A series of protein phosphatase gene disruptants inSaccharomyces cerevisiae

Yeast 15, 1669–1679 (1999)

A Series of Protein Phosphatase Gene Disruptants inSaccharomyces cerevisiae

NAOKO SAKUMOTO†, YUKIO MUKAI†, KOUJI UCHIDA, TOMOKO KOUCHI,JYOH KUWAJIMA, YOUJI NAKAGAWA, SHIGEMI SUGIOKA, EISHI YAMAMOTO,TOMOMI FURUYAMA, HIROYUKI MIZUBUCHI, NAOSHI OHSUGI, TAKESHI SAKUNO,KOUJI KIKUCHI, ITSUMI MATSUOKA, NOBUO OGAWA, YOSHINOBU KANEKO ANDSATOSHI HARASHIMA*

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita,Osaka 565-0871, Japan

Thirty-two protein phosphatase (PPase) genes were identified in the genome nucleotide sequence of Saccharomycescerevisiae. We constructed S. cerevisiae disruptants for each of the PPase genes and examined their growth undervarious conditions. The disruptants of six putative PPase genes, i.e. of YBR125c, YCR079w, YIL113w, YJR110w,YNR022c and YOR090c, were created for the first time in this study. The glc7, sit4 and cdc14 disruptants were lethalin our strain background. The remaining 29 PPase gene disruptants were viable at 30�C and 37�C, but only onedisruptant, yvh1, showed intrinsic cold-sensitive growth at 13�C. Transcription of the YVH1 gene was induced at13�C, consistent with an idea that Yvh1p has a specific role for growth at a low temperature. The viable disruptantsgrew normally on nutrient medium containing sucrose, galactose, maltose or glycerol as carbon sources. The ppz1disruptant was tolerant to NaCl and LiCl, while the cmp2 disruptant was sensitive to these salts, as reportedpreviously, and none of the other viable PPase disruptants exhibited the salt sensitivity. When the viable disruptantswere tested for sensitivity to drugs, i.e. benomyl, caffeine and hydroxyurea, ppz1 and ycr079w disruptants exhibitedsensitivity to caffeine. Copyright � 1999 John Wiley & Sons, Ltd.

— protein phosphatase; disruption mutation; YVH1; cold-sensitive growth; YCR079w; caffeinesensitivity; Sacharomyces cerevisiae

*Correspondence to: S. Harashima, Department of Biotech-nology, Graduate School of Engineering, Osaka University,2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-7420; fax: +81-6-6879-7421. e-mail: [email protected]†The first and the second authors contributed equally to thiswork.Contract/grant sponsor: Ministry of Education, Science,Sports and Culture, Japan; Contract/grant number: 08250210,

INTRODUCTION

Reversible phosphorylation of proteins is the keyregulator of a variety of cellular processes, includ-ing cell signaling, gene expression and mitosis

10168215.

CCC 0749–503X/99/151669–11$17.50Copyright � 1999 John Wiley & Sons, Ltd.

(Cohen, 1989; Stark, 1996). The protein kinasescatalyze the phosphorylation of the proteins andthe protein phosphatases (PPases) catalyze thedephosphorylation of proteins phosphorylated byprotein kinases in this regulation.

PPases were initially classified into six sub-families, PP1, PP2A, PP2B, PP2C, PTP and dual-specificity phosphatase. The PP1, PP2A, PP2B andPP2C subfamilies dephosphorylate serine andthreonine residues and are distinguished by theirsubstrate preference, metal cation requirement andsensitivity to inhibitors (Cohen, 1989). PP1, PP2A,PP2B and a number of other protein phosphatases
are more highly conserved in sequence than PP2C;
Received 5 March 1999Accepted 24 June 1999

1670 N. SAKUMOTO ET AL.

these are referred to as the PPP family, followingthe nomenclature for the human protein phos-phatases. The protein tyrosine phosphatases(PTPs) which dephosphorylate tyrosine residues,and dual-specificity phosphatases which dephos-phorylate serine and tyrosine residues, compriseother subfamilies (Walton and Dixon, 1993).

Completion of the budding yeast Saccharomycescerevisiae genome sequencing project has made itpossible to determine the number of genes of aparticular type (Goffeau et al., 1996). Conse-quently, 32 PPase genes were listed in the yeastproteome database (http://www.proteome.com;Table 1) among approximately 6000 genes en-coded in the genome. It is much less certain thatYNR022c is a protein phosphatase, since theregions of similarity to protein tyrosine phos-phatases do not include the active site, and theregion showing superficial resemblance to theactive site of tyrosine phosphatase lacks the keyconserved arginine at +6 relative to the catalyticcysteine. The function of 26 PPase genes is knownor expected, while that of the remaining six PPasegenes, YBR125c, YCR079w, YIL113w, YJR110w,YNR022c and YOR090c, is unknown.

Some PPases in S. cerevisiae are known todephosphorylate different substrates involved in adiverse cellular processes in combination with thespecific regulatory subunits. For example, Glc7p(PP1) is involved in cell cycle progression in com-bination with Egp1p, glycogen accumulation withGac1p, glucose repression with Reg1p and sporu-lation with Gip1p and Red1p (MacKelvie et al.,1995; Hisamoto et al., 1995; Ramaswamy et al.,1998; Tu and Carlson, 1995). Involvement of eachPPase in apparently unrelated biological processescould be unveiled by investigation of the variousphenotypes that each of PPase disruptantsdisplays.

In an effort to identify the involvement of eachPPase in various biological processes and under-stand their role in the cellular physiology ofyeasts, we have constructed a series of disruptionmutants for each of the PPase genes, includingsix of unknown function. The glc7, sit4 and cdc14disruptants were lethal in our strain background,while others were viable at 30�C. Disruption ofYVH1 resulted in cold sensitivity for growth.Only two disruptants, ppz1 and cmp2 among allviable 29 disruptants, were tolerant and sensitiveto NaCl and LiCl, respectively. The ppz1 andycr079w disruptants exhibited sensitivity tocaffeine.

Copyright � 1999 John Wiley & Sons, Ltd.

MATERIALS AND METHODS

Strains, plasmids and mediaThe S. cerevisiae wild-type strains used in this

study were HYP100 (MATa ura3-52 leu2-3, 112trp1� his3� ade2-101 lys2-801) (Mukai et al.,1993), its isogenic diploid HYP101d (MATa/MAT� ura3-52/ura3-52 leu2-3,112/leu2-3,112trp1�/trp1� his3�/his3� ade2-101/ade2-101 lys2-801/lys2-801), W303-1A (MATa ura3-1 leu2-3,112trp1-1 his3-11,15 ade2-1), W303-1B (MAT� ura3-1leu2-3,112 trp1-1 his3-11,15 ade2-1) and its iso-genic diploid W303 (MATa/MAT� ura3-1/ura3-1leu2-3,112/leu2-3,112 trp1-1/trp1-1 his3-11,15/his3-11,15 ade2-1/ade2-1) (Thomas and Rothstein,1989). Plasmid pCgHIS3 (Figure 1) was con-structed by the insertion of a 1·78 kb EcoRI–HindIII fragment containing the Candida glabrataHIS3 DNA (Kitada et al., 1995) into the EcoRI–HindIII gap of pUC19. Plasmid YCp19 is a YCp-based plasmid harbouring the TRP1 gene(Stinchcomb et al., 1982). Plasmid pYC-YVH1was constructed by ligating a 2·7 kb EcoRI–SphIfragment containing YVH1 gene (�1177 +1727)in YCp type vector pRS316 (Sikorski and Hieter,1989). YPDA medium consisted of 1% yeastextract, 2% polypeptone, 2% glucose and 0·04%adenine. SC-His medium consisted of 2% glucose,0·67% yeast nitrogen base without amino acids andthe required auxotrophic supplements except forhistidine. Agar (2%) was added to the plates.

Disruption of protein phosphatase genesPPase gene disruptants were constructed by

PCR (polymerase chain reaction)-mediated genedisruption (Baudin et al., 1993; Schneider et al.,1996) (Figure 1). Primer sets were designed suchthat 40 bases at the 5� end of the primers werecomplementary to those at the correspondingregion of the target gene, and 20 bases at their 3�end were complementary to the pUC19 sequenceoutside the polylinker region in plasmid pCgHIS3containing the C. glabrata HIS3 gene as a select-able marker. We used nutritional markers for genedisruption in this study due to their ease ofhandling. However, it is worth noting that genedisruption by nutritional markers could occasion-ally result in marker effects (Baganz et al., 1997).Primer sets for PCR were primarily designed todelete most of the open reading frame (ORF)(Table 1). When the length of ORF was largerthan 2 kb, primer sets were designed to delete

Yeast 15, 1669–1679 (1999)

1671PROTEIN PHOSPHATASE GENE DISRUPTION

approximately 1·5 kb DNA region within theORF, because the long separation between theregions of the target gene was assumed to reducedisruption frequency.

The disruption was verified by colony-PCRamplification (Huxley et al., 1990) using threeprimers: forward primer CCgH, corresponding toa sequence within the C. glabrata HIS3 gene(nucleotide positions 295–314 relative to ATG)(Figure 1); reverse primer Cr, corresponding to asequence downstream of the target gene; and athird primer Cf, corresponding to a sequenceupstream of the target gene. We verified the dis-ruption using the primer pairs of CCgH and Cr,and Cf and Cr. Cells from colonies of transform-ants grown overnight on YPDA plates weresubjected to this assay.

Northern blot hybridization analysisThe preparation of RNA and the method for

Northern blot hybridization were as described byRose et al. (1990). Total RNAs were preparedfrom cells of the wild-type strain growing in mid-exponential phase (A660=1·0) at 30�C, 13�C and37�C. In time-course experiments, when the cellswere grown to exponential phase (A660=0·8) inYPDA medium at 30�C and shifted to 13�C, totalRNAs were prepared from the cells at 0 h, 0·5 h,1 h, 2 h, 3 h, 4 h, 6 h and 9 h after the temperatureshift. DNA probes containing YVH1 ORF(nucleotide positions �48 to +1146 relative toATG) or ACT1 ORF (+450 to +776) were syn-thesized by PCR and labelled with 32P accordingto the protocols of the Random Primer DNALabelling Kit, Ver. 2 (TaKaRa Co.).

RESULTS

Construction of protein phosphatase genedisruptants

Thirty-two protein phosphatase (PPase) genes ofS. cerevisiae were listed in the yeast proteomedatabase (http://www.proteome.com). In an effortto understand the role of those PPases in thecellular physiology of S. cerevisiae cells, we con-structed disruptants for each of these PPase genesby PCR-mediated gene disruption (see Materialsand Methods) (Figure 1).

Since two PPase genes, GLC7 and CDC14, werereported to be essential for growth (Feng et al.,1991; Wan et al., 1992), we transformed the


diploid strain W303 (MATa/MAT� his3-11,15/his3-11,15) with the PCR products prepared fordisruption of GLC7 and CDC14. The resultanttransformants were sporulated and the asci weredissected. Only two spores from each ascus germi-nated among the 13 asci and seven asci, respect-ively, so far examined for each case and all of thegerminated colonies exhibited the His� pheno-type, confirming that GLC7 and CDC14 areessential for growth.

Since we did not obtain the sit4 haploid dis-ruptants, we transformed the diploid strainHYP101d with the PCR products generated forthe disruption of SIT4. The resultant transform-ants were sporulated and the asci were dissected.Only two spores from each ascus germinatedamong the six asci so far examined and all of thegerminated colonies exhibited the His� pheno-type. Therefore, we concluded that disruption ofSIT4 resulted in lethality in the HYP100 back-ground. It is known that viability of the mutantstrain having deletion of SIT4 is dependent on theallele of the SSD1 gene, which was identified as asuppressor of the lethality caused by the sit4mutant (Sutton et al., 1991) and was recentlysuggested to encode a cytoplasmic RNA-bindingprotein (Uesono et al., 1997). Several laboratorystrains have a different allele of SSD1. sit4 disrup-tion in combination with ssd1-d allele results inlethality, while that with ssd1-v allele is viable.Therefore, the version of SSD1 in strain HYP100might be ssd1-d. The disruptants of the remaining29 including six putative PPases, i..e YBR125c,YCR079w, YIL113w, YJR110w, YNR022c andYOR090c of unknown function, were viable at30�C.

Temperature-sensitive growthWe examined the effect of PPase gene disruption

on temperature-sensitive growth. Colonies of 29viable PPase disruptants grown on YPDA plates at30�C were replicated onto the same medium andincubated at 37�C and 13�C. All disruptants grewat 37�C similar to the wild-type strain (data notshown). However, 12 of these, the cna1, cna2,msg5, ppg1, ppz1, ptc1, ptp2, siw14, tep1, ycr079w,ynr022c and yvh1 disruptants, grew more slowly at13�C than the wild-type strain (Figure 2), althoughthe growth of these 12 disruptants was not com-pletely arrested at 13�C. To examine their slowgrowth phenotype at 13�C more carefully, each ofthe disruptants were streaked onto YPDA plates

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Table 1. S. cerevisiae protein phosphatase genes.

ORF Gene

Phenotypes ofdisruptantidentified

in this study* Function or homology

Regionremoved indisruptant† Reference

PPP familyPP1 family

YER133w GLC7/DIS2 Lethal S. cerevisiae PP1 44–1421 Cannon et al., 1994YML016c PPZ1 (Cs), Saltt,

CaffeinesRelated to PP1, involved insalt homeostasis

51–1490 Posas et al., 1995

YDR436w PPZ2 Related to PP1, involved insalt homeostasis

51–1510 Posas et al., 1995

YPL179w SAL6 Related to PP1, regulationof protein synthesis

44–1557 Vincent et al., 1994

PP2A familyYDL047w SIT4/PPH1 Lethal Involved in cell cycle

regulation44–893 Luke et al., 1996

YDL134c PPH21 Similar to mammalianPP2A

47–1067 Evans and Stark,1997

YDL188c PPH22 Similar to mammalianPP2A

47–1081 Evans and Stark,1997

YDR075w PPH3 Similar to Sit4 44–884 Evans and Stark,1997

YNR032w PPG1 (Cs) Involved in glycogenaccumulation

44–969 Posas et al., 1993b

PP2B familyYLR433c CNA1/CMP1 (Cs) Calcineurin catalytic

subunit47–1459 Nakamura et al.,

1993YML057w CMP2/CNA2 (Cs) Salts Calcineurin catalytic

subunit46–1440 Nakamura et al.,

1993Other

YGR123c PPT1 Human PP5-related PPase 44–1495 Stark, 1996PP2C family

YDL006w PTC1 (Cs) Down-regulate the Hog1pathway

44–803 Maeda et al., 1994

YER089c PTC2 Involved in the UPRpathway

44–1194 Maeda et al., 1994

YBL056w PTC3 Yeast homologue of PP2C 44–1364 Maeda et al., 1994YBR125c 44–1139YCR079w (Cs), Caffeines 47–1261YOR090c Protein with similarity to

Ser/Thr PPase44–1460

PTP family/Dual-specificity PPaseYDL230w PTP1 PTP with dual specificity

domain44–930 Wilson et al., 1995

YOR208w PTP2 (Cs) PTP with dual specificitydomain

44–1460 Maeda et al., 1993

YER075c PTP3 Involved in Hog1 MAPKpathway

44–1460 Wurgler-Murphyet al., 1997

YFR028c CDC14 Lethal Function at late in the cellcycle

44–1460 Wan et al., 1992

YMR036c MIH1 S. pombe cdc25+

homologue44–1460 Sia et al., 1996

Copyright � 1999 John Wiley & Sons, Ltd. Yeast 15, 1669–1679 (1999)


and incubated at 30�C and 13�C. Results of thistest were consistent with those obtained by thereplica-plate method (data not shown).

During the course of this study, we becameaware that some of the disruptants constructed byinsertion of the C. glabrata TRP1 gene instead ofthe CgHIS3 gene as a selectable marker exhibitednormal growth at 13�C, similar to the wild-typestrain. Therefore, we transformed the 29 dis-ruptants having the trp1 mutation and the inser-tion of CgHIS3 at the target locus of each PPasegene with the low-copy-number plasmid YCp19harbouring the S. cerevisiae TRP1 gene, and exam-ined their growth at 13�C by the replica-platemethod. The Trp+ transformants of all the PPasegene disruptants, except for yvh1, grew normally at13�C, as did the wild-type strain (Figure 2).

To see whether the disruption of the YVH1 genecauses cold-sensitive growth phenotype, the yvh1disruptant of W303-1A was crossed with thewild-type strain W303-1B and tetrad analysis wascarried out. Ten asci tested so far revealed 2:2segregation of histidine prototrophy (disruptionmarker of CgHIS3) and cold sensitive growth,and both phenotypes co-segregated in all tetrads.
Furthermore, we observed the suppression of cold-

sensitive growth phenotype of the yvh1 disruptantby the introduction of plasmid pYC-YVH1, har-bouring the YVH1 gene on the YCp-type plasmid.Therefore we concluded that the disruption ofYVH1 is the sole disruption that intrinsically leadsto cold-sensitive growth at 13�C.

Table 1. Continued.

ORF Gene

Phenotypes ofdisruptantidentified

in this study* Function or homology

Regionremoved indisruptant† Reference

PTP family/Dual-specificity PPaseYPR073c LTP1 Similar to phosphatase

from human placenta44–443 Ostanin et al., 1995

YBR276c PPS1 PTP with dual specificitydomain

44–1460 Ernsting and Dixon,1997

YIR026c YVH1 Cs Yeast homologue ofvaccinia virus PTP, VH1

44–998 Guan et al., 1992

YNL032w SIW14 (Cs) Involved in nutritionalcontrol of cell cycle

44–803

YNL053w MSG5 (Cs) Dephosphorylates Fus3 44–1427 Doi et al., 1994YNL128w TEP1 (Cs) Similar to human tumour

suppressor gene44–1262

YIL113w 56% identical to Msg5 44–583YJR110w 68–1983

OthersYNR022c (Cs) Weak similarity to PPase 44–377

*Cs (cold sensitive), slower growth than wild-type strain at 13�C; (Cs), trp1-dependent cold-sensitive growth; caffeines, sensitiveto caffeine; salts, sensitive to salts; saltt, tolerant to salts. †Indicates the deleted region in the respective disrupted alleles.Adenine of ATG initiation codon in ORF was taken as position 1.

Transcriptional regulation of YVH1 by lowtemperature

Transcription of YVH1 was reported to beinduced by nitrogen starvation (Guan et al., 1992).Since the yvh1 disruptant showed cold-sensitivegrowth, we examined the effects of low tempera-ture on transcription of YVH1. Total RNA wasprepared from wild-type strain HYP100 cultivatedin YPDA to mid-exponential phase at 30�C, 13�Cand 37�C and subjected to Northern analysis. Thelevel of transcription of YVH1 at 13�C was two-fold higher than those at 30�C and 37�C (Figure3A). We also examined the time course of tran-scription of the YVH1 gene at low temperature.Cells of the wild-type strain were grown to expo-nential phase in YPDA at 30�C and shifted to13�C. Total RNA was prepared from cells at0 h, 0·5 h, 1 h, 2 h, 3 h, 4 h, 6 h and 9 h after the
temperature shift and subjected to Northern
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analysis. The wild-type cells at 13�C were still inexponential phase even after 9 h because the cellsgrow slowly at 13�C, with a doubling time of 20 h.Transcription of YVH1 was induced eight-foldwithin 30 min after the temperature was shiftedfrom 30�C to 13�C (Figure 3B). The highest level ofYVH1 (10-fold) was observed 1 h after the tem-perature shift. After prolonged cultivation (9 h) at13�C the level of the transcription was decreasedback to the same level as that as 0 h. Theseobservations suggested that Yvh1 protein plays aspecific role for growth at low temperature.


Utilization of carbon sourcesTo investigate the utilization of carbon sources

in PPase disruptants, colonies of the 29 PPasedisruptants at 30�C were replicated onto nutrientmedia containing sucrose, maltose or galactose asfermentable carbon sources, and glycerol as anon-fermentable carbon source. All the PPase dis-ruptants grew on each of these media in the sameway as the wild-type strain did, indicating that thePPase genes are not indispensable for the uptakeand subsequent utilization of the above carbonsources.

Sensitivity to saltsIt was reported that disruption of PPZ1 and

CMP2 resulted in increase in the tolerance andsensitivity to salts, respectively (Nakamura et al.,1993; Posas et al., 1995). To see whether otherPPase disruptants exhibit alteration in the toler-ance or sensitivity to salts, colonies of the PPasedisruptants grown on YPDA plates were repli-cated onto YPDA plates containing LiCl at con-centrations of 100 m, 300 m and 600 m, andonto YPDA plates containing NaCl at concen-trations of 0·8 , 1·0 and 1·2 . Growth behav-iour of all disruptants except for ppz1 and cmp2 inthese plates was the same as that of the wild-typestrain: they grew in the presence of 100 m LiClslowly in the presence of 300 m LiCl and not atall in the presence of 600 m LiCl, and they grewwell in the presence of 0·8 and 1·0 NaCl butpoorly in the presence of 1·2 NaCl (data notshown). The ppz1 disruptants grew even in thepresence of 600 m LiCl or 1·2 NaCl where thewild-type strain displayed no or poor growth,respectively (data not shown). The growth of thecmp2 disruptant was severely impaired in the pres-ence of 300 m LiCl and 0·8 NaCl, whereas thewild-type strain did not exhibit significant growthimpairment at this concentration (data notshown). These observations indicated that amongall PPases only two, whose disruptions are viable,are involved in salt homeostasis, although thepossibility that any of the three essential phos-phatases are also involved in salt homeostasis isnot formally ruled out and should be noted.

Figure 1. Construction and verification of the PPase genedisruptants. (A) PCR disruption strategy. Primers weredesigned such that 40 bases of the target gene at the 5� end(solid region of the primer arrows) directed the PCR producttoward the target gene and 20 bases at the 3� end (strippedregion of the primer arrows) were complementary to thepCgHIS3 sequence. Following PCR amplification, the PCRproducts were used directly to transform the wild-type strainand histidine-prototroph transformants were selected. 5�-PPand 3�-PP represent the 5�- and 3�-portions of the target PPasegene, respectively. (B) Strategy for confirmation of PPase genedisruption through colony-PCR amplification. The primer pairof CCgH and Cr was expected to yield an amplified band ifdisruption had occurred at the target locus. The primer pair ofCf and Cr was expected to yield PCR products originating fromwild-type alleles of a particular size, depending upon therespective PPase genes, if disruption had not occurred; the sameprimer pair was expected to generate PCR products of adifferent size if disruption had occurred at the target locus.CgHIS3, C. glabrata HIS3 gene.

Sensitivity to drugsWe examined the sensitivity of the disruptants to

drugs known to inhibit the cell cycle, such ascaffeine, benomyl and hydroxyurea (HU). Growthsensitivity to caffeine is often associated with

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Figure 2. Cold-sensitive growth of protein phosphatase gene disruptants. Colonies ofwild-type HYP100 strain (WT) and all 29 disruptants grown on YPDA plates at 30�C werereplicated onto the same medium and incubated for 2 days at 30�C or for 7 days at 13�C.Colonies of transformants of the wild-type strain and the PPase disruptants with plasmidYCp19 containing the TRP1 gene was grown on SC-Trp plates at 30�C, replicated ontoYPDA and incubated for 7 days at 13�C.

Figure 3. Transcriptional regulation of YVH1 by low temperature. (A) Transcription of YVH1 geneat different temperatures. Cells of wild-type strain were inoculated to 0·2 of A660 in YPDA mediumand cultivated at 30�C, 13�C and 37�C. Total RNA was prepared from the cells in mid-log phase(A660=1·0) (lane 1, 30�C; lane 2; 13�C; lane 3, 37�C). (B) Time course of transcription of YVH1 geneat 13�C. Cells of wild-type strain were grown to log phase (A660=0·8) in YPDA medium at 30�C andshifted to 13�C. Total RNA was prepared from the cells at 0 h (lane 1), 30 min (lane 2), 1 h (lane 3),2 h (lane 4), 3 h (lane 5), 4 h (lane 6), 6 h (lane 7) and 9 h (lane 8) after temperature shift. The equalamounts of RNA (5 �g) were applied to lanes in agarose gel (1·5%) containing formaldehyde andseparated by electrophoresis. The gel was blotted onto a nylon filter. Filters were first hybridized witha 32P-labelled YVH1 DNA prepared by PCR as a probe and then rehybridized with a 32P-labelledACT1 DNA prepared by PCR as a probe.

defects in components of the MAP kinase andcAMP-dependent protein kinase pathways(Nickas and Yaffe, 1996). Colonies of all PPasedisruptants were replicated onto YPDA platescontaining 10 m caffeine. The ppz1 and ycr079wdisruptants did not grow at all in the presence of10 m caffeine, whereas other disruptants as wellas the wild-type strain exhibited normal growth.


To further confirm the caffeine sensitivity of ppz1and yrc079w disruptants, cells of these disruptantswere streaked onto 10 m caffeine plates. Theresult of this test agreed with those obtained bythe replica-plate method (Figure 4). The increasedsensitivity of ppz1 disruptant to caffeine wasconsistent with the results of a previous study(Posasa et al., 1993a). On the other hand, caffeine

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Figure 4. Sensitivity to caffeine of ppz1 and ycr079w dis-ruptants. Cells of wild-type HYP100 strain (WT) and ppz1 andycr079w disruptants were streaked onto a YPDA plates with orwithout 10 m of caffeine, and incubated at 30�C for 5 days.


DISCUSSION

We constructed disruptants for 32 PPase genesfrom a single parental strain in this study andfound that no disruptants other than glc7, sit4 andcdc14, whose lethality has already been deter-mined, were lethal. We also found that 11 dis-ruptants exhibited trp1-dependent cold-sensitivegrowth at 13�C. Therefore, this phenomenon indi-cated that double mutation of the TRP1 and thePPase gene caused a synthetic cold-sensitivegrowth. However, a caveat to the cold-sensitivephenotype of S. cerevisiae is that S. cerevisiae trp1mutants, when combined with other mutations,often exhibit a cold-sensitive growth defect(Hampsey, 1997). On the other hand, the saltsensitivity of ppz1 and cmp2 disruptants and thecaffeine sensitivity of ppz1 and ycr079w disruptantswere not dependent on the trp1 mutation (data notshown). The trp1-dependent cold-sensitive growthof the disruptants on a rich medium may beexplained by the defect of tryptophan transport atlow temperatures, as tryptophan permease in S.cerevisiae is important for cell growth even in arich medium (Chen et al., 1994). If this is the case,PPases, whose disruption led to cold sensitivegrowth in the trp1 background, might be involvedin uptake of the tryptophan.

The YVH1 was found to play some role ingrowth under the condition of cold stress. TheTIP1, TIR1, TIR2 and NSR1 genes are known ascold-induced genes (Kondo and Inouye, 1991;Munoz-Dorado et al., 1994). The nucleotide se-quence of the YVH1 promoter does not containsimilar sequences to those present in the upstreamregion of each of the above cold-induced genes.Transcription of YVH1 is induced by the nitrogenstarvation as well (Guan et al., 1992). Neithertranscription factors nor regulatory elements nec-essary for YVH1 induction by nitrogen starvationare known. It remains to be investigated whetherthe different transcription factors regulate YVH1expression responding to the signal of the lowtemperature and nitrogen starvation.

In contrast to 32 PPase genes, 117 protein kinasegenes are predicted in the whole genome of S.cerevisiae. This difference in the number of protein

sensitivity of the ycr079w disruptant has not beenreported.

To confirm that the caffeine-sensitive phenotypeis caused by the disruption of YCR079w markedwith His+ phenotype, tetrad analysis of a diploidconstructed between a ycr079w disruptant and thewild-type strain W303-1B was carried out. Eachtetrad of 20 asci tested revealed 2:2 segregation ofhistidine prototrophy and the caffeine-sensitivephenotype, and co-segregation of both phenotypeswas also observed. These results indicate thatdisruption of the YCR079w gene causes thecaffeine-sensitive phenotype.

Benomyl is an antimicrotubule drug and inhibitsmicrotubule-mediated processes. When colonies ofPPase disruptants were replicated onto YPDAplates containing 1 �g/ml, 10 �g/ml and 100 �g/mlbenomyl, growth of all of PPase disruptants, aswell as the wild-type strain, was severely inhibitedin the presence of 100 �g/ml benomyl, but not inthe presence of 1 �g/ml and 10 �g/ml (data notshown).

HU is an inhibitor of ribonucleotide reductasewhich blocks the synthesis of deoxyribonucleo-tides. The treatment of the wild-type cells with HUresults in cell cycle arrest in S phase (Johnstonand Williamson, 1978). When colonies of PPasedisruptants were replicated onto YPDA plates

containing 100 m and 200 m HU, we found nodifference in growth behaviour between the PPasedisruptants and the wild-type strains (data notshown).

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ACKNOWLEDGEMENTS

We thank members of the M. Arisawa group,Nippon Roche Research Center, for helpful adviceand information on PCR-mediated disruptionstrategies and colony-PCR amplification. Weare also grateful to K. Kitada, Nippon Roche
Research Center, for donating plasmid pCgHIS3.

This study was partially supported by Grants-in-Aid for Scientific Research on Priority Areas (Nos08250210 and 10168215) to S.H. from the Ministryof Education, Science, Sports and Culture ofJapan.

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kinase and PPase genes each have several interpre-tations. One possible interpretation is that a singlePPase can dephosphorylate multiple proteinsphosphorylated to oppose the effect of someprotein kinases. In fact, mammalian PPases havebroad substrate specificities in vitro and theirspecificity is strictly regulated by regulatory sub-unit in vivo (Cohen, 1989; Hubbard and Cohen,1993). The specificity of PPases that function indiverse processes is acquired by a variety of regu-latory subunits, directing the PPase to particularlocations and/or altering its activity upon particu-lar substrates. One typical example is the casefor Glc7p involved in cell cycle progression incombination with Egp1p, glycogen accumulationwith Gac1p, glucose repression with Reg1p andsporulation with Gip1p and Red1p (MacKelvieet al., 1995; Hisamoto et al., 1995; Ramaswamyet al., 1998; Tu and Carlson, 1995). Our obser-vation that the ppz1 disruption simultaneouslyconferred three different phenotypes, i.e. cold-sensitive, salt-tolerance and caffeine-sensitivephenotype, suggests that Ppz1p dephosphorylatesdifferent substrates acting in those different bio-logical processes. We suppose that Yvh1p mightalso dephosphorylate at least two different pro-teins, one acting in the sporulation regu-atory system and the other in the growth at lowtemperature.

Some PPases are known to have an overlappingor redundant function in a particular biologicalprocess. Well-known examples are Ppz1p andPpz2p (PP1-related), both of which have a role insalt homeostatsis (Lee et al., 1993; Posas et al.,1995). Ptp2p, Ptp3p (PTP) and Ptc1p (PP2C) takepart in dephosphorylation of Hog1p proteinkinase (Maeda et al., 1993; Wurgler-Murphy et al.,1997) and Pph21p, Pph22p and Pph3p (PP2A) areconcerned with bud morphogenesis and regulationof mitosis (Ronne et al., 1991; Lin and Arndt,1995). These facts imply that double or multipledisruption of PPases might be required to furtherunderstand the role of PPases in S. cerevisiae.Work with this line is in progress.

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A series of protein phosphatase gene disruptants inSaccharomyces cerevisiae