The Gene PPG Encodes a Novel Yeast Protein Phosphatase ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 2, Issue of January 15. pp. 1349-1354,1993 Printed in U.S.A.

The Gene PPG Encodes a Novel Yeast Protein Phosphatase Involved in Glycogen Accumulation*

(Received for publication, May 15, 1992)

Francesc PosasS, Josep ClotetQ, M. Teresa Muns, Josep Corominas, Antonio Casamayor, and Joaquin Arinoll From the DeDartament de Bioauimica i Biologia Molecular, Facultat de Veterimiria, Uniuersitat Autonoma de Barcelona, Bellaterra, Barcelona E-08193,’Spain

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Degenerate oligonucleotides were used to selectively amplify yeast genomic sequences related to Ser/Thr protein phosphatases. Among the sequences obtained, clone ST4-2 was found to code for a novel sequence related to previously known phosphatases. A size-selected yeast genomic library was constructed and screened using clone ST4-2 as probe, and one positive clone, named PPG, was isolated. DNA sequencing of a 1.8-kilobase pair fragment of this clone revealed an open reading frame of 1104 base pairs which codes for a 368-amino acid protein. On the basis of its amino acid sequence, the product of gene PPG would be an acidic protein, structurally more related to type 2A than to type 1 or 2B phosphatases, and is characterized by an extension of about 50 amino acids at the carboxyl terminus. The gene, which is located in chromosome XIV, is expressed as a 1.3-kilobase mRNA and is not essential for growth. Haploid mutants carrying a disrupted copy of the gene were able to grow in glucose as well as in other carbon sources, but they accumu- lated less glycogen than the wild type strain. However, the state of activation of glycogen synthase was essentially identical in wild type and mutant cells. The finding that, in early exponential phase, mutant cells contain higher levels of glycogen phosphorylase a, in addition to a lower amount of total glycogen synthase activity observed in medium-late exponential phase, could account for the difference found in glycogen accumulation.

One of the most relevant mechanisms for the control of cell functions is the reversible phosphorylation of proteins. Pro- tein phosphorylation regulates a number of critical cell proc- esses, including transcription and translation, cell metabo-

* This work was supported by Grant PB89/0313 from the Comision Interministerial de Ciencia y Tecnologia (to J. A.) and by an ‘‘Accibn Integrada Hispano-Alemana” (to J. A and F. K. Z). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

M94269. to the GenBankTM/EMBL Data Bank with accession number(s)

j: Recipient of a Formacion Personal Investigador Fellowship from the Ministry of Education (Spain).

Recipient of a fellowship from the Conselleria d’Ensenyament de la Generalitat de Catalunya.

ll To whom all correspondence should be addressed: Dept. de Bio- quimica i Biologia Molecular, Facultat de Veterinaria, Universitat

34-3-5812182; Fax: 34-3-5812006. Autbnoma de Barcelona, Bellaterra, Barcelona E-08193, Spain. Tel.:

lism, response to external stimuli, and entry into and pro- gression through meiosis. In yeast, more than 30 protein kinases have been identified by genetic and biochemical methods (see Refs. 1 and 2 for a recent review) and are now known to play different roles in the yeast biology.

Protein phosphatases reverse the reaction catalyzed by protein kinases. Ser/Thr protein phosphatases were initially characterized in mammalian tissues and, mostly after the work of Cohen and co-workers (3 ,4) , they were classified into type 1, 2A, 2B, and 2C phosphatases on the basis of their substrate specificity and sensitivity to cations and inhibitory proteins. Molecular cloning of cDNAs encoding the catalytic subunit of the different types of phosphatases revealed that types 1, 2A, and 2B are structurally related ( 5 ) . Yeast genes encoding proteins homologous to the type 1 (6-8), 2A (9-12), and 2B (13, 14) phosphatases have been cloned and found to be highly conserved when compared to their mammalian counterparts. In addition to those phosphatases, other related gene products do exists in yeast. For instance, the gene SIT4 (15) codes for a protein closer to type 2A than to type 1 phosphatases, as it is the case for the recently reported PPH3 gene (12).

The molecular cloning of several cDNAs from mammalian and Drosophila species reveals than Ser/Thr phosphatases could be quite a large family (16), perhaps approaching in complexity to the protein kinase family. To test this hypothesis, we carried out amplification experiments using degenerate oligonucleotides and genomic yeast DNA. We found, in addition to known phosphatase genes, two novel amplification sequences. The genes containing those sequences have been cloned and characterized. One of them corresponds to a phosphatase, named PPZl, which contains a large and unusual amino-terminal extension (17). The second one, named PPG, which is described in the present paper, has been found to be involved in glycogen accumulation.

EXPERIMENTAL PROCEDURES

Materials-Oligonucleotides MP-1s and MP-3as were synthesized on an Applied Biosystems synthesizer and were a generous gift of Dr. Gary L. Johnson (National Jewish Center, Denver, CO). The sequence of oligonucleotide MP-1s was GGGAATTCGA(CT)(CAT) T(GC)(CT)T(AGC)TGGTC(ATG)GA(TC)CCas CCAAGCTT (CT)(CA)(GA)CA(GA)TA(GA)TT(GT)GG(CAGT)GC w a m quence of MP-Bas (underlined sequences correspond to EcoRI and Hind111 restriction sites added to facilitate cloning). Those sequences are separated approximately 200-220 bp’ in type 1, 2A, and 2B phosphatases. Thermus aquaticus DNA polymerase (Taq polymerase) was obtained from Perkin-Elmer Cetus or Boehringer Mannheim. a- Factor came from Sigma. Restriction enzymes and the nonradioactive DNA detection system were purchased from Boehringer Mannheim.

Strains and Media-Saccharomyces cereuisiae strain M5 (MATa/

The abbreviations used are: bp, base pair(s); kbp, kilobase pair(s).

1349

1350 Novel Yeast Phosphatase Involved in Glycogen Accumulation

MATa, homozygous for leu2-3/112 urd-52 trpl) was used for genomic DNA preparation and gene disruption experiments. The haploid strain W303-1A (MATa leu2, 3-112 trpl-1 u r d - 1 his3-11, 15) was also used for gene disruption experiments and glycogen metabolism studies. Yeast cells were grown at 30 "C in YPD medium or in SD synthetic medium (18). Escherichia colicells were grown at 37 "C in LB medium containing 50 pg/ml ampicillin, when needed, for plasmid selection.

Recombinant DNA and Standard Genetic Methods-Bacterial cells were transformed as previously described (19) or by electrophoration. Yeast cells were transformed using the lithium acetate method (20). DNA probes were labeled by the random priming method using either [32P]dCTP or digoxigenin-labeled dUTP. Restriction reactions, DNA dephosphorylation and ligation, and other standard recombinant DNA techniques were performed essentially as described elsewhere (21). Tetrad analysis and scoring of markers were performed by standard methods (18).

Nucleotide Sequencing and Sequence Analysis-Nucleotide sequencing of the cloned DNA was performed using the dideoxynucle- otide chain termination method (22) and fluorescent primers in an Applied Biosystems 373A automatic DNA sequencer. Single-stranded M13 phage was used for subcloning. All the DNA sequences presented were determined by sequencing both strands. Nucleotide sequence manipulations were performed using the PCGENE package from Intelligenetics.

Genomic DNA Amplification by PCR-Yeast genomic DNA was prepared as previously described (18). In essence, 0.2 pg of DNA was amplified exactly as described by Posas et al. (17). After 30 cycles, the mixture was extracted with phenol/chloroform, ethanol-precipi- tated, and digested with a mixture of EcoRI and HindIII. The sample was loaded on a 1.5% agarose gel, and amplification fragments of the expected size (about 250 bp) were excised and purified using the GeneClean system (Bio 101). The purified DNA was ligated with either EcoRI-HindIII-digested M13mp18 or M13mp19 vectors and sequenced as described above.

Southern Blot and Chromosomal Analysis-For Southern blot experiments genomic DNA was prepared as above. Five to 10 pg of DNA were digested with different restriction enzymes, electrophoresed on 0.7% agarose gels, and transferred under vacuum to nylon membranes. Hybridization was performed at 65 "C in 5 X SSC, 0.1% SDS (1 X SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), and filters were washed at 55 "C in 0.1 X SSC, 0.1% SDS unless otherwise stated. DNA was cross-linked to the membranes using a UV Stratalinker (Stratagene), and detection of the hybridizing fragments was accomplished using the digoxigenin-based DNA detection system provided by Boehringer Mannheim, with minor modifications or, in some cases, using 32P-labeled DNA probes. Chromosomal assignment by pulse field gel electrophoresis was performed as previously described (17).

Northern Blot Analysis-Total yeast RNA was prepared as described by Sherman et al. (18), and poly(A)-selected RNA was obtained by oligo(dT)-cellulose chromatography. RNA samples were electrophoresed on 0.7% agarose-formaldehyde gels and transferred under vacuum to nylon membranes. DNA probes were 32P-labeled. Hybridization was carried out at 42 "C in 5 X SSC and in the presence of 50% formamide. Washing was carried out at 55 "C in 0.1 X SSC, 0.1% SDS.

Construction and Screening of Size-selected Yeast Genomic DNA Library-Genomic DNA (25 pg) was digested with PstI and electrophoresed on 0.7% agarose gels. The region of the gel containing DNA fragments of about 10 kbp was excised, and DNA was recovered from the gel, ligated into pUC19 plasmid previously digested with PstI, and dephosphorylated. Transformation into E. coli by electrophoration yielded about 1200 clones. The library was plated and colonies transferred to nylon membranes (Colony/Plaque ScreenTM, Du Pont). Membranes were soaked for 7 min in 1.5 M sodium chloride, 0.5 M sodium hydroxide, neutralized for 6 min in 1.5 M sodium chloride, 1 M Tris-HCI, pH 7.2,l mM EDTA, and briefly soaked in 95% ethanol and, finally, in pure chloroform. After a 2 X SSC wash, the membranes were baked at 80 "C for 2 h. Prehybridization was performed for 3 h as described by the manufacturer of the labeling and detection kit (Boehringer Mannheim). Filters were then incubated overnight at 65 'C with 2.5 ml/filter of the same solution containing 5-10 ng/ml of a random-primed digoxigenin-labeled ST4-2 fragment and finally washed at 55 'C in 0.1 X SSC, 0.1% SDS. Subsequent procedures were performed as described by the manufacturers. Positive clones were identified using the chromogenic substrates nitroblue tetrazo- lium salt and 5-bromo-4-chloro-3-indolyl phosphate.

Gene Dkruption Methods-Gene disruption experiments were carried out by the one-step gene disruption method (23). For such purpose a ppg::TRPl construct was created by removing an approximately 0.89-kbp BglII-BglII fragment corresponding to amino acid 51-351 from a PstI-XhoI fragment of about 5.6 kbp in length cloned into Bluescript and containing the entire coding region of the gene (pPPG-F). In parallel, plasmid YRp7 (18) was digested with EcoRI and the 1.45-kbp fragment containing the TRPl gene was cloned into Bluescript SK(+). The resulting plasmid was cleaved with BglII and BamHI. Then, the 0.85-kbp fragment (which carries the TRPl coding region but lacks the ARS sequences) was gel-purified and cloned into the BglII sites of pPPG-F, yielding plasmid pMM1. Plasmid pMMl was digested with ClaI and Sac1 (a site present in the poly-linker of the vector), and the -3.0-kbp insert obtained was purified and used to transform diploid M5 and haploid W303-1A cells.

Determination of Glycogen Phosphorylase and Glycogen Synthase- Glycogen synthase and phosphorylase a activities were measured in yeast extracts as described in a previous report (24). Total glycogen synthase activity was measured in the presence of 6.6 mM glucose 6- phosphate in the assay mixture. All assays were performed at 30 "C. One unit is defined as the amount of enzyme that catalyzes the incorporation of 1 pmol of ['*C]glucose into glycogen per min under the assay conditions.

Immunological identification of glycogen synthase in yeast extracts was carried out by Western blot techniques. Yeast extracts (about 200 pgllane) were electrophoresed on 10% polyacrylamide gels and transferred to membranes (Immobilon-P, Millipore). After blocking, filters were incubated overnight with a polyclonal antibody raised against purified yeast glycogen synthase (25) and then with lz5I- protein G (Amersham). Autoradiograms were obtained, and the amount of glycogen synthase was determined by scanning of the films.

Analytical Procedures-Qualitative determination of accumulation of glycogen was estimated by inverting plates containing patches of cells over iodine crystals. Quantitative determination of glycogen and glucose 6-phosphate was performed in acid extracts as previously described (26).

Protein was measured in cell extracts using the Biuret method (27). Glucose levels in the culture medium were measured, after filtration of the cells, using a Gluco-quant kit from Boehringer Mannheim adapted for a Cobas Bio-autoanalyser (Roche, Basel). Sensitivity to a-factor and heat-shock sensitivity was tested essentially as described in Sneddon et al. (IO).

RESULTS

Amplification of PPG Sequences from Yeast Genome-Yeast genomic DNA from strain M5 was amplified as described under "Experimental Procedures." Electrophoretic analysis of the different amplification products revealed a DNA band of about 250 bp in length, that is, slightly larger than the products expected for the previously known yeast phosphatase genes. However, this DNA fragment hybridized at low strin- gency with SIT4 and DISBSl probes, thus suggesting a rela- tionship with these yeast phosphatases. The amplification fragment, which was named ST4-2, was cloned into M13 phage and sequenced. Sequencing of clone ST4-2 revealed that it encoded an amino acid sequence clearly related to Ser/ Thr phosphatases. The ST4-2 fragment was used as probe in Northern blot experiments and found to hybridize with a 1.3- kbp message. Pulse field gel electrophoresis analysis of yeast chromosomes, followed by Southern blot analysis, indicated that the gene containing the ST4-2 sequence was located in chromosome XIV (Fig. 1, B and C ) .

Molecular Cloning and Sequencing of Gene PPG-In order to clone the gene containing the nucleotide sequence corresponding to fragment ST4-2, genomic yeast DNA was digested with different restriction enzymes and probed with the ST4- 2 clone (Fig. U). We found that the probe hybridized with a PstI-PstI fragment of about 10 kbp, and therefore we constructed a size-selected PstI-PstI genomic library. Probe ST4- 2 was used to screen the library, and one positive clone containing an insert of about 10.5 kbp was isolated. The

Novel Yeast Phosphatase Involved in Glycogen Accumulation 1351

A B C

FIG. 1. Southern blot analysis, Northern blot analysis, and chromosomal assignment of gene PPG. A, genomic yeast DNA (5-10 pgllane) was digested with different restriction enzymes and electrophoresed in a 0.7% agarose gel. DNA was transferred to nylon membranes, probed with a digoxigenin-labeled ST4-2 fragment, and washed at 60 "C in 0.1 X SSC, 0.1% SDS. Numbers on the left indicate the migration of standard DNA size markers and correspond to kbp. H , HindIII; Xb, XbaI; R, EcoRI; P , PstI; Bg, BglII. B, 10 pg of poly(A)- selected RNA from strain M5 were electrophoresed and transferred t o membranes. RNA was probed with a 32P-labeled 1.2-kbp HindIII- Hind111 fragment, and membranes were washed a t 55 'C in 0.1 X SSC and exposed for 24 h. Arrows indicate the migration of ribosomal 25 S and 18 S bands. C, yeast chromosomes were resolved by pulse field gel electrophoresis, transferred to membranes, and probed with a 32P-labeled ST4-2 fragment. Filters were washed in 0.1 X SSC, 0.1% SDS at 65 "C. Arrows on the left indicate the migration of chromosomes running in the vicinity of chromosome XIV, as observed by ethidium bromide staining.

- 1 kbp

FIG. 2. Restriction map of the PPG region. The restriction map of the PstI-XhoI fragment is depicted. The open reading frame corresponding to the PPG gene is shown as an arrow.

portion of the DNA fragment that hybridized to probe ST4-2 was found to be located at the 5' end of a 5.6-kbp PstI-XhoI fragment, which was cloned into Bluescript (pPPG-F) and subjected to restriction mapping (Fig. 2).

Nucleotide sequencing of approximately 1.8 kbp corresponding to the 5' end of pPPG-F revealed an open reading frame of 1104 bp which contained the sequence found in ST4- 2. As shown in Fig. 3A, this open reading frame codes for a 368-amino acid protein, with an estimated molecular mass of 42,416 daltons. A TATA-like motif (TAATAA) is found at position -171. The encoded protein is rather acidic (PI = 4.591, and it is clearly related to other yeast protein phosphatases. The predicted PPG protein can be aligned from the very beginning of the PPH3 and SIT4/PPH1 proteins, and from amino acid 68 and 76, respectively, of the PPH2l and PPH22 proteins. The level of identity found between PPG and the products of genes SIT4IPPH1, PPH21, PPH22, and PPH3 is rather similar, that is, 52-53%, when a large fragment of the PPG protein is used for comparison (amino acids 16- 292) and the internal extra amino acids present in PPG (residues 205-215) are not considered. The PPG protein is also rather close to the human PP2A (56% identity), although the highest level of identity (60%) is found when PPG is compared with PPX, a putative protein phosphatase deduced from a rabbit cDNA. When compared with those proteins, PPG has a carboxyl-terminal extension of almost 50 amino acids. This extension shows no significant identity with the

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FIG. 3. Nucleotide sequence and sequencing strategy of gene PPG. A, nucleotide sequence and predicted amino acid sequence of gene PPG. The stop codon is denoted by an asterisk. The sequence amplified by polymerase chain reaction (clone ST4-2) is underlined, and the extra amino acids found when compared with other type PA-related phosphatases are shadowed. B, restriction map and nucleotide sequencing strategy for gene PPG. The dark bar indicates the 1104-bp open reading frame. Nucleotide sequence was obtained by subcloning the appropriate restriction fragments in M13mp18 or M13mp19 vectors. Restriction sites relevant for subcloning are shown.

carboxyl-terminal extension of the mammalian or yeast type 2B phosphatases. However, the protein ends with the sequence. . . DYFL (stop) which has been found to be a common motif for the last four amino acids in any known PPZA or PP2A-related phosphatase. The PPG protein is less related to the yeast type 1 phosphatases DIS2S1 (about 39%) or to its counterparts in insects or mammals and to PPZl, a recently cloned yeast type 1 phosphatase (about 37%). Also, the protein is only distantly related to a type 2B yeast phosphatase (35% of identity to the product of gene CMPl on a 288- amino acid overlap).

PPG Is Not Essential for Cell Growth-To investigate the function of PPG in yeast cells we performed one-step gene disruption experiments. Diploid cells were transformed with a linear DNA fragment (Fig. 4) where most of the PPG-coding region had been removed and replaced by a functional copy of the TRPI gene. Stable tp+ cells were selected and shown by Southern analysis to carry a disrupted allele of PPG (Fig. 4). When cells were induced to sporulate the four spores were able to grow in all cases, indicating that the PPG protein was not essential for cell survival. Mutant spores were able to grow in glucose as well as in various carbon sources (glycerol, ethanol, and lactate). Because some heterogeneity in the genetic background of strain M5, gene disruption was also performed in the haploid W303-1A background. Under these conditions, ppg- cells did not show significant differences from wild type cells when tested for doubling time, rate of


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FIG. 4. Gene disruption of PPG. A 0.89-kbp BglII-BglII fragment containing most of the coding region of gene PPG was removed and replaced by S. cerevisiae gene TRPl (pane l A ) . W303-1A haploid cells were transformed and stable trp+ cells selected. Genomic DNA was prepared from wild type (panel B, lane I ) or trp+ colonies (panel B, lone 2), digested with XbaI, and transferred to membranes. A 1.2- kbp HidII-Hid11 fragment was used as probe. The disruption of the gene is confirmed by presence of a 1.8-kbp signal as a result of the XbaI site found in the TRPl gene and by the absence of the wild type 3.5-kbp band.

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FIG. 5. Accumulation of glycogen inppg cells. A, iodine staining of wild type and ppg- yeast cells. I and 2, ppg- and wild type W303-1A strain; 3 and 4, ppg- and wild type M5 strain. B, the effect of disruption of the gene PPG in glycogen accumulation. Haploid wild type W303-1A (W) and the isogenic ppg- strain (0- - - - -0) were grown in YPD at 30 "C. Cells were collected during the culture and glycogen content measured as described under "Experimental Procedures."

glucose consumption, accumulation of glucose 6-phosphate, heat-shock sensitivity, or a-factor sensitivity.

Disruption of Gene PPG Results in Decrease in Glycogen Accumulation-Iodine staining indicated that ppg- cells did accumulate less glycogen than wild type cells (Fig. 5A). This effect was clearly reversed when mutant cells were transformed with a multicopy plasmid containing the 1.8-kbp PstI- H i d 1 1 fragment depicted in Fig. 3B ( d a t a not shown). To measure this effect, wild type W303-1A cells and the same strain carrying a disruption in the PPG gene were grown in rich medium, and their content in glycogen was measured (Fig. 5B). As expected, mutant cells were found to accumulate less glycogen than wild type cells. The difference was specially marked at medium-late exponential phase.

In order to understand the reason for such difference, the active form of glycogen synthase, as well as the total amount of the enzyme activity (that is, the activity determined in the presence of exogenous glucose 6-phosphate) was measured (Table I). Surprisingly, we did not detect any significant difference in the state of activation of glycogen synthase when mutant and wild type cells were compared. Interestingly enough, the amount of total glycogen synthase activity in mutant cells, although not -differing from wild type cells at early exponential or stationary phase, was significantly lower (about 30%) at medium-late exponential phase (that is, during

TABLE I Glycogen synthase activity in wild type and ppg- cells

Wild type W303-1A cells and an isogenic strain carrying a dismp- tion of gene PPG were grown in YPD medium and collected at medium-late exponential phase. Glycogen synthase activity was measured in the absence (active form or synthase I) or the presence (total activity) of exogenous glucose 6-phosphate (G-6-P). The state of activation of glycogen synthase is expressed as the ratio between the active form and the total enzyme. Data is presented as mean f S.E. of fourteen indeDendent determinations.

Glycogen Total glycogen synthase synthase I synthase activity (-G-6-P,+G-6-P,

activity ratio . . ~~ ,~ ,

milliunits/mg protein PPG 8.1 f 0.4 10.1 f 0.6 0.78 f 0.02 DD~::TRPI 5.8 f 0.2" 7.3 f 0.2" 0.79 k 0.02

a The difference between the means is significant at the 0.05 level.

Syntbmme Immunoreacilve activity C. synthase

FIG. 6. Immunodetection of glycogen synthase in wild type and ppg cells. Extracts containing the same amount of protein (about 200 gg) were electrophoresed, transferred to nylon membranes, and incubated with antibodies against yeast synthase. Wild type (panel B, lanes I and 3) and ppg cells (lanes 2 and 4 ) were collected from early exponential cultures (lanes I and 2) or medium-late exponential cultures (lanes 3 and 4) . Lane 5 correspond to a preparation of purified glycogen synthase run in parallel. In panel A the total amount of synthase activity and the immunoreactive protein in mutant cells are presented as percentage of the corresponding values determined in wild type cells. Data correspond to the mean f S.E. of five independent experiments.

the period when major differences in glycogen content were detected). This finding was confirmed by quantifying of glycogen synthase by immunoblot experiments. For this purpose, wild type and ppg cells were collected at both early and medium-late exponential phase. Extracts were prepared, electrophoresed, and transferred to membranes. A polyclonal antisera raised in rabbits against purified yeast glycogen synthase was used to detect the amount of the enzyme in wild type and mutant cells. As observed in Fig. 6, A and B, the antibody recognized a 78-79-kDa protein which showed identical electrophoretic mobility that purified yeast glycogen synthase. Medium-late exponential phase ppg cells contained less immunoreactive glycogen synthase than wild type cells. The decrease in immunoreactive protein was quantitatively similar to that observed by measuring the total enzyme activity (Fig. 6A). Interestingly, early exponential cells were found to contain less glycogen synthase than medium-late exponential cells. The antibody did also recognize a slower migrating protein (about 85 kDa) which was essentially unaffected in ppg cells.

When the active form of glycogen phosphorylase was measured through the culture it was found that, at early exponential phase, the amount of active phosphorylase in mutant cells

Novel Yeast Phosphatase Involved in Glycogen Accumulation 1353

almost doubled the activity found in wild type cells (11.6 k 0.1 versus 6.8 2 0.5 milliunits/mgprotein, respectively). Phos- phorylase a activity was found to peak just before the onset of the stationary phase (about 50 milliunits/mg protein), and no significant differences were found in medium-late exponential or saturated cultures between wild type and ppg cells (data not shown).

DISCUSSION

The use of degenerate oligonucleotides encoding conserved amino acid sequences, which are characteristics of a given family of proteins, to specifically prime DNA extension has become in the last few years a very useful tool to uncover novel members of such families. In our laboratory, two novel members of the yeast Ser/Thr protein phosphatases have been recently found. The aim of this work has been to study one of these members, which we have named PPG. The gene PPG contains an open reading frame encoding an 368-amino acid protein and its amino acid sequence reveals that PPG can be considered a member of the type PA-like phosphatase family, rather than a type 1- or 2B-related protein.

In S. cerevisiue, four genes encoding proteins structurally related to type 2A phosphatases have been identified so far. Two of them, encoded by genes PPH21 and PPH22, appear to be the homologous forms of the mammalian type 2A phosphatases. Disruption of PPH21 or PPH22 alone has no effect, but PP2A-deficient cells present severe growth defects or are inviable (10, 12). The remaining two phosphatases can be considered as members of a different evolutionary branch and should better be considered as PP2A-like proteins. These proteins are encoded by genes PPHlISIT4 (15) and PPH3 (12). The SIT4 gene product performs an important function in the GI phase of the cell cycle ( l l ) , and mutations in SIT4 are lethal in certain genetic backgrounds. The PPH3 gene has been shown to provide some PP2A-complementing activity since, although disruption of PPH3 alone has no appreciable phenotypic effect, it is lethal in a pp2u background (12).

The PPG protein displays virtually the same degree of identity when compared with the PPH21, PPH22, PPHlI SIT4, or PPH3 gene products (however, it is not located in chromosome IV, as it is the case for those four genes, but in chromosome XIV). Therefore, the gene PPG represents the fifth member of the type 2A-related genes found in yeast. Interestingly enough, because of its carboxyl-terminal extension, the PPG protein also represents a novel type of yeast phosphatase, as depicted in Fig. 7. From this figure, it is evident that the structure of some S. cerevisiae phosphatases, as it is the case of the SIT4, PPH3, and DIS2Sl proteins, can - DIX2.91

u PPC

m4, PPH3,

Add ua r PPHz1, PPMZ - CNA2IChfF2 CNAIICMPI.

C!S.&&lBU

PPZI

I I I I I I ,

-400 -so0 -am -100 1 100 100 300 4m 500 600

FIG. 7. The different forms of yeast protein phosphatases. The known yeast phosphatases have been aligned to show their different structures. The black boxes denote the region containing the sequences which are common to type 1, 2A, and 2B phosphatases. Other relevant characteristics are also marked. Amino acid I is referred to the initial amino acid in the DIS2S1 protein. Negative values are assigned to amino-terminal extensions.

be considered as representative of a “catalytic core.” They are about 300 amino acids long, and their sequences can be aligned with the corresponding type 1 and 2A phosphatases from mammals, insects, or plants. However, PPH21 and PPH22, the actual type 2A phosphatases in budding yeast, contain additional, rather acidic sequences at their amino terminus (10, 12) and, therefore, represent a different kind of structure (it is noteworthy that the type 2A protein in the fission yeast Schizosaccharomyces pombe is similar in size and structure to the higher eukaryotic forms (9)).

Another example of yeast phosphatase containing an amino-terminal extension, but in this case about 350 amino acids in length, is the product of the recently reported gene PPZl (17). The catalytic region of this protein is more related to the type 1 phosphatases, whereas the amino-terminal extension is characterized by an abundance of Ser and Thr, as well as of basic residues. No related protein has been reported so far in other eukaryotic systems.

Two genes encoding type 2B phosphatases have been cloned recently in yeast (13,14). The proteins have the characteristic long (about 250-300 residues) carboxyl-terminal tail including a region able to interact with calmodulin. In this case, the structure of the proteins is very similar to their mammalian counterparts. Finally, the PPG protein described in this work shows a comparatively short (and apparently unrelated to type 2B proteins) carboxyl-terminal extension. However, as discussed above, PPG should be considered a member of the 2A-like family. With the exception of the type 2B proteins, the possible role of these extensions is not known.

Disruption of gene PPG reveals that the gene product is dispensable for survival and growth. Among the phenotypic traits checked in our laboratory in ppg- disruption mutants, only differences in the ability to accumulate glycogen were found. Initially, this was not an unexpected finding, since it has been known for many years that in yeast, as well as in higher cells, glycogen metabolism can be controlled by phosphorylation-dephosphorylation reactions (28, 29). It is ac- cepted that the phosphorylated forms of glycogen synthase would be inactive, whereas phosphorylation of phosphorylase would activate the enzyme. Evidence supporting the role of yeast phosphatases in the control of synthase activity and phosphorylation state was reported some time ago (30, 31) and has been reinforced recently. For instance, the glc7 mutant, a glycogen-deficient strain, was found to contain a glycogen synthase markedly inactivated, suggesting that the enzyme was present mostly in the phosphorylated forms. This mutant had a defect in the GLC7 gene (32) and, upon sequencing, the GLC7 gene was found to be identical to DIS2S1, the budding yeast type 1 phosphatase (8). Therefore, yeast type 1 phosphatase seems to be involved in the regulation of glycogen accumulation by controlling the state of activation of glycogen synthase. This idea is also supported by the recent discovery that disruption of the GACl gene, a putative regulatory subunit of the yeast type 1 phosphatase, also results in inactivation of glycogen synthase and failure to accumulate glycogen (33). On the other hand, phosphatase activity reminiscent in its enzymologic properties to the mammalian type 2A enzyme has been purified from yeast and found to dephosphorylate and activate yeast glycogen synthase “in vitro” (34). This suggests that yeast type 2A phosphatase could be also involved in the control of accumulation of glycogen through the dephosphorylation and activation of glycogen synthase. In this regard, experiments performed in our laboratory using PP2A- deficient mutants also point to a role of type 2A phosphatase in synthase activation and glycogen accumulation?

F. Posas, J. Clotet, and J. Ariiio, unpublished observations.


However, disruption of PPG, although affecting glycogen accumulation, did not result in inactivation of synthase. This effect on glycogen accumulation can be explained by the observed decrease in the amount of total synthase activity (in addition to some increase in the active form of phosphorylase found at early exponential phase). Therefore, a defect in PPG would affect glycogen metabolism not by inactivating synthase but by decreasing the amount of the catalytic protein. Recently, it has become evident that two isoforms of glycogen synthase exist in budding yeast (35, 36). The predominant isoform is encoded by gene GSY2 and migrates as a 77"kDa band in SDS-polyacrylamide gel electrophoresis. The GSY2 gene product accounts for up to 85% of total synthase activity and seems to be responsible for the accumulation of glycogen linked to nutrient depletion. The second isoform, which migrates as a 85-kDa protein, is encoded by the GSYl gene. Since disruption of gene PPG results in a decrease in the amount of both glycogen and total glycogen synthase activity, one might expect that the major isoform of synthase, the GSYP protein, would be affected in ppg cells. This hypothesis is in agreement with the decrease in the 79-kDa protein that we have observed. In addition, the fact that this protein is found in lower amounts in early exponential cultures provides further evidence regarding its identity with the GSY2 protein, which has been found to increase prior the onset of the stationary phase (36). Since the GSYl and GSY2 proteins are 80% identical, it is most likely that the 85-kDa protein recognized by our antibody might correspond to the GSYl protein, which has been shown to migrate as a 85-kDa band.

The possibility that, in yeast, glycogen metabolism could be controlled at two levels, by changes in the activity of synthase and phosphorylase through phospho-dephosphorylation reactions and by coordinate changes in the amount of the enzymes involved in the metabolism of the polysaccharide, is rather attractive. In fact, it is known that, in addition to the GSY2, the gene encoding yeast phosphorylase (GPHI) (37), DIS2SlIGLC7 (€9, and its putative regulatory subunit (33), as well as the glycogen branching enzyme (38) are induced at the time that glycogen begins to accumulate. Therefore, it could be the case that the PPG protein could be involved in the control of glycogen accumulation through the control of the amount of glycogen synthase. Whether or not the PPG protein is involved in the expression of other proteins remains to be determined.

Acknowledgments-We thank Dr. A. Sanchez for critical reading of the manuscript, Dr. C. Gancedo for yeast strains and technical advice, and B. Wadzinsky and G. L. Johnson for primers MP-1s and

MP3-as. We acknowledge the help of Dr. F. K. Zimmermann in the earliest steps of gene cloning and also A. Vilalta and M. Camps for skillful technical help.

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The Gene PPG Encodes a Novel Yeast Protein Phosphatase ...

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