabib E. J. Biol. Chem. 1974; 249: 3851-3857Abstract Full Text PDF PubMed Google Scholar, 7Hardy T.A. Roach P.J. J. Biol. Chem. 1993; 268: 23799-23805Abstract Full Text PDF PubMed Google Scholar). However, activity is restored even to fully phosphorylated enzyme by the presence of glucose-6-P, so the −/+ glucose-6-P activity ratio is frequently used as an index of the activation state of glycogen synthase. Glycogen synthase can be rate-determining for glycogen synthesis in yeast, and mutation of potential phosphorylation sites in Gsy2p results in overaccumulation of glycogen (7Hardy T.A. Roach P.J. J. Biol. Chem. 1993; 268: 23799-23805Abstract Full Text PDF PubMed Google Scholar). Mutations in GAC1 andGLC7, which encode type 1 phosphatase subunits, cause a decreased −/+ glucose-6-P activity ratio and concomitantly impaired glycogen deposition (8Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar, 9François J. Thompson-Jaeger S Skroch J. Zellenka U. Spevak W. Tatchell K. EMBO J. 1992; 11: 87-96Crossref PubMed Scopus (126) Google Scholar). Conversely, deletion of PHO85, which encodes a glycogen synthase kinase catalytic subunit, leads to glycogen hyperaccumulation (10Timblin B.K. Tatchell K. Bergman L.W. Genetics. 1996; 143: 57-66Crossref PubMed Google Scholar, 11Huang D. Farkas I. Roach P.J. Mol. Cell. Biol. 1996; 16: 4357-4365Crossref PubMed Scopus (73) Google Scholar). Another gene that influences glycogen storage is SNF1 (8Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar, 12Thompson-Jaeger S. François J. Gaughran J.P. Tatchell K. Genetics. 1991; 129: 697-706Crossref PubMed Google Scholar), which encodes a protein kinase essential for expression of glucose-repressible genes (13Celenza J.L. Carlson M. Mol. Cell. Biol. 1984; 4: 49-53Crossref PubMed Scopus (99) Google Scholar). Thus, snf1 mutants grow poorly on alternative carbon sources (14Carlson M. Osmond B.C. Botstein D. Genetics. 1981; 98: 25-43Crossref PubMed Google Scholar) and also fail to accumulate glycogen (8Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar, 12Thompson-Jaeger S. François J. Gaughran J.P. Tatchell K. Genetics. 1991; 129: 697-706Crossref PubMed Google Scholar). We had previously suggested that the inability of snf1 mutants to synthesize glycogen was caused by hyperphosphorylation, and hence inactivation, of glycogen synthase (15Hardy T.A. Huang D. Roach P.J. J. Biol. Chem. 1994; 269: 27907-27913Abstract Full Text PDF PubMed Google Scholar). In the present study, we searched for second site suppressors of the glycogen accumulation defect of snf1 cells. One of the resulting suppressor genes was identified as PFK2, which encodes one subunit of the glycolytic enzyme 6-phosphofructo-1-kinase (16Heinisch J. Ritzel R.G. Borstel R.C.V. Aguilera A. Rodicio R. Zimmermann F.K. Gene ( Amst. ). 1989; 78: 309-321Crossref PubMed Scopus (79) Google Scholar). We propose that the glycogen storage phenotype caused by this mutation results from high intracellular glucose-6-P levels and that glucose-6-P acts not only as a direct activator of glycogen synthase but also as an inhibitor of glycogen synthase kinase activity. The S. cerevisiae strains used are listed in Table I. Rich medium (YP) contains 1% yeast extract, 2% Bacto-peptone, and 2% concentration of the indicated carbon source. Synthetic medium consists of yeast nitrogen base (6.7 g/liter), lacking the appropriate amino acid, and 2% concentration of the indicated carbon source. Cells carrying apgi1 disruption were grown in rich medium with 2% fructose and 0.1% glucose (YPFD). Plasmids were maintained in Escherichia coli strain DH5α. Standard methods for yeast genetic analysis (18Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 3-863Crossref PubMed Scopus (2543) Google Scholar) and transformation (19Ito H. Fukada Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) were used.Table IYeast strainsStrainGenotypeSourceEG328-1AMATα trp1 leu2 ura3–52K. TatchellEG327-1DMATα trp1 leu2 ura3–52 glc7-1K. TatchellEG353-1CMATαtrp1 leu2 ura3–52 snf1::URA3K. TatchellF243MATa thr4R. C. WekDH2MATα trp1 leu2 ura3–52 gsy2::URA3Ref. 17Huang D. Chun K.T. Goebl M.G. Roach P.J. Genetics. 1996; 143: 119-127Crossref PubMed Google ScholarDH5MATα trp1 leu2 ura3–52 snf1::LEU2Ref. 17Huang D. Chun K.T. Goebl M.G. Roach P.J. Genetics. 1996; 143: 119-127Crossref PubMed Google ScholarDH22MATatrp1 ura3–52 thr4 snf1::URA3This studyDH23MATα trp1 leu2 ura3–52 snf1::URA3 ssg1-1This studyDH35-64MATa ura3–52 thr4 pho85::URA3This studyDH51MATa trp1 ura3–52 thr4 glc7-1This studyDH54-0MATα trp1 leu2 ura3–52 glc7-1 pfk2::URA3This studyDH54-102MATα trp1 leu2 ura3–52 glc7-1 ssg1-1This studyDH55-0MATα trp1 leu2 ura3–52 pfk2::URA3This studyDH56-0MATα trp1 ura3–52 pfk1::URA3This studyDH57-0MATαtrp1 leu2 ura3–52 glc7-1 pfk1::URA3This studyDH58-0MATα trp1 leu2 ura3–52 snf1::LEU2 pfk2::URA3This studyDH59-13MATa trp1 ura3–52 thr4 ssg1-1This studyDH60MATα trp1 leu2 ura3–52 ssg1-1-PFK2-URA3This studyDH61-0MATαtrp1 leu2 ura3–52 snf1::LEU2 pfk1::URA3This studyDH62-62MATαtrp1 leu2 ura3–52 glc7-1 pgi1::URA3This studyDH63-0MATα trp1 leu2 ura3–52 pgi1::URA3This studyDH64-42MATαtrp1 leu2 ura3–52 gsy2::URA3 pfk2::URA3This studyDH65MATathr4This studyDH66MATa ura3 thr4This studyEG328–1A is the wild type of this study. EG327-1D and EG353-1C are isogenic to EG328-1A. They were all kindly provided by Dr. Kelly Tatchell. DH65 and DH66 are the progeny of five and four backcrosses, respectively, to the wild type strain EG328-1A from F243. The DH strains are all related to EG328-1A. Open table in a new tab EG328–1A is the wild type of this study. EG327-1D and EG353-1C are isogenic to EG328-1A. They were all kindly provided by Dr. Kelly Tatchell. DH65 and DH66 are the progeny of five and four backcrosses, respectively, to the wild type strain EG328-1A from F243. The DH strains are all related to EG328-1A. For disruption of PFK2, the polymerase chain reaction was used to generate a DNA fragment that contained PFK2 sequences straddling a URA3 marker gene (20Lorenz M.C. Muir R.S. Lim E. McElver J. Weber S.C. Heitman J. Gene ( Amst. ). 1995; 158: 113-117Crossref PubMed Scopus (257) Google Scholar). The vector pRS306 (21Sikorski R.S. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) was used as the template for the polymerase chain reaction. The resulting polymerase chain reaction product contained the 5′ sequence (+242 to +287, referred to the open reading frame) and 3′ sequence (+2806 to +2851) of PFK2 at each end of a 1.1-kilobase pair sequence containing the URA3gene. This DNA fragment was then used to transform strains EG328-1A, EG327-1D, and EG353-1C to replace the PFK2 gene and generate strains DH55-0, DH54-0, and DH58-0. Gene disruption in Ura+transformants was confirmed by polymerase chain reaction. A similar strategy was employed to disrupt PFK1 and PGI1(Table I). For pgi1disruption, Ura+ transformants were selected in synthetic complete media without uracil and containing fructose (2%) and a low concentration of glucose (0.1%) as carbon source. Since diploids homozygous forsnf1 do not sporulate, we crossed an ssg1 snf1:: URA3 mutant to an isogenic wild-type strain DH66 (Ura−). Tetrad analysis revealed segregation ratios of 2+:2−, 3+:1− and 4+:0− for glycogen accumulation, with most tetrads 3+:1−, indicating that mutation in a single gene was responsible for the phenotype. The sporulation also yielded tetratypes consisting of one Ura− colony with normal glycogen (SSG1 SNF1), one Ura+ colony with normal glycogen (ssg1 snf1), one Ura+colony defective for glycogen (SSG1 snf1), and one Ura− colony with hyperaccumulation of glycogen (ssg1 SNF1). For spores segregating 2+:2−, the two glycogen-positive clones always hyperaccumulated and were Ura−, while the two glycogen negative clones were Ura+. These results suggest that, besides restoring glycogen in snf1 cells, ssg1 caused hyperaccumulation of glycogen in a wild-type background. We also tested whether ssg1 would overcome the glycogen deficit inglc7-1 cells, which are defective in the type 1 protein phosphatase catalytic subunit (8Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar). When an ssg1 strain (DH59-13) was crossed with EG327-1D (glc7-1), the resulting tetrads exhibited a 3+:1− segregation ratio for positive glycogen storage, suggesting that the ssg1mutation suppresses the glc7-1 glycogen-deficient phenotype. Multiple independent ssg1 glc7-1 double mutant strains were isolated and found to accumulate an intermediate level of glycogen. Thus, the ssg1 mutation partially suppressed the glycogen phenotype of glc7-1. A genomic library cloned in YCp50 (22Rose M.D. Novick P. Thomas J.H. Botstein D. Fink G.R. Gene ( Amst. ). 1987; 60: 237-243Crossref PubMed Scopus (830) Google Scholar), kindly provided by John Cannon (University of Missouri, Columbia), was used to transform a glycogen-accumulating ssg1 glc7-1 ura3strain (DH54-102). From approximately 5000 transformants, we obtained 14 colonies in which glycogen accumulation was eliminated as judged by iodine staining of colonies. We isolated a plasmid, YCSSG1-1, containing a 17.5-kilobase pair insert, that complemented thessg1 phenotype. Complementation was localized to a 2.9-kilobase pair EcoRI-EcoRI fragment, which, from sequence analysis, matched the PFK2 gene. The fragment began at +817 in the coding region of PFK2, exactly the sameEcoRI fragment as had previously been reported to complementpfk2 defects (23Heinisch J. Mol. Gen. Genet. 1986; 202: 75-82Crossref PubMed Scopus (88) Google Scholar). To determine that the cloned sequence contained the SSG1 locus, the 2.9-kilobase pair fragment was integrated into the genome of strain DH54-102. The transformants had a glycogen-deficient phenotype. Two such transformants were mated with DH66 (ura3) to yield a strain with the putativessg1-URA3-SSG1 integration in a GLC7 background (DH60). This strain was then crossed to DH65 (SSG1 GLC7 URA3) and DH59-13 (ssg1 GLC7 ura3). When crossed to DH65, 10 tetrads examined revealed 4:0 segregation for the Ssg1+ phenotype (normal glycogen) and 4:0, 3:1, or 2:2 for Ura+. In a cross with DH59-13, 10 tetrads segregated 2:2 for both Ssg1+ and Ura+, and Ssg1+was always linked to Ura+. The tight linkage between Ssg1+ and Ura+ suggests that the cloned DNA derived from the ssg1 locus. Yeast cells were collected at late log phase (5–8 × 107 cells/ml), unless indicated otherwise, and resuspended in a homogenization buffer containing 50 mmTris-HCl, 1 mm EDTA, 3 mm dithiothreitol, 50 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm N α-p-tosyl-l-lysine chloromethyl ketone, 5 mm benzamidine, 0.25 μg/ml leupeptin, and 0.5 μg/ml aprotinin, pH 7.4. The cells were broken with glass beads as described previously (7Hardy T.A. Roach P.J. J. Biol. Chem. 1993; 268: 23799-23805Abstract Full Text PDF PubMed Google Scholar). Glycogen synthase was assayed by the method of Thomas et al. (24Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar) as described by Hardy et al. (15Hardy T.A. Huang D. Roach P.J. J. Biol. Chem. 1994; 269: 27907-27913Abstract Full Text PDF PubMed Google Scholar) except that the extracts were first passed over a Sephadex G25 spin column to remove low molecular weight compounds as described below. A unit of activity is defined as the amount of enzyme that catalyzes the transfer of 1 μmol of glucose from UDP-glucose to glycogen per min under conditions of the standard assay. The total activity of glycogen synthase is that measured in the presence of 7.2 mm glucose 6-phosphate. The −/+ glucose-6-P activity ratio is defined as the activity measured in the absence of glucose-6-P divided by that measured in its presence. Each measurement was the average of duplicate assays. Glycogen synthase kinase activity in extracts was measured by two procedures. In one, the inactivation of added, purified recombinant Gsy2p protein was followed with time, monitoring the decrease in the −/+ glucose-6-P activity ratio. In the reaction, 20 μl of purified Gsy2p (0.76 mg/ml), 50 mm Tris-HCl, pH 7.6, 2 mm EDTA, 2 mm EGTA, 10 mmβ-mercaptoethanol, 20% (v/v) glycerol was mixed with 300 μl of yeast lysate (adjusted to ∼4 mg of protein/ml) prepared as described above. The reaction was started by the addition of 80 μl of 25 mm ATP in 750 mm Tris-HCl, 150 mmMgCl2, pH 7.4, together with any other additions. After incubation at 30 °C for different times, a portion (40 μl) was removed, mixed with 10 μl of 0.3 m EDTA, 0.4m NaF to terminate the reaction, and loaded onto a spin column (Sephadex G25, 1.5-ml volume). After centrifugation at 1000 rpm in an IEC Clinical Centrifuge for 3 min, the pass-through was collected, mixed with 50 μl of the same homogenization buffer described above but with 100 mm NaF, and assayed for glycogen synthase activity. In some experiments, cell extracts were pretreated with Sephadex G25 columns to remove endogenous glucose-6-P prior to initiating the protein kinase reaction. In control experiments in which we measured glucose-6-P levels, we found that the spin column removed over 95% of the glucose-6-P. To measure glycogen synthase kinase with this assay, we had to use gsy2 strains so that the endogenous glycogen synthase activity, which is reduced to about one-tenth of wild type, does not interfere significantly with that of the added Gsy2p. The amount of added Gsy2p gives a level of activity about twice that in a normal extract of wild-type cells. In the second assay for glycogen synthase kinase activity, the direct phosphorylation of purified Gsy2p added to cell extracts was determined by analyzing the incorporation of 32P into Gsy2p from [γ-32P]ATP. Yeast cells were grown and collected as described above. The cells were resuspended in a homogenization buffer containing 50 mm Tris-HCl, pH 7.4, 0.1% (v/v) Triton X-100, 2 mm benzamidine hydrochloride, 1 mmphenylmethylsulfonyl fluoride, 0.1 mm N α-p-tosyl-l-lysine chloromethyl ketone, and 1 mm β-mercaptoethanol. Cell extracts were prepared using glass beads and, in some cases, were passed over a Sephadex G25 spin column as described above. Yeast extract (5 μl, diluted to ∼2.5 mg of protein/ml with homogenization buffer) was combined with 2.5 μg of His6Gsy2p and 100 nm okadaic acid in a final volume of 20 μl. In some experiments, glucose 6-phosphate was also added from an aqueous stock solution. The reaction was initiated by the addition of 5 μl of [γ-32P]ATP mix (1 mm ATP, 25 mmMgCl2, ∼1200 cpm/pmol). After incubation at 30 °C for 15 min, 25 μl of a 1:1 slurry of nickel-nitrolotriacetic acid-agarose in wash buffer (50 mm Tris-HCl, pH 7.9, 0.1% (v/v) Triton X-100, 500 mm NaCl, 50 mm NaF, 50 mm imidazole, and protease inhibitors as above) was added, followed by 500 μl of ice-cold wash buffer, and the incubation continued on ice for a further 30 min with occasional gentle agitation. The nickel-nitrolotriacetic acid-agarose was collected by centrifugation, and the pellet was washed four times with 500 μl of wash buffer. Bound His6Gsy2p was eluted using 25 μl of wash buffer with the imidazole concentration increased to 500 mm. The eluted material was analyzed by polyacrylamide gel electrophoresis in the presence of SDS (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) and autoradiography. Determination of glucose-6-P was a variation of the method of Lang and Michal (26Lang G. Michal G. Bergmeyer H.U. Methods in Enzymatic Analysis. Academic Press, Inc., New York1974: 1235-1242Google Scholar) as described by Wilson (27Wilson, W. A. (1995) Characterization of a Homologue of the Mammalian AMP-activated Protein Kinase in Saccharomyces cerevisiae. Ph.D. Thesis, University of Dundee, Scotland.Google Scholar). Cells were harvested by rapid filtration, resuspended in perchloric acid (5%), and broken with glass beads. The acid extracts were collected and centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatant was extracted twice to remove perchloric acid with 10% excess (by volume) of a 1:1 mixture of tri-n-octylamine and 1,1,2-trichlorotrifluoroethane. Glucose-6-P was measured by using 0.5 units of glucose-6-phosphate dehydrogenase in the presence of 2 mm MgCl2 and 0.05 mm NADP. Typically, a relative value for glucose-6-P level was obtained by normalizing to the absorbance at 600 nm. To give an indication of the absolute concentrations, measurements of glucose-6-P in wild-type cells were also expressed in terms of the dry weight of cells. Glycogen was determined in extracts of cells as described by Hardy et al. (7Hardy T.A. Roach P.J. J. Biol. Chem. 1993; 268: 23799-23805Abstract Full Text PDF PubMed Google Scholar). Recombinant His6Gsy2p with an NH2-terminal poly-His tag was produced in E. coli and purified as described previously (11Huang D. Farkas I. Roach P.J. Mol. Cell. Biol. 1996; 16: 4357-4365Crossref PubMed Scopus (73) Google Scholar). Polyacrylamide gel electrophoresis in the presence of SDS was a variant of the method of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). Cells defective in the Snf1p protein kinase fail to accumulate glycogen, a deficit that we attributed to hyperphosphorylation of glycogen synthase (15Hardy T.A. Huang D. Roach P.J. J. Biol. Chem. 1994; 269: 27907-27913Abstract Full Text PDF PubMed Google Scholar). Several second site suppressors of this glycogen phenotype, designatedssg1-5 (suppressor of s nf1 for glycogen phenotype), were identified, and the wild-type allele of one, ssg3, was found to encode Pho85p, a protein kinase catalytic subunit (11Huang D. Farkas I. Roach P.J. Mol. Cell. Biol. 1996; 16: 4357-4365Crossref PubMed Scopus (73) Google Scholar). Another mutant allele, ssg1, also restored the wild-type glycogen synthase activity ratio to snf1 cells, suggesting that assg1 snf1 strain could have reduced glycogen synthase kinase activity. Also, the presence of the ssg1 allele in a wild-type background caused glycogen hyperaccumulation (Fig.1). Cloning ssg1 by complementation was complicated by the fact that transformation of thessg1 snf1 cells was extremely difficult, and we could not obtain sufficient numbers of transformants for effective library screening. Since ssg1 also complemented the glycogen defect in glc7-1 cells, which have an impaired type 1 phosphatase catalytic subunit (8Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar), we screened for loss of glycogen accumulation in an ssg1 glc7-1 ura3 strain (DH54-102) after transformation with a yeast genomic library (22Rose M.D. Novick P. Thomas J.H. Botstein D. Fink G.R. Gene ( Amst. ). 1987; 60: 237-243Crossref PubMed Scopus (830) Google Scholar). We isolated a plasmid that complemented the ssg1 phenotype and identified the responsible gene as PFK2, which encodes the β-subunit of phosphofructo-1-kinase (16Heinisch J. Ritzel R.G. Borstel R.C.V. Aguilera A. Rodicio R. Zimmermann F.K. Gene ( Amst. ). 1989; 78: 309-321Crossref PubMed Scopus (79) Google Scholar). As described under “Experimental Procedures,” we confirmed by genetic methods that the relevant phenotype was indeed linked to the PFK2 locus. Strains harboring deletions of PFK2 were found to hyperaccumulate glycogen in a wild-type background, and glycogen storage was restored to glc7-1 and snf1cells (Fig. 1). Deletion of PFK2 has been reported to increase glucose-6-P levels (28Breitenbach-Schmitt I. Schmitt H.D. Heinisch J. Zimmermann F.K. Mol. Gen. Genet. 1984; 195: 536-540Crossref Scopus (33) Google Scholar), and we therefore measured glucose-6-P in ssg1 and several other relevant strains. In wild-type cells, glucose-6-P decreases from about 2 nmol/mg, dry weight, during logarithmic growth to less than half of that level before the onset of stationary phase. Elevated levels of glucose-6-P were observed inssg1, pfk2, ssg1 glc7-1, andpfk2 glc7-1 cells (Table II). The double mutants ssg1 snf1 and pfk2 snf1contained less glucose-6-P than pfk2 cells but still more than wild type. In a snf1 mutant, the glucose-6-P concentration was significantly lower than that in wild-type cells, especially at late logarithmic phase (Table II).Table IIRelative glucose-6-P levelStrainMutant alleleGlucose-6-P2-aGlucose-6-P was measured in yeast cell extracts as described under “Experimental Procedures.” Cells were harvested during either logarithmic growth (∼107 cells/ml) or late logarithmic phase (∼8 × 107 cells/ml).LogarithmicLate logarithmic%EG328-1AWild type10034EG327-1Dglc7-1ND2-bND, not determined.59EG353-1Csnf17612DH56-0pfk1260194DH55-0pfk2538351DH59-13ssg1-1ND298DH57-0glc7-1 pfk1ND254DH61-0snf1 pfk1ND113DH54-0glc7-1 pfk2ND341DH54-102glc7-1 ssg1-1ND394DH58-0snf1 pfk2ND186DH23snf1 ssg1-1ND258EG328-1AWild type2-cStrains were grown in rich medium containing 2% fructose and 0.1% glucose.9131DH63-0pgi1 2-cStrains were grown in rich medium containing 2% fructose and 0.1% glucose.143161The glucose-6-P level is normalized to that in wild-type cells during logarithmic growth. The absolute value of glucose-6-P in wild-type cells under these conditions was 2 nmol/mg, dry weight, of cells.2-a Glucose-6-P was measured in yeast cell extracts as described under “Experimental Procedures.” Cells were harvested during either logarithmic growth (∼107 cells/ml) or late logarithmic phase (∼8 × 107 cells/ml).2-b ND, not determined.2-c Strains were grown in rich medium containing 2% fructose and 0.1% glucose. Open table in a new tab The glucose-6-P level is normalized to that in wild-type cells during logarithmic growth. The absolute value of glucose-6-P in wild-type cells under these conditions was 2 nmol/mg, dry weight, of cells. Since glucose-6-P is a potent activator of glycogen synthase, an obvious hypothesis is that enhanced glycogen storage by pfk2mutants is caused by this activation. In our initial analysis of glycogen synthase activity in extracts from ssg1 snf1 cells, we observed that the −/+ glucose-6-P activity ratio was significantly elevated as compared with snf1 cells (11Huang D. Farkas I. Roach P.J. Mol. Cell. Biol. 1996; 16: 4357-4365Crossref PubMed Scopus (73) Google Scholar). However, not knowing that glucose-6-P levels might be increased, we had taken no special measures in the earlier study to remove small molecules from the extract. Thus, there could have been carry-over of endogenous glucose-6-P from the cell extract into the glycogen synthase assay. For the assay in the absence of added glucose-6-P, this could cause increased activity and an erroneously high −/+ glucose-6-P activity ratio. Therefore, cell extracts were treated with a Sephadex G25 spin column to remove endogenous glucose-6-P prior to the assay. The −/+ glucose-6-P activity ratio in pfk2 mutants was still over twice that of wild-type cells (Fig. 2). In pfk2 snf1 cells, the activity ratio was restored to wild-type levels, whereas deletion of PFK2 in aglc7-1 background only partially restored the activity (Fig.2). This result indicates that the intrinsic activity, and by inference the phosphorylation state, of glycogen synthase was affected in these mutants. Since pfk2 mutants have elevated glucose-6-P, we explored the possibility that the high level of this metabolite caused the glycogen hyperaccumulation. Mutation of two other genes, PFK1 and PGI1, is also known to affect glucose-6-P levels, and we asked whether such mutations also influenced glycogen accumulation. Mutants defective in PFK1, which encodes the α subunit of phosphofructokinase, contain elevated glucose-6-P but less than in pfk2 strains (Ref. 28Breitenbach-Schmitt I. Schmitt H.D. Heinisch J. Zimmermann F.K. Mol. Gen. Genet. 1984; 195: 536-540Crossref Scopus (33) Google Scholar; see also Table II) and in general have a less severe phenotype thanpfk2 mutants. We found that pfk1 mutants did overaccumulate glycogen but to a lesser degree than pfk2cells. A pfk1 glc7-1 (DH57-0) double mutant was still glycogen-deficient (Fig. 1). However, glycogen accumulation was restored in pfk1 snf1 double mutants (Fig. 1), indicating that the defect in snf1 cells is more sensitive to the glucose-6-P level than the defect in glc7-1 cells. Similarly, the glycogen synthase activity ratio in extracts frompfk1 and snf1 pfk1 mutants was elevated beyond the wild-type values, whereas in a pfk1 glc7-1 strain it was still below wild type. Deletion of PGI1, which encodes phosphoglucose isomerase, prevents growth on glucose; therefore, pgi1 strains were grown in a fructose medium supplemented with trace amounts of glucose (YPFD; Ref. 29Corominas J. Clotet J. Fernandez-Banares I. Boles E. Zimmermann F.K. Guinovart J.J. Arino J. FEBS Lett. 1992; 310: 182-186Crossref PubMed Scopus (14) Google Scholar; see also “Experimental Procedures”). This mutant had an extremely high glucose-6-P level during" @default.
• W2056952404 created "2016-06-24" @default.
• W2056952404 creator A5048878179 @default.
• W2056952404 creator A5076217348 @default.
• W2056952404 creator A5089116639 @default.
• W2056952404 date "1997-09-01" @default.
• W2056952404 modified "2023-10-01" @default.
• W2056952404 title "Glucose-6-P Control of Glycogen Synthase Phosphorylation in Yeast" @default.
• W2056952404 cites W1529654145 @default.
• W2056952404 cites W1530881561 @default.
• W2056952404 cites W1533533640 @default.
• W2056952404 cites W1535912426 @default.
• W2056952404 cites W1537442027 @default.
• W2056952404 cites W1824573715 @default.
• W2056952404 cites W1943809043 @default.
• W2056952404 cites W1958996383 @default.
• W2056952404 cites W1973782344 @default.
• W2056952404 cites W1978074135 @default.
• W2056952404 cites W1993010754 @default.
• W2056952404 cites W2001866120 @default.
• W2056952404 cites W2003698297 @default.
• W2056952404 cites W2004390444 @default.
• W2056952404 cites W2009100500 @default.
• W2056952404 cites W2022611853 @default.
• W2056952404 cites W2052562460 @default.
• W2056952404 cites W2061292131 @default.
• W2056952404 cites W2063238607 @default.
• W2056952404 cites W2082442667 @default.
• W2056952404 cites W2097016419 @default.
• W2056952404 cites W2100837269 @default.
• W2056952404 cites W2117535209 @default.
• W2056952404 cites W2123995833 @default.
• W2056952404 cites W2141886669 @default.
• W2056952404 cites W2159103375 @default.
• W2056952404 cites W2169333620 @default.
• W2056952404 cites W2178458220 @default.
• W2056952404 cites W2346149142 @default.
• W2056952404 cites W2417924340 @default.
• W2056952404 cites W4240065971 @default.
• W2056952404 doi "https://doi.org/10.1074/jbc.272.36.22495" @default.
• W2056952404 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9278401" @default.
• W2056952404 hasPublicationYear "1997" @default.
• W2056952404 type Work @default.
• W2056952404 sameAs 2056952404 @default.
• W2056952404 citedByCount "41" @default.
• W2056952404 countsByYear W20569524042012 @default.
• W2056952404 countsByYear W20569524042013 @default.
• W2056952404 countsByYear W20569524042014 @default.
• W2056952404 countsByYear W20569524042018 @default.
• W2056952404 countsByYear W20569524042020 @default.
• W2056952404 crossrefType "journal-article" @default.
• W2056952404 hasAuthorship W2056952404A5048878179 @default.
• W2056952404 hasAuthorship W2056952404A5076217348 @default.
• W2056952404 hasAuthorship W2056952404A5089116639 @default.
• W2056952404 hasBestOaLocation W20569524041 @default.
• W2056952404 hasConcept C112243037 @default.
• W2056952404 hasConcept C11960822 @default.
• W2056952404 hasConcept C181199279 @default.
• W2056952404 hasConcept C185592680 @default.
• W2056952404 hasConcept C192118531 @default.
• W2056952404 hasConcept C2777499176 @default.
• W2056952404 hasConcept C2779222958 @default.
• W2056952404 hasConcept C55493867 @default.
• W2056952404 hasConcept C78245214 @default.
• W2056952404 hasConcept C9262364 @default.
• W2056952404 hasConceptScore W2056952404C112243037 @default.
• W2056952404 hasConceptScore W2056952404C11960822 @default.
• W2056952404 hasConceptScore W2056952404C181199279 @default.
• W2056952404 hasConceptScore W2056952404C185592680 @default.
• W2056952404 hasConceptScore W2056952404C192118531 @default.
• W2056952404 hasConceptScore W2056952404C2777499176 @default.
• W2056952404 hasConceptScore W2056952404C2779222958 @default.
• W2056952404 hasConceptScore W2056952404C55493867 @default.
• W2056952404 hasConceptScore W2056952404C78245214 @default.
• W2056952404 hasConceptScore W2056952404C9262364 @default.
• W2056952404 hasIssue "36" @default.
• W2056952404 hasLocation W20569524041 @default.
• W2056952404 hasOpenAccess W2056952404 @default.
• W2056952404 hasPrimaryLocation W20569524041 @default.
• W2056952404 hasRelatedWork W2033092971 @default.
• W2056952404 hasRelatedWork W2066823697 @default.
• W2056952404 hasRelatedWork W2088519859 @default.
• W2056952404 hasRelatedWork W2099748948 @default.
• W2056952404 hasRelatedWork W2123935034 @default.
• W2056952404 hasRelatedWork W2288930261 @default.
• W2056952404 hasRelatedWork W2526722150 @default.
• W2056952404 hasRelatedWork W2966767867 @default.
• W2056952404 hasRelatedWork W3172902043 @default.
• W2056952404 hasRelatedWork W4205531156 @default.
• W2056952404 hasVolume "272" @default.
• W2056952404 isParatext "false" @default.
• W2056952404 isRetracted "false" @default.
• W2056952404 magId "2056952404" @default.
• W2056952404 workType "article" @default.