Deletion of the gene encoding the cyclin-dependent protein kinase ...

Download PDF
2 downloads 0 Views 3MB Size Report
Comment
Pho85, a protein kinase with significant homology to the cyclindependent kinase, Cdc28, has been ...... cose-6-phosphate) and the total glycogen synthase activ-.
Copyright 0 1996 by the Genetics Society of America

Deletion of the Gene Encoding the Cyclin-Dependent Protein Kinase Ph085 Alters Glycogen Metabolism in Saccharomyces cereuisiae Barbara K. Timblin," Kelly Tatchell?and Lawrence W. Bergman* *Department of Microbiology and Immunology, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 191 02-1 192 and tDepartment of Biochemistry and Molecular Biology, Louisiana State University, Shreueport, Louisiana 71 130 Manuscript received October 2, 1995 Accepted for publication January 25, 1996 ABSTRACT Pho85, a protein kinase with significant homology to the cyclindependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (AF'ase, encoded by P H 0 5 ) in high phosphate (Pi) medium, aswellas in regulation of the cell cycle at Gl/S. We describe several unique phenotypes associated with the deletion of the p H 0 8 5 gene including growth defects on avariety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snfl kinase.However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the p H 0 8 5 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.

N

UTRIENT limitation or depletion results in numerous phenotypes in the yeast Saccharomyces cerevisiae. These phenotypes range from arrest in the G1 phase of the cell cycle (PRINGLE and HARTWELL 1981) to accumulation of the storage carbohydrates glycogen and trehalose (LILLIEand PRINGLE1980). Pathways signaling changes in nutrient levels such as glucose, nitrogen and phosphate and initiation of these stress responses are notwell characterized. Glycogen accumulation and degradation is regulated in part by changes in the phosphorylation state of glycogen synthase and glycogen phosphorylase (ROTHMAN-DENES and CABIB 1970; FRANCOISand HERS 1988; FRANCOIS et al. 1988; PENGet al. 1990). Glycogen synthase (GS) inyeast is encoded by two genes, GSYZ and GSY2, which encode glycogensynthase-1 (GSl) and glycogensynthase-2 (GS-2), respectively(FARKASet al. 1990, 1991). GSY2 expression is increased before entry into stationary phase in response to thedepletion of thecarbon source, and the encoded protein GS2 is additionally regulated by phosphorylation/dephosphorylation of three sites within the carboxy-terminus (FARKAS et al. 1990, 1991; HARDY and ROACH 1993;NI and LAPORTE 1995). Two protein kinases, Snfl and CAMP-dependent protein kinase(cAPK),have been implicated in the regulation of glycogen biosynthesis (BROEK et al. 1985; Correspondingauthm: Lawrence W. Bergman, Department of Microbiology and Immunology, Medical College of Pennsylvania and Hahnemann University, Mailstop 410, Broad and Vine, Philadelphia, PA 19102-1192. E-mail: [email protected] Genetics 143 57-66 (May, 1996)

TATCHELL et al. 1985; CANNON and TATCHELL 1987; CAMERON et al. 1988; THOMPSON~AEGER et al. 1991; HARDY et al. 1994). Null SNFI mutants are unable to initate the dephosphorylation of the carboxy-terminus in response to nutritional deprivation, rendering the cell deficient in glycogen accumulation (HARDY et al. 1994). It is likely that Snfl functions to signal PP-1, a type-1 protein phosphatase encoded by GLC7 (FENGet al. 1991). PP-1, in association with the Gacl regulatory subunit, is most likely involved in the direct dephosphorylation ofGS-2 (FENGet al. 1991; FRANCOIS et al. 1992). Mutations in GLC7 (glc7-I)and deletionof GACl result in a decrease in glycogen levels in nutritionally stressed cells (PENGet al. 1990; FRANCOIS et al. 1992). Truncation of the carboxy-terminus of GS2 suppresses the glycogendeficiency of a snfl mutant, aswellas the glc7-I and gacl mutants (HARDY and ROACH 1993; HARDY et al. 1994). Glycogen synthesisand degradation is also regulated through the cAPK in which the functionally overlapping catalytic subunits areencoded by TPKl, TPK2, and TPK? (CAMERON et al. 1988). During periods of nutritional abundance,cAPK is in its active form, which results in an increase in activity of the glycogen phosphorylase and a decrease in glycogen synthase activity (BROEKet al. 1985; TATCHELL et al. 1985; CANNON and TATCHELL 1987; CAMERON et al. 1988). Upon nutrient depletion, cAF'K is inhibited by the bindingof its regulatory subunit, encoded by BCYl (TODAet al. 1987; CANN O N et al. 1990). Deletion of BCYl constitutively acti-

58

B. K. Timblin, K. Tatchell and L. W. Bergman

vates the kinase, which results in the cell's inability to respond properly to nutritional stress including accumulation of glycogen (TODAet al. 1987). Bcyl mutants show a dramatic decrease in the amountof GSY2 mRNA present in nutritionally stressed cells (HARDYet al. 1994).Additionally, bcyl mutants that constitutively express GSY2 from a ADHl promoter still showa decrease in GS-2 activitysuggesting a posttranslational regulatory role for cAPK Bcyl mutants can respond normally to nutritional stress upon attenuationof the cAPK catalytic subunits, suggesting that aregulatory pathway independent of cAPKexists (CAMERON et al. 1988). This pathway has yet to be characterized. This laboratory is interested in the biochemical changes within the yeast cell after depletion of phosphatefromthe exocellular environment. These changes include anincrease in the expression of several acid and alkaline phosphatases (BOERand STEYN-PARVE 1966; ONISHIet al. 1979), accumulation of storage carbohydrates (LILLIEand PRINGLE 1980) and sporulation of a/a diploids (FREESEet al. 1982). Little is known about the signaling pathway between changes in the phosphate level and initiation of these stress responses, but these processes are known to be regulated by protein kinases and protein phosphatases (reviewed by ROACHet al. 1991; JOHNSTON and CARLSON 1992; MITCHELL1994). A well-studied regulatory system focuses on the transcriptional regulation of the p H 0 5 gene in response to inorganic phosphate(Pi) levels (BOSTIANet al. 1980; KRAMER and ANDERSEN 1980; LEMIRE et al. 1985). During periods of high Pi, p H 0 5 expression is repressed and derepression occurs upon the depletion of environmental phosphate(THILL et al. 1983; BAJWA et al. 1984). Five factors have been genetically defined that regulate APase expression. The positive regulatory factors are encodedby the PH04, PH02, and pH081 genes and deletion of any of these genes results in a nonderepressible phenotype (OSHIMA 1981; TAMAI et al. 1985). Deletion of either of the two negative factors, encoded by pH085 and PH080, results in constitutive expression of APase in high Pi medium (UEDA et al. 1975). pH085 encodes a protein kinase that has 51% identity tothe cyclin dependent protein kinaseCdc28 (UESONO et al1987; TOH-Eet al. 1988). Recently, it has been shown that Pho85 functions in conjunction with the cyclin Pho80 to regulate APase expression ( m F MAN et al. 1994), as well as with two additional cyclins, Pcll and Pc12, to function in regulating progression of the cell cycle(ESPINOZA et al. 1994; MEASDAYet al. 1994). What specific role Pho85 is playing in cell-cycle regulation is not known at this time. In this paper, we have identified a function for Pho85 outside of APaseregulation and cell-cycle control. Disruption of the pH085 gene results in several phenotypes in addition to the constitutive synthesis ofAPase. These include slower growth on rich media with glucose asthe carbonsource,

failure to grow on carbon sources such as glycerol/ lactate, suggesting a mitochondrial defect, inability of a / a homozygous diploids to sporulate and hyperaccumulation of glycogenin rich medium high in phosphate. Hyperaccumulation of glycogen is the result of the increased activity of glycogen synthase. Signaling for this increase inglycogenlevels does not directly involve the Snflprotein kinase, but hyperaccumulation of glycogen is suppressed by a constitutivelyactive cAPK. Mutations in the type-1 protein phosphatase encoded by GLC7 (glc7-1) only partially suppress the hyp eraccumulation of glycogen in a pho85 strain. Additionally, strains containing a deletion of the pH085 gene show an increase in expression of GSY2. The data we present hereindicate that Pho85 is involved inthe regulation of glycogen synthesis at least in part at the level of transcription MATERWS AND METHODS Strains and media: All yeast strains used in this study are listed in Table 1. Yeast strains were grown in rich medium (YEPA, 1.0% yeast extract, 2.0% peptone) or synthetic mediumsupplemented with 2.0% glucose, 2.0% galactose or 3.0% glycerol/2.0% lactate,whereindicated.Transformations of yeast cells were performed by the lithium acetate procedure (ITO et al. 1983) or by the lithium acetate/DMSO procedure (GIETZet al. 1992). Sporulation of various strains was tested in sporulation media(2.0% potassium acetate 0.1% glucose, 0.25% yeast extract, pH 7.0). Plasmid construction: The pH085 gene was cloned from the genome of yeast strain YF'98by amplification of a 1.27-kb fragment using a 5' oligonucleotidecontaining a SalI site and a 3' oligonucleotide containing a XbaI site corresponding to the pH085 sequences reported previously (UESONOet al. 1987). The amplified product corresponds to sequences -144 (relative to 1 of the ATG of the protein produced from the spliced mFWA) to 1114 (95 bp present3' of the termination codon). This fragment will complement a pho85::TRPl allele when introduced on a centromeric vector (data not shown). A partial deletion of the pH085 gene was constructed by digestion of the resulting plasmid with BgnI and EcoRI to remove 89 bp internal to the pH085 coding region (bases 257 to 346). An 860-bp EcoRI-BglII fragment containing the TRPl gene was ligated into this plasmid and the identity of the resulting plasmid, p85=TRP,was confirmed by restriction analysis. A 1.4kb XhoI-BstNI fragment (cleavage sites are located at base pairs 101 and 731, respectiveiy) from p85-TRP was purified and used to transform strain W98 to tryptophan prototrophy. TrpC transformants were screened for constitutive synthesis of APase on complete synthetic high Pi medium plates containing 50 pg/ml 5-bromo-4-chloro-3-indoyl-phosphate (X-P) and supplemented with 2% glucose. Genomic DNA was isolated from strain W98 anda putative pho85 deletion strain (85-TRP-A) and Southern analysis performed to confirm the partial deletion of the pH085 gene. This construction removes amino acids 53-83 of Pho85 with the insertion of the TRPl fragment. Subsequently, strain 85-TRP-A was mated to YP202 and sporulated to yield strain 85T-IC. A pho85::HZS3 gene disruption was constructed by ligating a 1782-bp BamHI fragment containing the HIS3 gene into the BglII site of pH085 resulting in plasmid p18-85SX::HZS3 A 3.1-kb Sua-BamHIfragment fromp18-85SX::HZS3was purified and used to transform strains W102, KT1113, KT1149, and (3'130 to histidine prototrophy. The SalI site was generated

59

Pho85 and Glycogen Metabolism

TABLE 1 Yeast strains Strain

Genotype

YP98 W102 YP198 YP202 85-TW-A 85T-1C

MATa ura?-52,lys2-801,ade2-101, trpl-AI, leu2-AI MATO ura?-52,lys2-801,ade2-101,his?-A200, leu2-AI MATa ura3-52, lys2-801, ade2-101, his?-A200, leu2-AI trpl-Al MATa ura?-52,lys2-801,ade2-101, his3A200, leu2-AI trpl-Al MATa ura?-52,lys2-801,ade2-101, leuZ-AI, trpl-AI, pho85::TRPl MATa ura?-52,lys2-801,ade2-101,his?-A200, leu2-AI, trpl-AI pho85 ::TRPl MATa ura?-52, lys2-801, ade2-I 01, his?-A200, leu2-AI pho85::HZS3 MATa ura?-52,lys2-801,ade2-101,his?-A200, leu2-AI, trpl-AI pho80::LEU2 MATaura?-52,lys2-801,ade2-101,his?-A200, leu2-A1, trpl-AI pho85::TRPl pho4::LEU2 MATa ura?-52,lys2-801,ade2-101,his?-A200, leu2-AI, trpl-AI, pho4 ::LEU2, PHO4'.': :TRPl MATa ura?-52,lys2-801,ade2-101,his?-A200, trpl-AI, PH081' Mata/a diploid of 85-HISA X YP198 Mata/a same as YBTl10 except snfl ::Urn? MATa haploid from YBT165 snfl:: URA? MATa haploid from YBT165 snfl:: URA?, pho85::HZS? MATa same as YP102 except bcyl::URA? MATa same as YBT168 except pho85::HZS? MATa same as 85T-1C except ph085 K36R-URA? MATa same as 85T-1C except PHO85-URA? MATa his?, leu2, ura3-52 MATa same as KT1113 except pho85::HZS? MA Ta his3, leu2, ura?-52 gacl ::ura? MATa same as KT1149 except pho85::HZS? MATa glc7-1, his?, leu2, ura3-52 MATa same as CV130 except pho85::HZS? Same as YBTllO except GSY2-lac2 Haploid from YBT318 GSy2-1~~2, pho85::HZS? Haploid from YBT318 GSY2-hcZ

85-HIS-A h-A CY38 CY71 CY81 YBTllO YBTl65 YBT166 YBT167 YBTl68 YBT169 YBTl75 YBTl76 KT1113 YBTl99 KT1 149 YBTPOO (3'130 YBT20 1 YBT318 YBT319 YBT320

in the original amplification and the BamHI site is located 3' of the XbaI (from the inital amplification) in the pUC18 vector. His' transformants were screened for constitutive synthesis of APaseon complete synthetic high Pi medium X-P plates supplemented with 2% glucose plates. Genomic DNAwas isolated from His+ strains and Southern analysis performed to confirm disruption of PH085. The snfI::crRA? and bcyl::URA? alleles were constructed by replacement of the entire coding region of each gene with URA?. Briefly, fragments from the 5' flanking region of each gene were amplified using the PCR and primers containing a XhoI site at the 5' end and a HindIII site at the 3' end. Fragments from the 3' flanking region of each gene were amplified with primers containing aHindIII site at the 5' end and a BamHI site at the 3' end. The amplified fragments were digested with the appropriate enzymes and ligated into pBluescriptI1, whichhad been digested with XhoI and BamHI. Appropriate clones were digested withHzndIII and ligated with the 1.1-kb HindIII fragment containing URA?. The resulting clones were digested with XhoI and BamHI and used to transform various strains. The BCYl gene was disrupted in wild-type strain W102 to yield strain YBTl68. Deletion of the BCYl gene was confirmed by Southern analysis. Strain YBT168was subsequently disrupted for the pH085 gene (pho85::HZS?) as described above. The SNFl gene wasdisrupted in theheterozygous pho85::HZS? diploid YBTllO. Ura+ colonies were obtained, DNA isolated and deletion of SNFl

Source P. HIETER P. HIETER L. W. BERGMAN L. W. BERGMAN L. W. BERGMAN L. W. BERGMAN L. W. BERGMAN S. MADDEN

c. CREASY c. CREASY c. CREASY This study This study This study This study This study This study This study This study K. TATCHELL This study J. FRANCOIS This study K. TATCHELL This study This study This study This study

confirmed by Southern analysis. Heterozygous snfl::URA3 diploids were subjected to tetrad analysis to yield strains YBT166 and YBT167. A pH085 "kinase dead" (pho85-iV6R) mutation was constructed by site directed mutagenesis (SANTOSet al. 1995). A 1.3-kb Sall-XbaI fragment from either the wild type or pho85K36R mutant of pH085 was subcloned into pRS306 (SIKORSKI and HIETER1989). The resulting plasmids were digested with NcoI (within the URA?gene) to target the integration to ura352 in strain 85T-1C and Ura+ colonies screened for APase expression. Southern analysiswas performed to confirm UFW3 targeting and integration of only a single copy of the plasmid (data not shown). A GSY2-lacZfusion was constructed by fusing a Sall-BamHI GSY2 fragment with the laczstructural gene in plasmid YIp367R (MYERS et al. 1986). The 830-bp Sua-BamHI fragment (-821-9 relative to 1 of the ATG) was obtained by PCR amplification in whichthe BamHI restriction site was created within the oligonucleotide with the fusion containing only the first three amino acids of GS. The resulting plasmid was digested with XhoI to target the integration to GSY2, which is used to transform strain YBTllO. Southern analysiswas performed to confirm GSY2 targeting and subsequently a Leu+ diploid was subjected to tetrad analysis. Iodine staining: Yeastcellswere replica plated to a rich medium plate, grown for 48 hr at 30" and stained with 10 ml of the iodine solution (0.1% iodine and 1%potassium iodide) for 3 min at room temperature (FENG et al. 1991).

B.

K. Timblin, IC Tatchell and L. W. Bergman

60

Quantitative analysis of glycogencontent:Yeast strains were inoculate to an ODmoof 0.1 into 50 ml of rich medium supplemented with 2.0% glucose and grown to an OD6o0 of -0.8 at 30"with shaking. A 30-ml sample was collected by filtration onto cellulose acetate filters (0.45 p~ HA Millipore) and stored at -20" until glycogen content was analyzed [GUNJA-SMITH et al. (1977) with modifications by LILLIEand PRINCLE(1980)l. Enzyme assays: Strains were grown in rich medium supplemented with 2% glucose to an OD6o0of -0.8. To assay glycogen synthase, a 30-ml sample was collected by filtration, flash frozen in a dry-ice ethanol bath and stored at -80" until the enzymeassaywas performed. Samples were washed in 1 ml of extraction buffer (50.0 mM TrisC1, pH 7.5, 2.0 mM EDTA, 2.0 mM D m , 1.0 pg/ml aprotinin, 2.0 mM PMSF, 1.0 mM benzamidine, 0.5 pg/ml leupeptin, and 1.0 pl/ml pepstatin) and pellets were resuspended in 300 pl cold extraction buffer and lysed by vortexing in the presence of glass beads. Homogenates were spun through a 3-pl syringe/glass wool spin column and 30p1 of sample was used inanalysis of enzyme et al. 1968). /%galactosidasewas measured activity (THOMAS as described previously ( CREASYet al. 1993).

loo0

A

1

uQ

C

RESULTS

Disruption of the PH085 generesults in marked growth defects Previous studies have shown that the deletion of the pH085 gene results in constitutive synthesis of AE'ase (MADDEN et al. 1990). In addition, we have observed altered growth properties of the pho85 mutant cell, therefore, we have examined the growth rates of strain 85-TRP-A (pho85::TRPI) on a variety of carbon sources. Growth of the pho85 strain was significantly slower with glucose as a carbon source as compared with the isogenic wild-type strain W98 (Figure 1A).Furthermore, strain 85-TRP-A fails to grow in galactose or in glycerol/lactate as a carbon source (Figure 1, B and C, respectively) aswellas in other nonfermentable carbon sources (data not shown). The slow growth or lack of growth is complemented by transformation of strain 85-TRP-A with a centromeric plasmid containing the pH085 gene (data not shown).Genetic analysis reveals the Gal- phenotype, but not the Gly/ Lac- phenotype is strain specific and results from an altered GAL2 allele present in this genetic background (S288c; data not shown). Strains containing the gal2 allele aswellas a deletion of pH085 are unable to express an integrated GALIO-lacZ fusion when grown in rich medium supplemented with 2.0% galactose (data not shown). In addition to these growth defects, diploid strains that are homozygous for the deletion of the pH085 gene fail to sporulate as there are no asci detected under several conditions that induce sporulation for either a wild-type strain or a strain heterozygous for the pho85 deletion (data not shown). The inability of the mutant to sporulate or to use glycerol/lactate as a carbon source suggests that deletion of the pH085 gene results in a mitochondrial defect. However, the nature of this defect is unknown. Deletion of pH085 gene resultsin the hyperaccumu-

l lo 0 W 10 20 3W 0

Time, hr. FIGURE1.-Deletion of the pH085 gene results in growth defects in a variety of carbon sources. Growth curves were performed for the strains YP98 (PH085)(A)and 85-TW-A (pho85 TRPl) (W) in rich media supplemented with either 2.0% glucose (A), 2.0% galactose (B) or 3.0% glycerol/2.0% lactate ( C ).

lation of glycogen: In a screen for high copy suppressors of the Gal- growthdefect resulting from the disruption of thepH085 gene, we have isolated the GLC7 and TPK2 genes (data not shown). Since PP-1 and cAPK are both involved in regulation of glycogen accumulation and degradation (BROEKet al. 1985; TATCHELL et al. 1985; CANNON and TATCHELL 1987; CAMERON et al. 1988; FENGet al. 1991; FRANCOISet al. 1992; HARDY et al. 1994), we have examined glycogen levels in the pho85::TRPl strain. Cells were grown in rich medium supplemented with 2.0% glucose to mid-logarithmic phase (an ODeooof -0.8), and glycogen content was analyzed. Figure 2A shows that wild-type strain W202 (PH085) contains a minimal level of glycogen when grown in a rich medium and deletion of the pH085 gene resultsin an -3.Gfold increase in the levelof glycogen (as compared with the isogenicwild-type strain) when grown under identical conditions. Growth of the wild-type strain in low Pi media also results in a significant increase in glycogen levels(data notshown). Similar levels of glycogen were seen in a ph085::HIS (strain 85-HIS-A) mutant as compared with the pho85::TRPI mutant (strain 85-TRP-A, data not shown).

Pho85 and Glycogen Metabolism

FIGURE2.-Effect of various mutations on glycogen levels. (A) Glycogen accumulation of wild-type strain W202 (PH085)and strains containing various mutations in thetranscription regulators of the pH05 gene that result in either constitutive expression of acid phosphatase in high phosphate media (strain 85T-lC, pho85::TRPI; strain h-A, pho80::LEUZ; strain CY71, PHO4; strain CY81, PH081'), a nonderepressible phenotype (strain ( 3 3 8 , pho85::TRPl, pho4::LEU2 or a pho85::TRPI strain containing an intregrated copy of the wildtype pH085 gene (strain YBTl76, pho85::TRP1, PH085URA3) or thekinase deadpho85-K36Rmutant (strainYBTl75, pho85::TRPI, pho85-K?bR-URA?). (B) Glycogen accumulation of awild-type strain (strainW102, PH085, S W l , B C Y I ) , pho85 mutant (strain85-HISA, pho85::HZS?), snfr mutant (strain (strain YBT166, snfl::URA?), pho85, snfl doublemutant YBT167, pho85::HZS3 snfl::URAi), bcyl mutant (strain YBT168, bcylr:URA?) and pho85, bcyl double mutant (strain YBT169,phoSS::HIS3bcyl::URA?). (Complete genotypes of the strains are listed in Table 1 and construction of the strains is described in the MATERIALSAND METHODS). All strains were grown in rich media supplemented with 2.0% glucose and containing high Pi levels. Cells were harvested atan of -0.8 by filtration and glycogen levels were analyzed as described in MATERIAIS AND METHODS. The glycogen levels presented are the average of four isolates assayed in duplicate.

To ensure glycogen accumulation was not the result of constitutive synthesis ofAPase, we have examined the glycogen levels in strains that constitutively expressed APase as a result of mutations within other APase regulatory factors. Dominant constitutive mutations in the

61

positive transcription factor pH04 (strain m1) (OGAWA and OSHIMA 1990) or in the mediator pH081 (strain (3'81) (CREASY et al. 1993) resulting in constitutive APase synthesis show essentiallyequivalent levels of glycogen when grown in rich medium supplemented with 2.0% glucose and high Pi (Figure 2A) as compared with the wild-type strain. This suggests that the increase in APase levelsis not triggering the hyperaccumulation of glycogen in the pho85 mutant. Figure 2A also showsthat deletionof the gene encoding the negative regulatory factor pH080 (strain h-A), which also results in constitutive expression of APase, does not result in the hyperaccumulation of glycogen when cells are grown in rich medium supplemented with 2.0% glucose and high Pi. This suggests the Pho80 cyclin in association with Pho85 does not regulate glycogen synthesis. Interestingly, strain h-A(pho8O:rLEU2) fails to grow in medium when glycerol/lactate is used as the sole carbon source (data not shown) suggesting additional regulatory roles of the Pho80/Pho85 complex. The glycogen levelin a pho4::URA?, pho85::TRPI mutant (strain CY38) was examined to ensure production of APase is not required for initiation of glycogen synthesis. Deletion of pH04 results in a nonderepressible phenotype for pH05 expression even in a pho85::TRPI background (OSHIMA1981). As seen in Figure 2A, strain CY38 shows an increase in glycogen levels when grown in ahigh phosphate rich medium, indicating the synthesis of APase isnot required in glycogen accumulation in the pho85 background. To ensure thatit is the kinase function of Pho85 that is required for glycogen regulation, we have used a "kinase dead" pho85-K36R mutation within the putative ATP-binding site of Pho85 (SANTOSet al. 1995). This mutation or the wild-type gene was subclonedinto pRS306 and integrated into strain 85T-1C at the u r d 52 locus, and glycogen levels weredetermined. As seen in Figure 2A, the glycogen levels seen in the "kinase dead" mutant are similar to that seen in a pho85 deletion strain. Glycogen levelsare significantly lowerupon integration of the wild-type PH085gene intostrain 85T1C. This indicates that thePho85 kinase activity is essential for the negative regulation of glycogen synthesis. Ph085 does not function through Snfl in glycogen regulation: The Snfl protein kinase is involved in activation of glycogen synthase in response to depletion of glucose or phosphate in the medium (THOMPSONJAEGER et at. 1991). To examine if Pho85 is involved in the regulation of Snfl or vice versa, we deleted SNFI by replacement of the coding region of SNFI with the URA? geneina pho85 heterozygous diploid (strain YBT110) as described in MATERIALS AND METHODS. Disruption of SNFI was confirmed by Southern analysis (data not shown). After sporulation and dissection of the snfl, pho85 heterozygous diploid (strain YBT165), four spore tetrads were screened for auxotrophic re-

62

B. K. Timblin, K. Tatchell and L. W. Bergman

t

t

t

FIGURE 3.-Dissection of a heterozygous PH085/ pho85::HZS3, SA?Fl/snfl::URA3 diploid (strain YBTl65). D i p loids were sporulated on potassium acetate plates, then dissected onto rich media (YEPA)containing 2.0%glucose and grown for 96 hr at 30".Subsequent auxotrophicmarker analysis confirmed the genotypeof the resulting haploids. Arrows indicate snfl::URA3 pho85::HIS3 strains.

quirements and several tetra-types were selected and screened initially for glycogen content by iodine vapor stain. Darker stainingcells were shown to be eitherHis+ (pho85::HZS3) or His+ Ura+ (pho85::HZS3, snfl::URA3) (data notshown) suggesting deletion of SNFl does not suppress the hyperaccumulation phenotype of a pho85 strain. Quantitative analysis of glycogen levels of strains resulting from a tetra-type ascus show the pho85::HZS3 and pho85::HZS3, snfl::URA3 mutants hyperaccumulated glycogen in rich medium high in phosphate to 2.4 or 2.8-fold, respectively, over that of the wild-type strain and 6.3- or 7.5-fold, respectively, over that of the strain containing the snfl::URA3 deletion alone (Figure 2B). Since deletion of the SNFI gene is unable to sup press the hyperaccumulation of glycogenresulting from deletion of the p H 0 8 5 gene, itis likely that Pho85 does not function to negatively regulate Snfl. Although, this experiment does not rule out that Pho85 functions downstream of Snfl to regulate glycogen metabolism. Interestingly, dissection of strain YBT165 resulted in growth differences of the spore colonies (Figure 3). The smaller colonies, indicated by the arrows, were all shown to contain both thepho85 deletion and the snfl deletion (His+ Ura'). In every spore tested, those that were His' Ura+ manifested the small colony phenotype. The largest colonies were alwayswild type for both genes (His- Ura-), while the spores containing either the snfl deletion or the pho85 deletion showed an intermediate colony size that is smaller than that of the wild type. The time required for germination of spores that were subsequently shown to be His+ Ura' was significantly longer thanspores shown to bewild typeor single mutants alone. An activated CAPK suppressesthe hyperaccumulation of glycogen in a pho85mutant: Strains that containmutations that result in the constitutive activation of cAPK are unable to accumulate glycogen in response to nutritional stress (MATSUMOTO et al. 1983; TODAet al. 1985, 1987; MARSHALL et ul. 1987, CANNON et ul. 1990). We

have followeda similar strategy as that of the SNFI gene by deleting the RCYl gene resulting in constitutive activation of cAPK to see if Pho85 functions as part of the cAPK signaling pathway. The BCYl gene was deleted in the haploid strain YP102 by replacement of the coding region with URA3 and deletion of BCYl (resulting in strain YBT168)was confirmed by Southern analysis. The p H 0 8 5 gene was subsequently deleted in strains containing the disruption of BCYl (YBT169). Iodine vapor stain (data notshown) and quantitative examination ofglycogenlevels revealed the wild-type strain (YP102) and the bcyl::URA3mutant (YBT168) accumulated little glycogen in a rich medium (Figure 2B). Additionally, the bcyl::URA3 mutant was unable to accumulate glycogenwhen phosphate stressed (datanot shown). Deletion of the pH085 gene in the bql::URA3 mutant resulted in the suppression of the hyperaccumulation of glycogen seen in a pho85 mutant (Figure 2B). This suggests that Pho85 may function upstream of cAPK in regulation of glycogen levels in the cell. However, it should be noted that increased cAPKactivity affects GSY2 expression and negatively regulates GS activity, which may mask the effect that the deletion of the p H 0 8 5 gene may have on glycogen synthase regulation. Hyperaccumulation of glycogen of a pho85 strain is partially suppressed by glc7-1, but not by a gacl::um3 deletion: To investigate if Pho85 functions to regulate proteins involved in the activation of the glycogen synthase, we have examined glycogen levels instrains containing both a pho85::HZS3 deletion and a glc7-I mutation or a gacl::uru3 deletion. The wild-type strain (KT1113), parentof the gk7-I and gacl::uru3 mutants, is not isogenic to the wild-type strain previously studied (YP98), thus we have also deleted the p H 0 8 5 gene in that strain (resulting in strain YBT199). Wild-typestrain KT1 113 and strains with either the glc7-I mutation (CV130) or disrupted for gacl::uru3 (KT1149) accumulate low levels of glycogen when grown in high Pi rich media (PENGet al. 1990; FRANCOIS et al. 1992). Quantitative analysis of glycogen levels show the deletion of the p H 0 8 5 gene in the wild-type strain KT11 13 increases the glycogen level of the cell by 4.8-fold (Figure 4). In addition, both strain CV130 (gk7-I) and strain KT1149 (gacl::uru3) contain higherlevels of glycogen when the pho85::HZS3 deletions (strains YBT201 and YBT200, respectively) are also present (2.3- and 1.7-fold, respectively). However, glycogen levels strain of YBT201 (glc7I , pho85::HZS3) are approximately twofold lower when compared with strain YBT199 (pho85::HZS3). These results suggest the glc7-I mutation can partially suppress the hyperaccumulation of glycogen of a pho85 mutant. There is still a significant increase in the glycogen levels of the strain YBT201 (gk7-I, pho85::HZS3) over that of the wild-type strain KT1 113 and of strain CV130 (gk7-I) suggesting that Pho85 does not function in a epistatic manner to PP-1, but that the gk7-I mutation may be masking the hyperaccumulation of glycogen phenotype

63

Pho85 and Glycogen Metabolism ''O'O

FIGURE4.-Effect of mutations in GIX7 and GACl on glycogen levels. The wild-type strain (KT1113), gk7-1 mutant (CV130) and gucl::uru? mutant (KT1149) containing a wildtype PH085gene ora pho85::HIS3disruption (strainsYBT199, YBT201,YBT200, respectively) were grown to an ODMHI of -0.8 in rich medium supplemented with 2.0% glucose, harvested by filtration and glycogen levels of the strains were determined as described in MATERIAIS AND METHODS. The glycogen levels presented are the average of four isolates assayed in duplicate.

of the pho85 mutant strain. This could indicate that Pho85 may be directly regulating glycogen synthase in a posttranslational manner. Glycogen synthase activityis increased upon deletion of the pH085 gene: Since mutations within PP-1 (glc71) fail to completely suppress the hyperaccumulation phenotype of a pho85 mutant, we examined whether the deletion of the p H 0 8 5 gene affected the enzymatic activity of glycogen synthase and whether deletions of the snfl or bcyl genes resulted in a change in this activity. As seen in Figure 5, deletion of the p H 0 8 5 gene (pho85::HK?) increases both the level of active glycogen synthase (activity measured in the absence of glucose-6-phosphate) and thetotal glycogen synthase activity in the cell(activity measured in theabsence of glucose4phosphate). Consistent with the observation that thebcyl, pho85 double mutantfailed to accumulate glycogen, glycogen synthase activity is low in the pho85, bcyl mutant strain (strain YRTl69). However, in contrast, there is a significant increase in both active and total glycogen synthase activity in the snfl, pho85mutant (strain YRT167) as compared with either the wild-type or pho85 strains (Figure 5). At present, the mechanism responsible for this observation is not clear. Deletion of the pH085 gene results in the increase of expression of Gm2: We have investigated whether Pho85 is regulating the expression of the inducible glycogen synthase gene GSY2 by examining the expression of a GSY2::lacZ fusion gene, which contains 820 bp u p stream of the translational start site and only the first 3 bp of the coding region. This fusion gene was integrated into the genome of a pho85 heterozygous diploid

-Glc6P +GMP

FIGURE 5.-Effect of pH085 deletion on glycogen synthase activity. Awild-type strain (strainW102, PH085, SWl, RCYI), pho85 mutant (strain 85-HIS-A,pho85::HI.V). snfl mutant (strain YBT166, snfI::URA?), pho85, snfl doublemutant (strain YBT167, pho85::HI.V snfl::URA?), bcyl mutant (strain YBTl68, bql::UIU?) and pho85, bql double mutant (strain YBTl69, pho85::HIS bcyl::URA3) were grown toan ODo, of -0.8 in rich medium supplemented with 2.0% glucose, harvested by filtration, flash frozen in a dry-ice ethanol bath and stored at -80" until glycogen synthase activity was tested in the absence (stippled bar) or presence (solid bar) of 7.2 mM glucose-6phosphate (Glc6P) as described in MATERIL5 AND METHODS. The activities presented are theaverage of four isolates assayed in duplicate.

(YBT110)as described in MATERIALS AND METHODS. After sporulation and dissection of a Leu+diploid, pho85::HIS3 haploids and wild-type haploids that all contained the GSY2-lncZfusion gene were grownin rich medium until an ODfiooof -0.8 and P-galactosidase activity was measured (Fi