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INTRODUCTION

Saccharomyces cerevisiae possesses a single, essential gene(GLC7) encoding the catalytic subunit of type 1 proteinphosphatase PP1, a member of the PPP family of proteinserine/threonine phosphatases found ubiquitously in eukaryoticcells (Cohen, 1989). Glc7p is involved in the regulation ofglycogen metabolism, general amino acid control and glucoserepression (see Stark, 1996, for a review). However, theinvolvement of Glc7p in these processes is dispensable forviability and hence it is of interest to determine which functionsof Glc7p are absolutely required by the cell. We have thereforegenerated novel conditional lethal glc7 alleles with the aim ofdefining essential functions of S. cerevisiae PP1.

A number of conditional glc7 alleles have already beendescribed. The cold-sensitive glc7Y–170 allele was isolated as anextragenic suppressor of a temperature sensitive (Ts−) cdc24mutant, which displays a defect in polarized growth (Hisamotoet al., 1994). glc7Y–170 cells arrest cell division in mitosis at thenon-permissive temperature with high histone H1 kinaseactivity (Hisamoto et al., 1994). The glc7-12 Ts− allele(MacKelvie et al., 1995) also arrests with a mid-mitotic

phenotype, as do cells depleted of Glc7p in a MET3 promotershut-off experiment (Black et al., 1995). Alanine-scanningmutagenesis of GLC7 generated the cold-sensitive glc7-129allele that also has a mitotic arrest phenotype (Baker et al.,1997; Bloecher and Tatchell, 1999). These results are thereforeconsistent with an essential requirement for PP1 in progressionthrough mitosis in a variety of eukaryotic systems includingfission yeast, Aspergillus nidulans, Drosophila melanogasterand mammalian cells (Axton et al., 1990; Doonan and Morris,1989; Fernandez et al., 1992; Ishii et al., 1996; Ohkura et al.,1989). In addition, the bimG11 PP1 mutant in A. nidulans showsa morphology defect in which mutant conidia fail to producegerm tubes but instead grow spherically and lyse (Borgia, 1992;Doonan and Morris, 1989).

Here we report the isolation and characterization of a newtemperature-sensitive glc7 allele (glc7-10) which causes cellsto arrest at the restrictive temperature with replicated DNA priorto the metaphase to anaphase transition. Elsewhere we haveshown that this arrest is dependent on the spindle checkpointand results from a defect in the ability of kinetochores to attachto microtubules (Sassoon et al., 1999). Here we demonstratethat the glc7-10 mutation also causes morphological and cell

507Journal of Cell Science 113, 507-520 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1036

GLC7 encodes the catalytic subunit of type 1 proteinserine/threonine phosphatase (PP1) in the yeastSaccharomyces cerevisiae. Here we have characterized thetemperature-sensitive glc7-10 allele, which displaysaberrant bud morphology and an abnormal actincytoskeleton at the restrictive temperature. At 37°C glc7-10 strains accumulated a high proportion of budded cellswith an unmigrated nucleus, duplicated spindle polebodies, a short spindle, delocalized cortical actin and 2CDNA content, indicating a cell cycle block prior to themetaphase to anaphase transition. glc7-10 was suppressedby growth on high osmolarity medium and exhibitedtemperature-sensitive cell lysis upon hypo-osmotic stress.Pkc1p, the yeast protein kinase C homolog which is thoughtto regulate the Mpk1p MAP kinase pathway involved incell wall remodelling and polarized cell growth, was foundto act as a dosage suppressor of glc7-10. Although neitheractivation of BCK1 (MEKK) by the dominant BCK1-20

mutation nor increased dosage of MKK1 (MEK) or MPK1(MAP kinase) mimicked PKC1 as a glc7-10 dosagesuppressor, extra copies of genes encoding upstreamcomponents of the Pkc1p pathway such as ROM2, RHO2,HCS77/WSC1/SLG1 and MID2 also suppressed glc7-10effectively. Conversely, mpk1∆∆ glc7-10 and bck1∆∆ glc7-10double mutants displayed a synthetic cell lysis defectcompared with each single mutant and glc7-10 washypersensitive to reduced PKC1 function, displaying highlyaberrant morphologies and inviability even at the normallypermissive temperature of 26°C. Dephosphorylation byPP1 therefore functions positively to promote cell integrity,bud morphology and polarization of the actin cytoskeletonand glc7-10 cells require higher levels of Pkc1p activity tosustain these functions.

Key words: GLC7, Protein phosphatase 1, PKC1, Yeast cell wall

SUMMARY

Type 1 protein phosphatase is required for maintenance of cell wall integrity,

morphogenesis and cell cycle progression in Saccharomyces cerevisiae

Paul D. Andrews and Michael J. R. Stark*

Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK*Author for correspondence (e-mail: [email protected])

Accepted 26 November 1999; published on WWW 19 January 2000

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integrity defects and interacts genetically with the Pkc1p-Mpk1p MAP kinase pathway and its upstream activators. Thephenotypes of glc7-10 are therefore quite distinct from thoseof other glc7 alleles and define additional, essential functionsfor PP1 in yeast.

MATERIALS AND METHODS

Strains, plasmids, media and general methods All yeast strains used in this work are listed in Table 1 and details ofthe plasmids used and generated in this work are given in Table 2.Basic yeast methods and growth media were as described by Kaiseret al. (1994) and yeast transformation was carried out according to themethod of Gietz et al. (1992). Routine recombinant DNAmethodology was performed as described by Sambrook et al. (1989).All restriction enzymes were from New England Biolabs (Beverly,MA, USA).

PCR mutagenesis, gapped plasmid repair and mutantisolationPCR mutagenesis of GLC7 and gapped plasmid repair to generate

glc7 Ts− alleles were carried out as previously described (MacKelvieet al., 1995), generating glc7-6 and glc7-8. The DNA sequence ofglc7-6 and glc7-8 was determined on both strands by the dideoxymethod using SequenaseTM version 2.0 (Amersham Pharmacia),custom synthetic oligonucleotide primers and double-strandedplasmid DNA template according to the manufacturer’s instructions.DNA sequence determination of the glc7-6 allele demonstrated thatone mutation lay in the intron (TrG at position +288, numbering fromthe ATG start codon) and a second in exon 2 of GLC7 (TrC atposition +928, causing an F135L substitution in Glc7p). The intronmutation was outside the consensus splice donor, acceptor andbranch-point sites, but to eliminate any possible contribution of thismutation to the Ts− phenotype of the strain the intron mutation wasreplaced with the wild-type sequence by ligating a 460 bp SalI-ApaI(carrying the F135L mutation) from YCplac22-glc7-6 to the 7.3 kbSalI-ApaI fragment of YCplac22-GLC7, generating YCplac22-glc7-10. The glc7-10 allele thus generated was reintroduced into SBY-SSαby plasmid shuffling (Sikorski and Boeke, 1991) and tested fortemperature-sensitivity: removal of the intron mutation was found notto affect the Ts− phenotype. glc7-8 was found to carry four alterations:ArG at +143 (Q48R in exon 1); TrA at +467 (intron); TrC at +1049(L175P in exon 2) and ArG at +1366 (M281V in exon 2). The singlepoint mutation in the intron of YCp-glc7-8 was replaced with the

P. D. Andrews and M. J. R. Stark

Table 1. Yeast strains usedStrain Genotype Source/reference

AY925 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 Gal+ Kim ArndtAY926 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 Gal+ Kim ArndtAYS927 MATa/α ade2-1/ade2-1 his3-11/his3-11 leu2-3,112 /leu2-3,112 Kim Arndt

trp1-1/trp1-1 ura3-1/ura3-1 can1-100 can1-100 ssd1-d2/ ssd1-d2 Gal+

SBY-SSα MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCpGLC7(URA3)] (Black et al., 1995; MacKelvie et al., 1995)

SBY-SSa MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCpGLC7(URA3)] This studyPAY3 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCplac22-glc7-10] This studyPAY4 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCplac22-glc7-13] This studyPAY5 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCplac22-GLC7] This studyPAY22-1 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 bck1∆::URA3 Gal+ This study; from PAY5

[YCplac22-GLC7]PAY38 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 Gal+ [YCplac22-glc7-12] (MacKelvie et al., 1995)PAY61 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 bck1∆::URA3 Gal+ This studyPAY150 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 mpk1∆::TRP1 Gal+ This study

[YCpGLC7(URA3)]PAY151 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 mpk1∆::TRP1 Gal+ This study

[YCpHAglc7-10, HIS3]PAY154 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 mpk1∆::TRP1 Gal+ This study

[YCpHA-GLC7, HIS3]MCY15-3A* MATa ade2 trp1 ura3 LEU2 pkc1ts (cly15-1) (Paravicini et al., 1992)PAY700-4 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1::glc7-10::TRP1 Gal+ This studyPAY704-1 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1::GLC7::TRP1 Gal+ This studyPAY715-1C MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 bck1∆::URA3 Gal+ This studyPAY720-1A MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 This study

trp1::GLC7::TRP1 bck1∆::URA3 Gal+

PAY720-3D MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 This studytrp1::GLC7::TRP1 bck1∆::URA3 Gal+

PAY725-9D MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 This studytrp1::glc7-10::TRP1 bck1∆::URA3 Gal+

PAY725-7A MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 This studytrp1::glc7-10::TRP1 bck1∆::URA3 Gal+

PAY725-5B MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 glc7::LEU2 trp1::glc7-10::TRP1 Gal+ This studyPAY760D MATa/MATα ade2-1/ade2-1 his3-11/his3-11 leu2-3,112/leu2-3,112 ura3-1/ura3-1 PAY725-9D ×

can1-100/can1-100 ssd1-d2/ssd1-d2 glc7::LEU2/glc7::LEU2 trp1::glc7-10::TRP1/ PAY725-7Atrp1::glc7-10::TRP1 bck1∆::URA3/bck1∆::URA3 Gal+

PAY755D MATa/MATα ade2-1/ade2-1 his3-11/his3-11 leu2-3,112/leu2-3,112 ura3-1/ura3-1 PAY720-3D × can1-100/can1-100 ssd1-d2/ ssd1-d2 trp1::GLC7::TRP1/trp1::GLC7::TRP1 PAY720-1Abck1∆::URA3/bck1∆::URA3 Gal+

PAY770D MATa/MATα ade2-1/ade2-1 his3-11/his3-11 leu2-3,112/leu2-3,112 ura3-1/ura3-1 PAY700-4 ×can1-100/can1-100 ssd1-d2/ ssd1-d2 glc7::LEU2/glc7::LEU2 trp1::glc7- PAY725-5B10::TRP1/trp1::glc7-10::TRP1 Gal+

*Of uncertain genetic background; all other strain are W303 derivatives

509Glc7p and morphogenesis in S. cerevisiae

wild-type sequence by ligating a 471 bp NdeI-SalI fragment fromYCplac22-GLC7 to the 7.18 kb NdeI-SalI fragment of YCplac22-glc7-8 to create YCplac22-glc7-9. A further derivative of glc7-9(deleting the C-terminal M282V mutation) was generated byreplacement of the 3′ 1.04 kb EcoRI fragment of YCplac22-glc7-9with the equivalent fragment from YCplac22-GLC7, generatingYCplac22-glc7-13. The presence or absence of the relevant mutationsin these constructs was verified by DNA sequencing. Neither removalof the intron mutation nor the additional removal of the ArG changeat +1369 affected the temperature-sensitive phenotype of the originalglc7-8 mutant.

Strains in which GLC7 or glc7-10 were integrated at the trp1 locuswere made by transferring the HindIII-BamHI insert of YCplac22-GLC7 and YCplac22-glc7-10 to YIplac204 (Sikorski and Hieter,1989), generating YIplac204-GLC7 and YIplac204-glc7-10respectively. These plasmids were linearized with BstXI andintegrated into SBY-SSa, followed by eviction of the residentYCpGLC7(URA3) plasmid using 5-fluoro-orotic acid (Sikorski andBoeke, 1991). Correct integration of a single copy of the YIplac204-based plasmid at trp1was verified by Southern blot analysis (notshown). One mutant strain (PAY700-4) and one wild-type strain(PAY704-1) were selected for further use. PAY700-4 (glc7-10) wasretested for temperature-sensitivity, suppression by sorbitol andmorphological abnormalities and found to behave identically to theplasmid-based strain described above.

Deletion of MPK1 and BCK1To generate strains carrying an MPK1 deletion allele (in which theprotein kinase domain is replaced by TRP1), the 2.1 kb SalI-EcoRIfragment from pBR322-mpk1∆::TRP1 was used to transform SBY-SSα to tryptophan prototrophy (generating PAY150). YCpHA-GLC7(PAY151) or YCpHA-glc7-10 (PAY154) were then introduced byplasmid shuffling on medium containing 1 M sorbitol. All gene

replacements were confirmed by Southern hybridization analysisand/or PCR analysis. BCK1 was deleted in the diploid W303 wild-type strain AYS927 by one-step gene replacement (Rothstein, 1991)using the 4.9 kb SalI fragment from pUC18-[bck1∆::URA3] (Lee andLevin, 1992) to transform the strain to uracil prototrophy. Genedisruption was verified by PCR using primers flanking the BCK1gene. Haploid BCK1 disruptants were obtained by sporulation andtetrad dissection onto YPD plates containing 1 M sorbitol. One Ts−,sorbitol-remedial isolate (PAY715-1C) was selected for further use.Double glc7-10 bck1∆ mutants were obtained by mating PAY700-4(glc7-10 BCK1) with PAY715-1C (GLC7 bck1∆), followed bysporulation of the diploid in the presence of 1 M sorbitol and tetraddissection onto YPD plates containing 1 M sorbitol. All Ura+ Trp+

Leu+ segregants were found to be slow-growing even on sorbitolplates at 26°C. Two such strains, PAY725-7A and PAY725-9D, wereselected for further study. GLC7 bck1∆ strains (PAY720-1A andPAY720-3D) were obtained in a similar way by crossing PAY704-1with PAY715-1C. Haploid bck1∆ mutants are known to be unstable,resulting in the generation of a high frequency of spontaneoussuppressor mutations. In order to circumvent this problem, ahomozygous glc7-10 bck1∆ diploid (PAY760D) was generated bymating PAY725-7A (MATa glc7-10 bck1∆) and PAY725-9D (MATαglc7-10 bck1∆) followed by zygote dissection on YPD sorbitol plates,checking several candidate diploids to ensure selection of arepresentative isolate. Homozygous GLC7 bck1∆ (PAY755D) andglc7-10 BCK1 (PAY770D) diploid strains were similarly obtained (seeTable 1).

Trypan blue exclusionA trypan blue exclusion assay (Karpova et al., 1993) was used toassess cell wall integrity. Cells were grown in liquid SCD mediumlacking tryptophan, leucine (and also lacking uracil if necessary toselect for YEp-PKC1), with or without 0.5 M NaCl. Mid-log phase

Table 2. PlasmidsName Description/relevant markers Source/Reference

pBR322-mpk1∆::TRP1 mpk1∆ deletion construct (Lee et al., 1993b)pC-186::RHO2 2µ RHO2, URA3 M. N. HallpDS131 HCS77 in YEp24, URA3 D. StirlingpDS143 MID2 in YEp24, URA3 D. StirlingpGAL-PKC1 PKC1 gene under control of GAL1,10 promoter, CEN, URA3 (Watanabe et al., 1994)pGAL-PKC1-K853R PKC1-K853R allele under control of GAL1,10 promoter, CEN, URA3 (Watanabe et al., 1994)pJO1 CLN1 gene in YEp24 (URA3) (Gray et al., 1997)pJO16 SWI4 gene in YEp24 (URA3) (Gray et al., 1997)pJO21 CLN2 gene in YEp24 (URA3) (Gray et al., 1997)pPS967 NUF2-GFP YIp (URA3) (Kahana et al., 1995)pRB1438 CEN, URA3 GAL1,10 cloning vector K. AyscoughpRS316-[BCK1-20] pRS316 (Sikorski and Hieter, 1989) carrying BCK1-20, URA3 (Lee and Levin, 1992)pSEY18::TOR2 2µ TOR2, URA3 M. N. HallpUC18-[bck1∆::URA3] pUC18 carrying bck1∆::URA3 (Lee and Levin, 1992)YCp-HA-GLC7 YCp50 derivative with HA-tagged GLC7, HIS3 Kim ArndtYCp-HA-glc7-10 YCp50 derivative with HA-tagged glc7-10, HIS3 This studyYCp-HA-glc7-12 YCp50 derivative with HA-tagged glc7-12, HIS3 This studyYCplac22-GLC7 YCplac22 carrying GLC7 (2.8 kb HindIII-BamHI fragment) (MacKelvie et al., 1995)YCplac22-glc7-6 YCplac22-GLC7 harboring glc7-6ts allele, TRP1 This studyYCplac22-glc7-10 YCplac22-glc7-6 lacking point mutation in intron, TRP1 This studyYCplac22-glc7-12 YCplac22-GLC7 harboring glc7-12ts allele, TRP1 (MacKelvie et al., 1995)YEp-BCK2 YEp352 with a 5.5-kb SphI-XbaI BCK2 gene fragment, URA3 (Lee et al., 1993a)YEp-BRY1 YEp24 with the entire BRY1/SKN7 gene, (Morgan et al., 1995)YEp-PKC1 YEplac195 (Gietz and Sugino, 1988) with a 4.3 kb SphI fragment encoding PKC1 This study

from pGY62 (Paravicini et al., 1992), URA3YEp-PPZ1 YEplac195 (Gietz and Sugino, 1988) with a 3.0 kb BamHI-SacII fragment encoding PPZ1 This study

derived from pPPZ1 (Hughes et al., 1993), URA3YEp24-KRE6 YEp24 with the 4.6 kb KRE6 BamH1-SalI fragment, URA3 (Roemer and Bussey, 1991)YEp352-[MKK1] YEp352 (Hill et al., 1986) carrying MKK1, URA3 (Irie et al., 1993)YEp352-[MPK1] YEp352 (Hill et al., 1986) carrying MPK1, URA3 (Irie et al., 1993)YEplac195::ROM2 2µ ROM2, URA3 M. N. HallYIplac204-GLC7 YIplac204 carrying GLC7 (2.8 kb HindIII- BamHI fragment) This studyYIplac204-glc7-10 YIplac204-GLC7 harboring the glc7-10ts allele This study

510

cultures were split and incubated at either 26°C or 37°C for a further1 hour, then stained 30 minutes before microscopic examination,either in growth medium or following transfer to water at roomtemperature. The proportion of stained cells was determined forsamples of 200-300 cells.

Tubulin, actin and SPB stainingMicrotubules were visualized by indirect immunofluorescenceessentially as described previously (Pringle et al., 1991). Briefly, cellswere fixed with formaldehyde (3.7%) and then incubated successivelywith anti-α-tubulin rat monoclonal antibody (YOL1/34, Sera-Labs,UK) and FITC-conjugated anti-rat IgG (ICN, UK). The mountingmedium contained 1 mg/ml 4′, 6-diamidino-2-phenylindoledihydrochloride (DAPI) to permit visualization of the DNA. Thespindle pole body (SPB) was visualized by transforming strains withplamid pPS967, which encodes a Nuf2p-GFP fusion protein thatlocalizes to the SPB (Kahana et al., 1995). Actin was visualized informaldehyde-fixed cells using rhodamine-phalloidin (MolecularProbes Inc.) essentially as described by Kaiser et al. (1994), exceptthat cells were fixed for 2 hours at room temperature and stainedovernight at 4°C. DIC and fluorescence images were acquired usinga System 300 cooled CCD camera (Digital Pixel Advanced ImagingSystems, Brighton, UK) with IPlab spectrum software (Scanalytics,Fairfax, VA, USA) on an Olympus BX60 fluorescence microscope(Olympus, UK).

FACS analysis of DNA contentDNA content of ethanol-fixed cells was assessed as described byButler et al. (1991) with the following modifications. RNA wasdigested by incubation of cells in 0.5 ml 50 mM sodium citratecontaining 0.1 mg/ml DNase-free RNase A (Sigma) for 2 hours at37°C. DNA was then stained by addition of an equal volume of 50mM sodium citrate containing 12 µg/ml propidium iodide (Sigma).

DNA content was measured using a Becton Dickinson FACScan anddata analysed using LysII software.

RESULTS

glc7-10 arrests in G2/M with an elongated large-budded morphology and loses viabilityA novel temperature-sensitive (Ts−) allele of GLC7 (glc7-10)was isolated by random PCR-based mutagenesis and gappedplasmid repair as described previously (MacKelvie et al.,1995). The glc7-10 allele is recessive (not shown) and encodesa phenylalanine to leucine replacement at position 135 in theGlc7 protein. This phenylalanine residue is invariant in type 1,2A and 2B serine/threonine protein phosphatases and lies inhelix E/α5 in the N-terminal sub-domain of PP1 (Egloff et al.,1995). The growth characteristics of the glc7-10 strain at 26°Cand 37°C are shown in Fig. 1A. Analysis of the budding indexin glc7-10 cultures showed a sustained rise to give over 95%budded cells after 4 hours at the restrictive temperature (Fig.1B). However, bud formation in glc7-10 at 37°C was highlyaberrant, with a high proportion of cells possessing extendedbuds that continued to elongate on further incubation (Fig. 2A).Despite the large bud size the nucleus failed to migrate into thebud in the majority of cells (Fig. 2B). Quantitation of themorphologies and nuclear position in the shifted culture isshown in Fig. 3. Over one-third of the cells exhibitedhyperpolarized growth of the bud with an unmigrated nucleusat 37°C, while a further one-fifth of the cells displayedabnormal, small-budded morphologies. A significant


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Fig. 1. Growth arrest characteristics ofglc7-10 strain. Synchronous, mid-logarithmic cultures of the glc7-10 strain(PAY3) growing at 26°C were split andeither shifted to 37°C (j) or returned to26°C (u) for continued incubation.(A) Cell numbers were monitored overthe time course of the incubation toassess the effect of temperature shift ongrowth rate. (B) The percentage andmorphology of budded cells in eachculture was also assessed. The glc7-10strain shifted to 37°C arrested with avery high proportion of budded cells(j), many of which showed aberrantmorphology (m). By comparison, thepercentage budded cells in the glc7-10strain at 26°C (u) remained constantover the course of the experiment andessentially no aberrant budmorphologies were observed (n).(C) glc7-10 arrests with a 2C DNAcontent at the restrictive temperature. Atselected time points samples wereremoved and processed for FACSanalysis of DNA content. (D) glc7-10loses viability at the restrictivetemperature. At selected times followingtemperature shift, viability (% survival)was determined by removing samplesfrom cultures growing at 26°C (u) and37°C (j) for cell number determinationand plating at 26°C to assess colonyforming ability.


proportion of the large-budded cells also hada second, small bud, suggestive of acytokinesis defect. This notion is supportedby the behaviour of glc7-10 cultures at thepermissive temperature, which comparedwith the GLC7 control showed a largeincrease in the fraction of large-budded cellsin which nuclear division was complete butcytokinesis and cell separation had notoccurred (Fig. 3).

Staining of microtubules in glc7-10 cellsincubated for between 3 and 4 hours at therestrictive temperature revealed the presenceof short spindles, coincident with the DAPIstaining (Fig. 2B). Compared with the wild-type control, a high proportion of glc7-10cells at 37°C showed misalignment of thespindle (Fig. 2C). FACS analysis of theseasynchronous glc7-10 cultures growing at26°C or after shifting to 37°C revealed amarked accumulation of cells with replicated DNA at therestrictive temperature (Fig. 1C). Visualization of the spindlepole bodies (SPBs) using Nuf2p-GFP (Kahana et al., 1995)showed that the arrested cells contained two closely-spacedSPBs (Fig. 2D), consistent with the short mitotic spindle.Taken together, these results indicate an arrest of the celldivision cycle after completion of DNA replication and SPBduplication but prior to the metaphase to anaphase transition.Cell viability was also assayed by measurement of colony-forming ability at 26°C, and found to drop to roughly 50% ofthe starting value after incubation for four hours at therestrictive temperature (Fig. 1D).

glc7-10 is rescued by sorbitol and displays a celllysis phenotype at low osmolarityIt was observed that during prolonged incubation at the

restrictive temperature that a proportion of the glc7-10 cellsunderwent lysis, especially if incubated in water duringmicroscopic examination. We therefore examined the effect ofhigh osmolarity on the temperature-sensitivity of glc7-10 andfound that growth of the glc7-10 strain at 37°C on YPDmedium was completely rescued by inclusion of 1 M sorbitol(Fig. 4), 0.5 M NaCl or 0.5 M KCl (not shown). Growth inhigh osmolarity medium also restored the normal cellmorphology (not shown). Suppression of glc7-10 by highosmolarity was allele-specific since neither of two quitedistinct glc7 alleles, glc7-12 and glc7-13, was suppressed inthis way (Fig. 4). High osmolarity might suppress a Ts−

mutation for nonspecific reasons, but analysis of the glc7-10strain by Trypan Blue exclusion confirmed that the mutationdid confer a temperature-sensitive cell lysis defect. When glc7strains and the wild-type GLC7 control grown in osmotically

Fig. 2. The glc7-10 strain arrests with anelongated bud, an unmigrated nucleus,duplicated SPBs, and a short mitotic spindle.Exponentially growing glc7-10 cells (PAY3)were incubated for 4 hours at the restrictivetemperature (37°C) and then fixed informaldehyde. Fixed cells were viewed bydifferential interference contrast (DIC)microscopy to show the distinct morphologiesof the arrested cells (A). Parallel fixed sampleswere subjected to indirect immunofluorescenceusing anti-tubulin antibodies to visualizemicrotubules and DAPI to visualised DNA(B; tubulin in green and DNA in blue overlaid inAdobe PhotoShop such that nuclearmicrotubules appear turquoise). The elongatedbud morphology of the arrested glc7-10 cultureis accompanied by a defect in nuclear migrationand a very short mitotic spindle which wasfound to be frequently misaligned with respectto the mother-daughter bud axis (C). Spindlepole body (SPB) duplication was monitored byvisualising a Nuf2p-GFP fusion in live glc7-10cells grown at 37°C for 3 hours (D; SPBs ingreen overlaid over a DIC image using AdobePhotoShop).

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non-stabilized conditions (i.e.without added salt) at thepermissive temperature, theyshowed little difference in theirability to exclude Trypan Bluewhen subsequently stained ineither water or growth medium(Fig. 5A). However, glc7-10cells grown for 1 hour at 37°C showed a noticeable lysis defectwhen transferred to water (Fig. 5A), while microscopicexamination showed that there was cell swelling and asignificant proportion of cells with abnormal bud morphology,together with some non-refractile cell ghosts (not shown).Since strains defective in PKC1 (encoding the yeast proteinkinase C homolog; Levin et al., 1990) show a cell lysis defectbut are hypersensitive to hypo-osmotic stress (Levin andBartlett-Heubusch, 1992; Paravicini et al., 1992), we alsoexamined Trypan Blue staining of cells pre-grown inosmotically stabilized conditions (0.5 M NaCl) and thentransferred to salt-free growth medium or water. glc7-10 cellsgrown in this way showed a large increase in Trypan Bluestaining on transfer to water, an effect markedly enhanced incultures grown at the nonpermissive temperature (37°C) forone hour prior to analysis (Fig. 5B). Indeed, up to 25% of glc7-

10 cells transferred to water after growth at 37°C stainedstrongly with Trypan Blue, while microscopic examinationrevealed a high level of non-refractile ghosts and cell debris,further indicative of a cell integrity defect. The cell lysis defectof glc7-10 cells evident from this analysis was allele-specific,since the equivalent glc7-12 strain behaved indistinguishablyfrom the wild-type control (Fig. 5B). The cell integrity defectof glc7-10 cells seen on hypo-osmotic shock was comparableto that of cells lacking BCK1, deletion of which leads to atemperature-sensitive cell lysis defect (Lee and Levin, 1992:see below), although bck1∆ null cells also showed significantlysis when grown at the permissive temperature or whentransferred to growth medium rather than water (Fig. 5C). Thefact that a significant cell lysis defect could be measured in theglc7-10 strain by the Trypan Blue assay after as little as 1 hourunder restrictive conditions strongly supports the notion thatthis is a primary defect of the glc7-10 mutation rather than asecondary effect. In common with other mutants that affect cellwall biosynthesis or cell integrity (Costigan et al., 1992; Ramet al., 1994), the glc7-10 mutant also showed hypersensitivityto caffeine even at 26°C (~3-fold; data not shown) but was notobviously sensitive to cell wall damage (0.001% SDS) at thepermissive temperature (not shown).

glc7-10 is suppressed by high-copy PKC1 and itsactivators but not by components of the BCK1-MPK1 MAP kinase pathwayThe temperature-sensitive cell lysis defect of the glc7-10 allelewas reminiscent of that shown by strains carrying mutations incomponents of the MAP kinase pathway activated by Pkc1p(Levin and Bartlett-Heubusch, 1992; Levin et al., 1990;Paravicini et al., 1992). Loss of function at each level in thispathway results in conditional cell lysis, a phenotype which issuppressed by elevated gene copy (or mutational activation) ofany of the downstream protein kinases in the pathway (Irie etal., 1993; Lee et al., 1993b; Lee and Levin, 1992). We thereforeinitially tested the effect of increased dosage of PKC1, MKK1(encoding the MEK) and MPK1 (encoding the MAP kinase)on the growth of glc7ts strains, as well as the effect of BCK1-20 (a dominant mutant allele encoding the MEKK which


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Fig. 4. The temperature sensitivity of the glc7-10 strain is suppressedby sorbitol. Haploid glc7-12 (PAY38: sector A), glc7-10 (PAY3:sector B), glc7-13 (PAY4: sector D) mutant strains as well as thewild-type GLC7 strain (PAY5: sector C) were streaked onto YPD orYPD supplemented with 1 M sorbitol as an osmotic support at theindicated temperatures for 4 days. Only the glc7-10 strain wasobserved to be osmotically-remedial.

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partially suppresses deletion of PKC1: Lee and Levin, 1992).Interestingly, we found that elevated dosage of PKC1 fullysuppressed the temperature-sensitivity of glc7-10, as shown inFig. 6A. This effect was allele-specific, since increased PKC1dosage failed to suppress the non-sorbitol remedial glc7-12 orglc7-13 alleles (not shown). As well as suppressing thetemperature-sensitivity of the glc7-10 strain, increased PKC1dosage completely suppressed the cell integrity defect asjudged by Trypan Blue staining (Fig. 5C). The effect ofelevated PKC1 dosage on glc7-10 was non-reciprocal, sinceGLC7 in high copy failed to suppress the temperature-sensitivity of a pkc1ts strain (MCY15-3A: not shown). Nosuppression of the glc7-10 phenotype was detected withmulticopy MKK1or MPK1, suggesting that the effect of PKC1might not be mediated by activation of this MAP kinasepathway (Fig. 6A). Although the activated BCK1-20 alleleimproved the growth of glc7-10 at 37°C very slightly (Fig. 6A),it was very poor compared with the effect of high-copy PKC1and we do not consider it to be significant. Pkc1p in S.cerevisiae appears to possess multiple functions, only one ofwhich may be to regulate the activity of the Bck1p-Mkk1/2p-Mpk1p MAP kinase module involved in cell wall constructionand polarized growth (Levin and Errede, 1995). Thus pkc1

mutants show a more severe phenotype than mutations in thedownstream protein kinases, while there is evidence for othergenes which also play roles in cell wall biosynthesis actingdownstream of PKC1, including BCK2 and KRE6 (see DiComo et al., 1995; Epstein and Cross, 1994; Lee et al., 1993a;Lee and Levin, 1992; Roemer et al., 1994). Interestingly, KRE6

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Fig. 5. The glc7-10 mutation causes a cell integrity defect on hypo-osmotic stress. glc7-10 (PAY3), glc7-12 (PAY38) or GLC7 wild-type(PAY5) strains were grown at 26°C in selective medium to mid-exponential phase in the absence (A) or presence of 0.5 M NaCl (B,C) as anosmotic stabiliser. Each culture was split, incubating one half at 26°C (open bars) and the other half at the restrictive temperature of 37°C(hatched bars) for 1 hour prior to trypan blue staining in either growth medium (M) or after transfer to water (W). glc7-10 showed a smalldegree of cell wall lysis when grown in the absence of salt at 37°C (A) especially when transferred to water. However, a high degree of celllysis was observed for the glc7-10 strain grown in 0.5 M NaCl at 37°C following transfer to water (B). A glc7-10 strain (PAY3) carrying amulticopy PKC1 plasmid (which was wild-type for growth) and a control Ts− bck1∆::URA3 strain (PAY22-1) were also assessed for cell wallleakiness after growth in osmotically stabilized medium (C). Multicopy PKC1 abolished the cell wall lysis defect of the glc7-10 strain, whereasthe bck1∆::URA3 (PAY22-1) strain displayed significant lysis at both permissive and restrictive temperatures as expected.

Fig. 6. Dosage suppressors of glc7-10. The glc7-10 strain PAY700-1was transformed with multicopy plasmids YEp-PPZ1, YEp352-[MPK1], YEp352-[MKK1], YEp-PKC1, YEp-BCK2, YEp24-KRE6and control vector YEplac195 or low-copy pRS316-[BCK1-20] (A);or multicopy plasmids pDS131 (HCS77/WSC1/SLG1), pDS143(MID2) and YEp-PKC1 (B); or multicopy plasmids pSEY18::TOR2,YEplac195::ROM2, pC-186::RHO2 (C). Cells were streaked ontoYPD medium at 26°C and 37°C for 4 days before photographing.

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(which is thought to be involved in β(1-6) glucan synthesis:Roemer et al., 1993) was an efficient high-copy suppressor ofglc7-10, while high-copy BCK2 only marginally stimulatedgrowth of glc7-10 at 37°C (Fig. 6A). The PP1-related proteinphosphatases, Ppz1p and Ppz2p have been show to interactgenetically with the PKC1 pathway (Lee et al., 1993a) andPPZ1 was also a partial high-copy suppressor of glc7-10 (Fig.6A). Significantly, other genes which interact with the PKC1-MPK1 pathway failed to suppress glc7-10. SWI4 is involved ininvolved in expression of G1 cyclins and cell wall biosynthesisgenes (Igual et al., 1996; Madden et al., 1997; Morgan et al.,1995) and suppresses the Ts− phenotype of both mpk1∆ andbck1∆ in high copy (Madden et al., 1997), but increased dosageof SWI4 failed to suppress glc7-10 (not shown). Similarly, lossof the G1 cyclins CLN1 and CLN2 is lethal in combination withmpk1∆ and Cln function can become limiting for the functionof the PKC1-MPK1 pathway under some conditions (Gray etal., 1997), but neither CLN1 nor CLN2 in high copy suppressedthe glc7-10 defect (not shown). SKN7 is a high copy suppressorof the lysis defect of pkc1∆ cells and skn7∆ is syntheticallylethal with pkc1∆ (Brown et al., 1994). In high copy, SKN7 canstimulate G1 cyclin expression (Morgan et al., 1995) and it hasbeen shown to be activated by Mid2p (Ketela et al., 1999),possibly through the mediation of Rho1p (Alberts et al., 1998).However, like SWI4 and the CLNs, SKN7 also failed to suppressglc7-10 in high copy.

Pkc1p is a target for the GTPase Rho1p (Drgonova et al.,1996; Kamada et al., 1996; Nonaka et al., 1995), which in turnis regulated by the phosphatidylinositol-3-kinase homologTor2p via activation of the Rho-GTP exchange factor (GEF)Rom2p (Helliwell et al., 1998a; Schmidt et al., 1997). Rom2pis also thought to act as GEF for Rho2p, a Rho-GTPase closelyrelated to Rho1p and with which it has overlapping function(Madaule et al., 1987; Ozaki et al., 1996; Schmidt et al., 1997).Rho1p functions in cell integrity and the organization of theactin cytoskeleton through Pkc1p (Helliwell et al., 1998b;Yamochi et al., 1994) and also acts as a subunit of β(1-3)glucan synthase to regulate cell wall synthesis directly(Drgonova et al., 1996; Qadota et al., 1996). We thereforetested whether increased dosage of ROM2 or TOR2 suppressedthe glc7-10 growth defect and found that ROM2 was a verygood dosage suppressor but that TOR2 was a weak suppressor(Fig. 6C). Since RHO1 can be toxic in high copy, we tested theRHO1 homolog RHO2 (Madaule et al., 1987; Ozaki et al.,1996) rather than RHO1 and found that high copy RHO2suppressed glc7-10 very well (Fig. 6C). Recently, a family ofgenes encoding putative membrane proteins (WSC1-3) havebeen shown to be required for the maintenance of cell wallintegrity via activation of the Pkc1p-Mpk1p MAPK module(Gray et al., 1997; Jacoby et al., 1998; Verna et al., 1997). Oneof these genes, HCS77/WSC1/SLG1, was also isolated as a highcopy suppressor of a swi4 deletion and as an upstream activatorof Pkc1p (Gray et al., 1997; Jacoby et al., 1998; Rajavel et al.,1999). We found that in high copy HCS77/WSC1/SLG1 fullysuppressed the growth defect of a glc7-10 strain (Fig. 6B).Deletion of the related gene MID2 is phenotypically additivewith hcs77/wsc1/slg1∆ (Ketela et al., 1999, D. Stirling and M.J. R. Stark, unpublished; Rajavel et al., 1999) and likeHCS77/WSC1/SLG1, MID2 is therefore likely to encode anupstream activator of the PKC1 pathway. MID2 was also agood suppressor of glc7-10 temperature sensitivity (Fig. 6B).

Thus in summary, several high-copy plasmids which arepredicted to lead to increased signalling through Pkc1p eachsuppressed the temperature-sensitivity of glc7-10.

glc7-10 is synthetically lethal with deletions of MPK1and BCK1 and hypersensitive to overproduction ofinactive Pkc1pWhile the above data argue that dosage suppression of glc7-10by PKC1 was not due to activation of the Bck1p-Mpk1ppathway, it was still conceivable that the osmotic-remedialdefect associated with glc7-10 was due to inactivation of thissignalling cassette or its downstream targets. We thereforeconstructed a glc7-10 mpk1∆ double mutant to test whether thetwo defects were additive, assessing the temperature-sensitivityof the double mutant in the presence and absence of 1 Msorbitol. While the Ts− phenotype of either glc7-10 or mpk1∆single mutant strains was in each case rescued by sorbitol, bothstrains were viable without sorbitol at 26°C. However, thedouble glc7-10 mpk1∆ mutant displayed an unconditionalgrowth defect in the absence of sorbitol (Fig. 7). Taken togetherwith the previous results, this synthetic lethality suggests glc7-10 may be defective in a parallel, Pkc1p-regulated pathway andthe additivity of the defects argues against a defect inregulation of the Mpk1p module itself. To extend thisobservation, a double glc7-10 bck1∆ mutant was generated andwas also found to be dependent on sorbitol for growth at 26°C(Fig. 8), demonstrating that bck1∆ is also synthetically lethalwith glc7-10. In fact the double mutant proved very difficult togrow at all in liquid medium containing 1 M sorbitol, even atthe reduced temperature of 23°C. Such cultures contained largenumbers of swollen and lysed cells (not shown), emphasisingthe enhanced cell integrity defect in the double mutant. Thusthe reduced Glc7p function of glc7-10 in combination with


Fig. 7. The glc7-10 defect is additive with a mpk1 deletion. Haploidyeast strains bearing either wild-type GLC7 or glc7-10 wereconstructed which additionally contained an mpk1∆::TRP1 deletion,streaked on YPD (with or without 1 M sorbitol) and incubated at theindicated temperature for 3-4 days. The glc7-10 mpk1∆::TRP1 strain(PAY151) required sorbitol for growth even at 26°C (sector A). Incontrast, the GLC7 mpk1∆::TRP1 control strain (PAY154) (sector D)and the glc7-10 MPK1 strain (sector C) displayed Ts− growth whichwas rescued by the presence of 1 M sorbitol, but grew withoutsorbitol at 26°C. A wild-type haploid strain (AY925) was included asa control (sector B).


deletions of components of the Bck1p-Mpk1p MAP kinasepathway generates a phenotype reminiscent of a pkc1∆ nullmutant, which requires sorbitol for growth even at lowtemperatures (Levin and Bartlett-Heubusch, 1992; Paraviciniet al., 1992).

Since loss of MPK1 or BCK1 functionwas highly detrimental to glc7-10 cells, wenext determined whether inhibition of thePkc1p pathway by expression of acatalytically-inactive Pkc1p (Watanabe etal., 1994) was also was inhibitory forgrowth of the glc7-10 strain. Althoughexpression of Pkc1K853Rp from theGAL1,10 promoter did not greatly inhibitgrowth of wild-type cells (Watanabe et al.,1994; Fig. 9A) it was found to be lethalfor glc7-10 cells even at the normally

permissive temperature of 26°C (Fig. 9A). Expression of wild-type PKC1 from the GAL1,10 promoter had no effect on thegrowth of either strain at this temperature (Fig. 9A).Microscopic examination of glc7-10 cells expressingPkc1K853Rp revealed the presence of a high proportion of cellswith extremely elongated buds (Fig. 9B), somewhatreminiscent of the phenotype of glc7-10 strains grown at 37°C.This phenotype was in contrast to the effect of overexpressionof the PKC1K853R allele in a swi4∆ strain which resulted inpredominantly large, unbudded cells (Gray et al., 1997). Thesynthetic lethality of glc7-10 and PKC1K853R overexpressionat 26°C was suppressed by 1 M sorbitol, demonstrating thatboth the cell integrity and morphology defects of glc-10 wereenhanced by reduced Pkc1p signalling.

glc7-10 displays aberrant actin localizationSince Pkc1p has been shown to mediate the cell-cycle-dependent organization of the actin cytoskeleton controlled byTor2p (Helliwell et al., 1998b) we examined actin organizationin the glc7-10 strain grown at the permissive and restrictivetemperatures. A diploid strain homozygous for glc7-10 wasused to facilitate actin visualization. To analyse the effect ofincreased dosage of PKC1, HCS77/WSC1/SLG1 or MPK1 onactin localization, the homozygous glc7-10 diploid strain wastransformed with either the control vector (YEplac195) orcomparable plasmids carrying the appropriate gene. In controlexperiments, the presence of YEplac195 was found not to alteractin localization (not shown). We found that at 26°C, actincables were visible in glc7-10 cells and the polarization of

Fig. 8. The glc7-10 defect is additive with a bck1 deletion. Diploidstrains homozygous for glc7-10 (PAY770D; sector C), bck1∆::URA3(PAY755D; sector A), glc7-10 bck1∆::URA3 (PAY760D; sector D)or a wild-type control (AYS927; sector B) were plated onto YPD(with or without 1 M sorbitol) for 4 days at the indicatedtemperatures. Double glc7-10 bck1∆::URA3 strains required sorbitolfor growth even at 26°C.

Fig. 9. glc7-10 is super-sensitive tooverproduction of catalytically inactive Pkc1p.In A, haploid glc7-10 (PAY700-4) or wild-typeGLC7 (PAY704-1) strains were transformedwith either pGAL-PKC1, pGAL-PKC1K853R orthe empty vector pRB1438 and streaked onsynthetic complete selective mediumcontaining either galactose or glucose for 4days at 26°C. In the presence of galactose,pGAL-PKC1 and pGAL-PKC1K853R induceoverexpression of wild-type or catalytically-inactive Pkc1p respectively from the GALpromoter. In B, the morphologies of glc7-10 orwild-type GLC7 cells harbouring pGAL-PKC1K853R and plated on galactose wereanalysed by differential interference contrastmicroscopy. Highly aberrant, elongated budswere noted in the glc7-10 strain overexpressingthe ‘kinase-dead’ K853R version of Pkc1p.

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cortical actin patches throughout the cell cycle was largely asseen in wild-type strains (Fig. 10A). We also noted a higherthan normal proportion of large-budded cells which had actinpatches in the mother and daughter cells but hardly everobserved any large-budded cells with an actin ring at the budneck. In contrast, after growth at 37°C for 4 hours actin cableswere rarely visible (Fig. 10A). Some cells with small- or

medium-sized elongated buds displayed polarized localizationin the bud, but the majority of cells had large, aberrantly-shaped buds and showed completely depolarized cortical actinpatches, with similar densities of actin patches in both motherand bud and absence of an actin ring at the bud neck (Fig. 10A,upper panel). Similar results were obtained on shifting cells tothe non-permissive temperature for only 2 hours (data not


Fig. 10. Actin localization in glc7-10 strains.(A) PAY770D, a homozygous diploid glc7-10/glc7-10 strain carrying YEplac195, wasgrown at 26°C in synthetic complete selectivemedium and the culture split, either continuingincubation at 26°C or shifting the temperatureto 37°C for a further 4 hours. Cells were fixedwith formaldehyde and actin was stained withrhodamine-phalloidin. (B) PAY770Dtransformed with high-copy plasmids carryingeither HCS77/WSC1/SLG1 (2µ HCS77/SLG1)or PKC1 (2µ PKC1), both of which suppressthe Ts− phenotype of glc7-10 (Fig. 6), and alsowith the non-suppressing MPK1 gene (2µMPK1). Cells were grown in selective mediumprior to temperature shift to 37°C for 4 hoursbefore treating as in A.


shown). In glc7-10 cells harbouring the suppressing multicopyPKC1 plasmid, actin cables were visible and the cortical actinpatches localized in the usual cell cycle-dependent manner(Fig. 10B). High-copy PKC1 also fully suppressed theelongated bud morphology of the glc7-10 cells. In contrast,actin cables were not visible in glc7-10 cells harbouring themulticopy MPK1 plasmid and the distribution of actin patchesin these cells resembled that in the glc7-10 strain transformedwith the control plasmid (Fig. 10A). In this strain we also noteda significant proportion of enlarged, small-budded cells witha completely depolarized actin cytoskeleton. In furtherexperiments where the glc7-10 strain was transformed withthe suppressing multicopy HCS77/WSC1/SLG1 plasmid, weobserved a much more normal polarization of the actincytoskeleton, although some buds were still somewhatabnormal in their morphology (Fig. 10B). Like high-copyHCS77/WSC1/SLG1, suppression of glc7-10 by increaseddosage of ROM2 was similarly incomplete compared withPKC1 suppression (not shown).

DISCUSSION

We have generated a novel, temperature-sensitive, GLC7 allele(glc7-10) which results from a relatively conservativesubstitution of leucine in place of phenylalanine 135, a residuethat is conserved in all members of the PPP family of proteinphosphatases. At the restrictive temperature, this mutationleads to a number of defects in cell growth and divisionincluding an elongated bud morphology, delocalized corticalactin, an osmoremedial cell integrity defect and an arrest of thecell division cycle after DNA replication but before themetaphase to anaphase transition. Thus dephosphorylation byPP1 is required for normal cell wall integrity, bud developmentand cell cycle progression. In Aspergillus nidulans, PP1 hasalso been shown to play a role in cell morphology and cell wallintegrity (Borgia, 1992; Doonan and Morris, 1989). However,in contrast to the glc7-10 phenotype which showshyperpolarised bud growth, A. nidulans bimG11 mutants failto establish germ tube polarity and lyse after excessiveswelling. The glc7-10 growth defect is efficiently suppressedby PKC1 in high copy, while elevated dosage of other genesknown to act in a positive manner upstream of Pkc1p are alsogood suppressors. Conversely, the glc7-10 defect issynthetically lethal with loss of function mutations in the MAPkinase pathway that lies downstream of Pkc1p. In addition, glc-10 cells are highly sensitive to expression of a ‘kinase-dead’mutant Pkc1p that is predicted to reduce Pkc1p pathwaysignalling. These genetic interactions with a pathway wellknown to be involved in promoting cell integrity and polarizedgrowth underline the importance of Glc7p dephosphorylationfor cell wall integrity and morphology.

The cell integrity defect of glc7-10 was less severe than thatof mutations that abolish signalling through MAP kinasepathway downstream of Pkc1p. Thus glc7-10 showed a clearcell integrity defect only on transfer from high to lowosmolarity conditions and after a short period of growth underthe restrictive conditions. By comparison, loss of BCK1function led to a significant cell lysis defect under less harshconditions and without the necessity for growth at highertemperatures (Fig. 5). This difference may in part relate to the

difference between complete loss of function (in the case ofbck1∆) compared with progressive loss of function of a mutantprotein at higher temperatures. Since GLC7 is an essentialgene, it is unfortunately not possible to test the effect ofcomplete loss of function on cell integrity. Although PKC1 andits upstream activators were good dosage suppressors of theglc7-10 mutant, it failed to be suppressed by high copy MKK1,MPK1 or the dominant activated BCK1-20. This pattern ofsuppression has been noted in at least two other instances. Thusthe double hcs77/wsc1/slg1∆ wsc3∆ mutant is suppressed byhigh-copy PKC1 but not MPK1 (Verna et al., 1997), while the‘mid’ (α-factor induced death) phenotype of mid2∆ is alsosuppressed by elevated dosage of PKC1 and its upstreamactivators but not by MPK1 or BCK1-20 (Ketela et al., 1999).By comparison, other genes such as PPZ1, PPZ2 and KRE6,which have been proposed to lie on additional pathwaysdownstream of PKC1, were able to suppress (at least partially)the glc7-10 temperature-sensitivity. The most straightforwardinterpretation of these data is that the dosage suppressors donot act by enhancing Bck1p-Mkk-Mpk1p signalling but ratherthat other pathways downstream of Pkc1p may mediate thiseffect. However, we cannot rule out the possibility that theupstream elements are simply more potent activators of Bck1p-Mkk-Mpk1p signalling when present in high copy and that theglc7-10 suppression is at least in part signalled via Mpk1p.

Dephosphorylation by PP1 might promote cell integrity in anumber of different ways. Thus PP1 might activate enzymesinvolved in cell wall synthesis, stimulate secretion of new cellwall material, be involved in polarization of secretion, activatetranscription of cell wall genes or play a role in the signallingpathways that regulate these processes. Interestingly, PP1 isrequired for vesicle fusion in yeast (Mayer et al., 1996; Peterset al., 1999) and may therefore be important for supporting thelevel of secretion required for proper cell wall synthesis andremodelling during growth. Since the severity of the defect inglc7-10 appears to depend on the level of Pkc1p pathwayfunction, it could alternatively be that PP1 dephosphorylationstimulates signalling through Pkc1p and its downstreampathways. However, the synthetic lethality of glc7-10 mpk1∆and glc7-10 bck1∆ argues that the phosphatase is not a positiveactivator of the MAP kinase module itself. In addition to actingon the other candidate pathways downstream of Pkc1p, glc7-10 might reduce the cell’s ability to activate Pkc1p bypromoting hyperphosphorylation of activators such as Rho1por Rom2p.

The glc7-10 mutant also showed delocalization of thecortical actin cytoskeleton at the restrictive temperature. Asimilar actin delocalization is seen in mpk1 MAP kinasemutants, while the synthetic lethality seen between mpk1mutations and either act1-1 or myo2-66 underline thecontribution of signalling through the Mpk1p MAP kinasepathway to the proper organization of the cytoskeleton(Mazzoni et al., 1993). Taken together with the proposal thatthe effects of tor2 mutations on the actin cytoskeleton aremediated via Pkc1p and Mpk1p (Helliwell et al., 1998b), ourresults are consistent with effects of the glc7-10 mutation onPkc1p-Mpk1p-mediated signalling, although we cannot ruleout effects which are independent of this pathway. The glc7-10 strain additionally showed an unconditional loss of the actinring at the bud neck in cells late in the cell division cycle,although cortical patches clustered around the site of septation

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could sometimes be seen (e.g. Fig. 10A). This is consistentwith the findings of Bi et al. (1998) that the actin ring is notessential for septation.

Over half the cells in glc7-10 cultures shifted to therestrictive temperature developed elongated buds (Fig. 3).Interestingly, a rom2∆ mutant (Manning et al., 1997), annhp6A∆ nhp6B∆ (Costigan et al., 1994) double mutant, and abro1∆ mutant (Nickas and Yaffe, 1996), all of which are knownto play a role in Pkc1p-Mpk1p signalling, show a proportionof cells with elongated buds, but never at the high levels seenin glc7-10. Elongated bud morphology can result from anumber of different perturbations to the cell. Cells deficient inClb•Cdc28p CDK function or which cannot degrade the B-typecyclin inhibitor Sic1p develop elongated buds (Schwob et al.,1994; Surana et al., 1991), but this is associated withhyperpolarisation of the actin cytoskeleton (Lew and Reed,1993) which is not seen in glc7-10 strains. Similarly, theelongated bud morphology shown by pseudohyphal cells isalso associated with hyperpolarized cortical actin (Cali et al.,1998) and is therefore distinct from what is seen in the glc7-10 mutant. Interestingly, another glc7 mutation (glc7Y–170) hasbeen found to suppress loss of function mutations in the smallGTPase Cdc42p (cdc42-1) and in Cdc24p (cdc24-4), itsguanine nucleotide exchange factor (Hisamoto et al., 1994),while an effector domain cdc42 mutant (cdc42V44A) alsoshowed an osmotically-remedial, elongated bud morphologydefect (Richman et al., 1999). Cdc42p is required for properlocalization of the septins, which form a ring structure at thebud neck and which also lead to an elongated bud phenotypewhen their function is compromised (Longtine et al., 1996).Thus perhaps the elongated bud morphology of glc7-10 reflectsan involvement of PP1 in some aspect of Cdc42p or septinregulation that is required for proper bud formation.

It is clear from our data that the morphology and cell lysisdefects of the glc7-10 mutant are intimately linked. Thus lossof cell integrity and elongated bud morphology are promotedby expression of PKC1 K853R in glc7-10, while high-copyPKC1 suppresses both defects. It is also striking that theproportion of inviable glc7-10 cells after 4 hours at 37°Cclosely mirrors the fraction of cells with elongated buds(compare Figs 1D and 3). It is therefore possible that the celllysis defect is a response of the mutant cells to the developmentof abnormally elongated buds. However, since the conditionsthat suppress glc7-10 correct the morphology defect as well, itseems more likely that glc7-10 affects a common step requiredfor both proper morphological development and cell integrity.

In addition to the cell lysis defect, an important feature ofthe glc7-10 phenotype is that cells arrest at the G2/M phase ofthe cell cycle, with fully replicated DNA and duplicated SPBsbut before spindle elongation and chromosome segregation.Consistent with this we have recently shown that the glc7-10mutation is associated with a kinetochore defect and that thiscell cycle arrest is dependent on the spindle checkpoint(Sassoon et al., 1999). The glc7-10 mutation is associated withreduced ability of kinetochores to bind microtubules in vitro,accompanied by a severe chromosome missegregation defectat higher temperatures in vivo. In addition, we have shown herethat the glc7-10 mutation adversely affects spindle orientationin a significant fraction of the mutant cells. The kinetochoreand cell lysis/morphology defects of glc7-10 appear not toresult from a defect in the dephosphorylation of a single critical

substrate, since neither 1 M sorbitol nor high-copy PKC1 orHCS77/WSC1/SLG1, each of which are effective suppressorsof the growth and cell lysis defect of glc7-10, suppress thechromosome missegregation phenotype of the phosphatasemutant (not shown). Thus analysis of the glc7-10 mutation hasrevealed essential functions of yeast PP1 in regulation of cellintegrity (this work), membrane fusion (Peters et al., 1999) andkinetochore function (Sassoon et al., 1999).

The authors thank David Levin, Gerhard Paravicini, Anja Schmidtand Mike Hall, Howard Bussey, Brian Morgan and Lee Johnston, JoeGray, Jason Kahana and Doug Stirling for plasmids. Thanks are alsodue to Kathryn Ayscough for help with actin staining and use of theCCD camera. We thank Doug Stirling and Kathryn Ayscough forhelpful discussions. This work was supported by the Wellcome Trust(Project Grants 036234, 046956 and 049778 to M.J.R.S.).

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