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Systematic Identification of the Genes Affecting Glycogen Storage in the Yeast Saccharomyces cerevisiae

2002; Elsevier BV; Volume: 1; Issue: 3 Linguagem: Inglês

10.1074/mcp.m100024-mcp200

1535-9484

Wayne A. Wilson, Zhong Wang, Peter J. Roach,

Endoplasmic Reticulum Stress and Disease

At the onset of nutrient limitation, the yeast Saccharomyces cerevisiae synthesizes glycogen to serve as a carbon and energy reserve. We undertook a systematic survey for the genes that affect glycogen accumulation by taking advantage of the strain deletion set generated by the Saccharomyces Genome Deletion Project. The strain collection analyzed contained some 4600 diploid homozygous null deletants, representing ∼88% of all viable haploid disruptants. We identified 324 strains with low and 242 with elevated glycogen stores, accounting for 12.4% of the genes analyzed. The screen was validated by the identification of many of the genes known already to influence glycogen accumulation. Many of the mutants could be placed into coherent families. For example, 195 or 60% of the hypoaccumulators carry mutations linked to respiratory function, a class of mutants well known to be defective in glycogen storage. The second largest group consists of ∼60 genes involved in vesicular trafficking and vacuolar function, including genes encoding 13 of 17 proteins involved in the structure or assembly of the vacuolar ATPase. These data are consistent with our recent findings that the process of autophagy has a significant impact on glycogen storage (Wang, Z., Wilson, W. A., Fujino, M. A., and Roach, P. J. (2001) Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol. Cell. Biol. 21, 5742–5752). Autophagy delivers glycogen to the vacuole, and we propose that the impaired vacuolar function associated with ATPase mutants (vma10 or vma22) results in reduced degradation and subsequent hyperaccumulation of glycogen. At the onset of nutrient limitation, the yeast Saccharomyces cerevisiae synthesizes glycogen to serve as a carbon and energy reserve. We undertook a systematic survey for the genes that affect glycogen accumulation by taking advantage of the strain deletion set generated by the Saccharomyces Genome Deletion Project. The strain collection analyzed contained some 4600 diploid homozygous null deletants, representing ∼88% of all viable haploid disruptants. We identified 324 strains with low and 242 with elevated glycogen stores, accounting for 12.4% of the genes analyzed. The screen was validated by the identification of many of the genes known already to influence glycogen accumulation. Many of the mutants could be placed into coherent families. For example, 195 or 60% of the hypoaccumulators carry mutations linked to respiratory function, a class of mutants well known to be defective in glycogen storage. The second largest group consists of ∼60 genes involved in vesicular trafficking and vacuolar function, including genes encoding 13 of 17 proteins involved in the structure or assembly of the vacuolar ATPase. These data are consistent with our recent findings that the process of autophagy has a significant impact on glycogen storage (Wang, Z., Wilson, W. A., Fujino, M. A., and Roach, P. J. (2001) Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol. Cell. Biol. 21, 5742–5752). Autophagy delivers glycogen to the vacuole, and we propose that the impaired vacuolar function associated with ATPase mutants (vma10 or vma22) results in reduced degradation and subsequent hyperaccumulation of glycogen. One attractive feature of the budding yeast Saccharomyces cerevisiae as an experimental organism has been its ease of genetic manipulation, including the ability to perform genetic screens by which yeast strains with a phenotype of interest can be recognized. Experimentally, the next phase involves identification of the gene(s) responsible, a task that can be time-consuming and sometimes non-trivial. The S. cerevisiae genome contains some 6,200 open reading frames (ORFs), 1The abbreviations used are: ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used are: ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride. and these have been disrupted systematically in the Saccharomyces genome deletion project (1.Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.Science. 1999; 285: 901-906Google Scholar). The availability of the resulting set of deletion strains, each carrying a deletion of one specific ORF, permits a totally different type of screen or survey for genes linked to a particular phenotype. Most importantly, the survey is systematic. The first report of using a partial strain deletion set analyzed growth rates in rich and minimal medium (1.Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.Science. 1999; 285: 901-906Google Scholar). Recently, an analysis of the rapamycin sensitivity of 2,216 haploid disruptants was reported (2.Chan T.F. Carvalho J. Riles L. Zheng X.F. A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR).Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13227-13232Google Scholar), and a systematic study of fluid phase endocytosis using around 700 strains generated by the European Functional Analysis Network has been conducted (3.Wiederkehr A. Meier K.D. Riezman H. Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis.Yeast. 2001; 18: 759-773Google Scholar). The first truly genome-wide screen of a defined null mutant collection was reported by Ni and Snyder (4.Ni L. Snyder M. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae.Mol. Biol. Cell. 2001; 12: 2147-2170Google Scholar) who analyzed over 4000 strains in a study of polarized growth. Information is also available about viability, based on analysis of the deletion strain set, and is available from the Saccharomyces Genome Database (5.Cherry J.M. Adler C. Ball C. Chervitz S.A. Dwight S.S. Hester E.T. Jia Y. Juvik G. Roe T. Schroeder M. Weng S. Botstein D. SGD: Saccharomyces Genome Database.Nucleic Acids Res. 1998; 26: 73-79Google Scholar) (genome-www.stanford.edu/Saccharomyces). We report here the use of a specific metabolic end point, the ability to store glycogen, as the basis for a screen of ∼4600 homozygous diploid mutants to identify genes that affect glycogen accumulation. Glycogen serves as a reserve of glucose. Its accumulation is initiated under conditions of nutrient limitation, such as the approach to stationary phase in liquid culture. Limitation for carbon, nitrogen, phosphorous, or sulfur all act as triggers for increased glycogen synthesis (6.Lillie S.H. Pringle J.R. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.J. Bacteriol. 1980; 143: 1384-1394Google Scholar). Our laboratory has been interested in glycogen as an example, in mammals and yeast, of a compound whose synthesis and utilization is under complex and intricate controls linked to the intracellular energy state, as well as the nutritional status of the environment (see Ref. 7.Francois J. Parrou J.L. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae.FEMS Microbiol. Rev. 2001; 25: 125-145Google Scholar for a review). Synthesis of glycogen requires the activities of glycogenin, a self-glucosylating initiator protein (encoded by GLG1 and GLG2; see Ref.8.Cheng C. Mu J. Farkas I. Huang D. Goebl M.G. Roach P.J. Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.Mol. Cell. Biol. 1995; 15: 6632-6640Google Scholar), glycogen synthase (GSY1 and GSY2; see Ref.9.Farkas I. Hardy T.A. Goebl M.G. Roach P.J. Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled.J. Biol. Chem. 1991; 266: 15602-15607Google Scholar), which catalyzes bulk synthesis, and the branching enzyme (GLC3; see Ref.10.Rowen D.W. Meinke M. LaPorte D.C. GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme.Mol. Cell. Biol. 1992; 12: 22-29Google Scholar), which introduces the branches characteristic of the mature polysaccharide. Glycogen breakdown requires glycogen phosphorylase (Gph1p; see Ref. 11.Hwang P.K. Fletterick R.J. Convergent and divergent evolution of regulatory sites in eukaryotic phosphorylases.Nature. 1986; 324: 80-84Google Scholar) and debranching enzyme (Gdb1p; see Refs. 12.Nakayama A. Yamamoto K. Tabata S. High expression of glycogen-debranching enzyme in Escherichia coli and its competent purification method.Protein Expr. Purif. 2000; 19: 298-303Google Scholar and 13.Teste M.A. Enjalbert B. Parrou J.L. Francois J.M. The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.FEMS Microbiol. Lett. 2000; 193: 105-110Google Scholar) or, under certain conditions, glucoamylase (Sga1p; see Ref. 14.Yamashita I. Fukui S. Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 1985; 5: 3069-3073Google Scholar). However, the enzymes of glycogen metabolism are under a variety of transcriptional and post-translational controls, and so genes encoding a number of other proteins affect glycogen accumulation (for a review, see Ref. 7.Francois J. Parrou J.L. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae.FEMS Microbiol. Rev. 2001; 25: 125-145Google Scholar). For example, the cyclic AMP pathway controls both gene expression and phosphorylation of key proteins (15.Smith A. Ward M.P. Garrett S. Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation.EMBO J. 1998; 17: 3556-3564Google Scholar–17.Hardy T.A. Huang D. Roach P.J. Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae.J. Biol. Chem. 1994; 269: 27907-27913Google Scholar). Starvation, as sensed by the Tor pathway, stimulates glycogen accumulation (18.Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. TOR controls translation initiation and early G1 progression in yeast.Mol. Biol. Cell. 1996; 7: 25-42Google Scholar). Signaling through the Snf1p and Pho85p protein kinases has antagonistic effects on glycogen storage (19.Huang D. Farkas I. Roach P.J. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.Mol. Cell. Biol. 1996; 16: 4357-4365Google Scholar). The genes mentioned above have been identified by a combination of conventional biochemical and genetic approaches, and there is no guarantee that all relevant genes have been found or any indication as to how many genes affect glycogen storage. The systematic survey described in this work indicated that 566 of ∼4600 strains from the homozygous diploid release of the deletion library had glycogen levels that differed from wild type. Of these genes, a surprising fraction, about 10%, had functions related to vesicular trafficking or vacuolar function. The homozygous diploid deletion series (BY4743 strain background) was purchased from Research Genetics. The yeast deletion series comprises a set of mutants where each open reading frame has been disrupted from start to stop codon, and a kanMX marker cassette (conferring resistance to the antibiotic G418) has been inserted. The library of deletions was supplied frozen in 96-well microtiter plates. Each well contained 200 μl of YPD (2% peptone, 2% glucose, 1% yeast extract) medium supplemented with G418 (200 μg/ml) and 15% glycerol. For keying, contamination checks and orientation purposes, two wells per plate contained only medium. One empty well defines the bottom left hand corner, and the other serves as an identifier. Complete details regarding series construction and availability can be found at sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html and www.resgen.com/products/YEASTD.php3. The deletion set contained 4,639 different strains, of which 82 were flagged by Research Genetics as quality-control failures. Excluding these, the collection is of 4,557 strains that correspond to 88% of all possible viable mutants. Additionally, we constructed strains derived from BY4741 and BY4742, which are also available from Research Genetics and are the MATa and MATα parents, respectively, of BY4743. A polymerase chain reaction strategy (20.Wach A. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae.Yeast. 1996; 12: 259-265Google Scholar) was used to generate apg1::URA3 in BY4741 and vma10::LEU2 in BY4742. These strains were mated, and tetrad dissection was performed to isolate apg1::URA3 vma10::LEU2 double mutants of both mating types. The genotypes of the strains isolated were MATahis3 leu2 met15 lys2 apg1::URA3 vma10::LEU2 and MATα his3 leu2 met15 lys2 apg1::URA3 vma10::LEU2. These MATa and MATα strains were then crossed to generate a diploid strain. For analysis, the master plates were thawed, and the cells were resuspended by pipetting up and down using a 12-channel automatic pipette. A 96-pin microplate replicator was used to transfer an aliquot from each well to a fresh microtiter plate containing 200 μl of appropriate medium per well. To assess glycogen accumulation, SC medium (0.67% yeast nitrogen base, 0.079% Complete Supplement Mix (Bio101 Inc.), 2% glucose) was used. Growth on YPG medium (2% peptone, 1% yeast extract, 3% glycerol) was used to assess respiratory competence. For glycogen accumulation studies, plates were prepared in duplicate. The parental wild-type strain (BY4743) was inoculated into well H1 on each plate to serve as an internal control. Plates were incubated at 30°C for 48 h without shaking. The 54 microtiter plates that constitute the deletion series were analyzed for both glycogen accumulation and ability to grow using glycerol as a carbon source. Preliminary studies with strains known to over- or underaccumulate glycogen were used to optimize the growth and staining conditions. Growth of cells in the microplate format was slower than in standard liquid culture in shaking flasks, likely because of a lack of aeration. Thus, to obtain enough cells for analysis of glycogen content by iodine staining, we grew cultures for 48 h. Cells were resuspended by pipetting up and down using a 12-channel automatic pipette. The cell suspensions were then transferred to a 96-well vacuum manifold (Bio-Dot microfiltration unit; Bio-Rad) and filtered onto a nitrocellulose membrane. The membrane was removed and stained for 2 min by exposure to iodine vapor, a time that was found to be suitable for uniform and reproducible results. Each membrane was photographed using a digital camera to create a permanent record, and cultures that stained either more or less intensely than wild type were scored (Fig. 1). Images were downloaded from the camera to an Apple Macintosh G4 microcomputer and processed with Adobe PhotoShop LE. In each case, duplicate plates were grown, harvested, and stained to control for any variability in staining intensity, and only wells that gave the same result on both plates were scored in the final tally. Growth in glycerol was assessed by visual inspection of plates and comparison to the congenic wild-type strain. The deleted gene responsible for the aberrant glycogen phenotype in each case was identified by reference to a spreadsheet compiled from data available at www.resgen.com/products/YEASTD.php3 where a text file detailing the ORF deletion present in each well of each microtiter plate can be found. Information from the Saccharomyces Genome Database (5.Cherry J.M. Adler C. Ball C. Chervitz S.A. Dwight S.S. Hester E.T. Jia Y. Juvik G. Roe T. Schroeder M. Weng S. Botstein D. SGD: Saccharomyces Genome Database.Nucleic Acids Res. 1998; 26: 73-79Google Scholar) (genome-www.stanford.edu/Saccharomyces)and from the YPD database (21.Costanzo M.C. Hogan J.D. Cusick M.E. Davis B.P. Fancher A.M. Hodges P.E. Kondu P. Lengieza C. Lew-Smith J.E. Lingner C. Roberg-Perez K.J. Tillberg M. Brooks J.E. Garrels J.I. The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information.Nucleic Acids Res. 2000; 28: 73-76Google Scholar, 22.Costanzo M.C. Crawford M.E. Hirschman J.E. Kranz J.E. Olsen P. Robertson L.S. Skrzypek M.S. Braun B.R. Hopkins K.L. Kondu P. Lengieza C. Lew-Smith J.E. Tillberg M. Garrels J.I. YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information.Nucleic Acids Res. 2001; 29: 75-79Google Scholar) (www.proteome.com/databases/index.html) was used to pair ORF numbers with gene names and functional properties where known. For quantitative determination of glycogen levels, cells were grown for 24 h to early stationary phase (∼1 × 108 cells/ml) in 10 ml of SC medium at 30°C. The cell density was checked by counting using a hemocytometer. An aliquot (1 ml) of culture was removed, and the cells were collected by centrifugation (14,000 × g, 1 min, 4°C). The supernatant was aspirated, and the cell pellet was immediately frozen on dry ice and stored at −80°C until use. The culture was maintained for another 24 h, and a second sample (48 h time point) was taken as described. Cell pellets were thawed by addition of 200 μl of 20% (w/v) KOH and boiled in a water bath for 1 h. The solution was neutralized by addition of HCl and processed as described previously (23.Parrou J.L. Francois J. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells.Anal. Biochem. 1997; 248: 186-188Google Scholar). The data shown are the mean of at least two independent determinations performed in duplicate. Glycogen synthase was assayed in extracts prepared from yeast cells by lysis with glass beads using the method of Thomas et al. (24.Thomas J.A. Schlender K.K. Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose.Anal. Biochem. 1968; 25: 486-499Google Scholar) as described previously (25.Hardy T.A. Roach P.J. Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation.J. Biol. Chem. 1993; 268: 23799-23805Google Scholar). Phosphorylation of glycogen synthase converts the enzyme into a less active form that requires the presence of the allosteric activator glucose 6-phosphate to elicit full activity. Thus, the ratio of activity without and with glucose 6-phosphate (−/+ glucose 6-phosphate activity ratio) is an index of the phosphorylation state of the enzyme with high values indicating that dephosphorylated and active glycogen synthase predominates. To induce autophagy, cells were grown to late logarithmic phase in YPD medium and then starved for nitrogen as described by Takeshige et al. (26.Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.J. Cell Biol. 1992; 119: 301-311Google Scholar) either in the presence or absence of 1 mm phenylmethylsulfonyl fluoride (PMSF). For analysis of vma10, vma22, and apg1 vma10 mutants, cells were grown to logarithmic phase or to saturation in SC medium. Cells were examined under a Nikon Microphot-FXA microscope, equipped with Nomarski optics, using a ×60 oil-immersion objective at a magnification of ×1200. Images were captured using a Pulnix TM-745 digital camera and the public domain NIH Image 1.62 software (developed at the United States National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image/) running on a Macintosh G4 microcomputer. Movies were prepared and edited using QuickTime Pro 5.0.1 software from Apple Computer. The strain deletion set was surveyed, in duplicate, for glycogen accumulation using iodine staining of cells harvested after growth in microtiter plates and filtration through nitrocellulose filters, as described under “Materials and Methods” (see Fig. 1). In addition, the ability to grow on glycerol was monitored. The rationale for including this assay was the well established observation that cells unable to grow using non-fermentable carbon sources cannot store glycogen, usually because of respiratory defects in what are termed petite mutants (27.Chester V.E. Heritable glycogen-storage deficiency in yeast and its induction by ultra-violet light.J. Gen. Microbiol. 1968; 51: 49-56Google Scholar, 28.Enjalbert B. Parrou J.L. Vincent O. Francois J. Mitochondrial respiratory mutants of Saccharomyces cerevisiae accumulate glycogen and readily mobilize it in a glucose-depleted medium.Microbiology. 2000; 146: 2685-2694Google Scholar). In this way, we could place mutants in this category, even if the corresponding gene had no known function. From the 4557 strains examined, we recovered 242 with high glycogen and 324 with low glycogen so that a total of 12.4% of the gene deletions influenced glycogen. We attempted to cluster the mutants into logical families (Table I). In some cases the families are clearly meaningful, with the presence of multiple genes of related function reinforcing the validity of their identification in the survey. In other instances, we grouped proteins according to biochemical function, such as “protein kinases,” where common biological functions were not obvious. A large group of 100 mutants, representing almost 18% of the genes identified, corresponded to ORFs of unknown function, about which little or nothing is known. Thirty-five genes involved in general transcription or RNA processing were identified as were 18 that had to do with DNA structure or maintenance of structural integrity. Our thought is that the mutations in these strains are likely to be only very indirectly linked to glycogen metabolism. Glycogen storage is in part a response to stress, which may be an aspect of the phenotype of these groups of mutants. Such could also be true for the genes encoding small ribosomal subunits, all 12 of which were associated with glycogen hyperaccumulation. Because of the large number of mutants identified, it is not possible to give a thorough description of each (refer to the Supplemental Material for a table containing all of the strains identified and all of our annotations).Table IGene familiesDescriptionTotal numberGlycogen phenotypeLowHighMitochondrial or respiratory20719512Vesicular transport or vacuolar function581642Carbohydrate metabolism1239Amino acid metabolism1019Adenine metabolism404Inositol metabolism761Miscellaneous metabolism221012Protein kinases1468Protein phosphatases853WD-40 repeat proteins734Other signaling1046Ubiquitination312Cytoskeleton523Transport, pore proteins817Sporulation413Transcription, RNA processing351718Small ribosomal subunit12012Chromatin, DNA structure18711Known function, miscellaneous22715Unknown function, little, or nothing known1003961Total566324242 Open table in a new tab A number of mutants were chosen for additional analyses. The selection was made to represent several of the gene families of Table I, together with some subjective choices (Table II). The further analyses included assessment of glycogen levels as judged by iodine staining of colonies on either YPD or SC plates, enzymatic determination of glycogen in liquid cultures, and measurement of glycogen synthase activity. These secondary measures of glycogen usually reproduced the original screening result, especially noting that yeast accumulate generally much less glycogen when grown in rich as opposed to synthetic medium. Thus, a number of mutants scored as high glycogen on the original survey with synthetic medium were wild type when grown on YPD plates. A notable exception is provided by some of the vacuolar and vesicular trafficking mutants (see below). Of the mutants selected for further study, few exhibited very great changes in total glycogen synthase activity. Glycogen synthase phosphorylation, and activation state, can be monitored via the −/+ glucose 6-phosphate activity ratio, lower values correlating with greater phosphorylation (see “Materials and Methods”). Deletion of genes implicated in direct phosphorylation of glycogen synthase, such as PHO85, leads to elevated activity ratio. Of the genes tested, only one, a WD-40 repeat protein of unknown function, had a significantly elevated activity ratio, making it a candidate to be a constituent of a glycogen synthase kinase.Table IIFurther analysis of selected deletion mutants isolated in the screenOriginal callGlycogen (iodine) onGlycogen (enzymatic)Glycogen synthaseActivity ratio (24 h)YPDSC24 h48 h24 h48 hProtein kinases KIN1Low+/−+/−0.8821.50.970.11 PTK2Low+/−+0.912.41.30.40.15 RIM15Low−−0.270.4110.450.07 CTK1High++++ND7.40.43 DBF2High+++2.14.20.820.920.23 NPR1High++1.60.7310.720.04 TOR1High++++1.43.41.110.22 YDL025CHigh+++0.7911.80.520.15 YDR247WHigh+++1.11.70.831.40.28 YNL099CHigh++0.91.910.640.18 YOL045WHigh+++1.13.30.761.80.36 YPL150WHigh++++1.55.81.21.80.18Protein phosphatases PIG2Low++/−0.550.261.60.980.09 RTS1High (very)++++++28.51.11.30.17 YCR079WHigh++++1.63.61.31.40.17WD-40 repeat proteins YAR003WLow+++0.510.271.10.40.18 YPL247CHigh+++1.40.531.30.870.17 YKL121WHigh+++10.961.20.740.19 YOL087CHigh++0.591.721.60.70.21 YOL138CHigh (very)+++++0.790.521.10.680.55Vesicular transport and vacuolar function BST1High (very)+++2.11.3 SEC22High (very)++++2.11.3 VID21High (very)++++++6.56.8220.22 VID22High (very)++2.22.8 VMA10High (very)+++1.85.7 VMA22High (very)++1.23.3 VMA3High+++ND3.5Inositol metabolism INO1Low+/−+/−0.350.631.200.04 IPK1Low+/−++0.61.51.21.40.09Carbohydrate metabolism RPE1High (very)++++11.10.891.20.12 YHR204WHigh (very)+++1.21.61.71.90.09Others ADO1High (very)++++++151.61.70.31 GIS4Low+++/−0.711.31.31.90.09 Open table in a new tab A number of the genes that were isolated in the screen have been implicated previously in the control of glycogen accumulation (Table III), which we view as a validation of the screening methodology. In an earlier genetic screen for aberrant glycogen accumulation, Cannon et al. (29.Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae.Genetics. 1994; 136: 485-503Google Scholar) had characterized eight so-called glc mutants and identified the corresponding genes. Five GLC genes were found in our screen (Table III). Of the three remaining glc mutants, two were not represented in the deletion series (GLC7 is essential, and GLC5/IRA1 was missing) and one, glc1/ras2, was present but had wild-type levels of glycogen in this genetic background under the growth conditions used.Table IIIGenes isolated by the screen and that had been implicated previously in glycogen accumulationGeneGlycogen phenotypeDescriptionReferenceGAC1LowRegulatory subunit for protein serine/threonine phosphatase Glc733GLC3Lowα-1,4-glucan branching enzyme29GLC8LowModulator of protein serine/threonine phosphatase Glc7p29GSY2LowMajor isoform of glycogen synthase9IRA2 (GLC4)LowGTPase activating protein for Ras1p and Ras2p29PIG2LowProtein interacting with Gsy2p; possible regulatory subunit for the protein serine/threonine phosphatase Glc7p35RIM15LowSerine/threonine protein kinase; positive regulator of IME2 expression and sporulation40SNF1 (GLC2)LowSerine/threonine protein kinase essential for derepression of glucose-repressed genes29GDB1HighGlycogen debranching enzyme13GPH1HighGlycogen phosphorylase11PFK1HighPhosphofructokinase α subunit32REG1HighRegulatory subunit for protein phosphatase Glc7p, required for glucose repression38TPS1 (GLC6)HighTrehalose 6-phosphate synthase29 Open table in a new tab Genes encoding four enzymes involved directly in glycogen metabolism were identified. Strains defective for the degradative enzymes, glycogen phosphorylase, Gph1p, and debranching enzyme, Gdb1p, were hyperaccumulators. The strain lacking GSY2, which encodes the predominant isoform of glycogen synthase, had low glycogen storage as did the strain lacking the glycogen branching enzyme encoded by GLC3. The PFK1 and TPS1 genes encode components of phosphofructokinase and the trehalose synthase complex, respectively. Both pfk1 and tps1 mutations result in elevated intracellular glucose 6-phosphate (30.Heinisch J. Construction and physiological characterization of mutants disrupted in the phosphofructokinase genes of Saccharomyces cerevisiae.Curr. Genet. 1986; 11: 227-234Google Scholar, 31.Thevelein J.M. Hohmann S. Trehalose synthase: guard to the gate of glycolysis in yeast?.Trends Biochem. Sci. 1995; 20: 3-10Google Scholar). It is probable that the increased glucose 6-phosphate levels bypass the phosphorylation control of glycogen synthase resulting in deregulated, hyperactive glycogen synthase (see for example Ref. 32.Huang D. Wilson W.A. Roach P.J. Glucose-6-P control of glycogen synthase phosphorylation in yeast.J. Biol. Chem. 1997; 272: 22495-22501Google Scholar). Four putative or actual targetin