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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.8.3693–3701.2001

Aug. 2001, p. 3693–3701 Vol. 67, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretionof Mouse a-Amylase from Saccharomyces cerevisiae

BI-DAR WANG1 AND TSONG-TEH KUO1,2*

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115,1 and Institute of Botany,National Taiwan University, Taipei 106,2 Taiwan

Received 27 December 2000/Accepted 30 May 2001

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycledependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels ofmouse a-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cellmitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activitiesof PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in a-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, comparedto the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as acdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and preventsthe defective bud morphology induced by overproduction of a-amylase. Our data also suggest that high levels ofa-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous a-amy-lase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.

The eukaryotic cell cycle is controlled by members of thecyclin-dependent kinase (Cdk) protein family (30). The CdkCdc28 plays an important role in the initiation of mitosis inSaccharomyces cerevisiae (34, 35, 41), and its association withB-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 isrequired for entry into mitosis (15, 16, 22, 37, 41). Inactivationof the cyclin B (Clb)-Cdc28 kinase, also known as maturation-promoting factor (MPF), is a key regulatory event in mitosis(39). Multiple pathways for regulation of MPF activity existand can affect cell mitosis. For example, Cdc55 is a regulatorysubunit of protein phosphatase 2A (PP2A) and has been im-plicated in a variety of cell procresses, including exit frommitosis (17, 19). The Cdk inhibitor Sic1 may also play a role inmitotic exit (38, 50).

Cell cycle progression may be correlated with protein pro-duction in yeast or other eukaryotic cells. For example, anti-body synthesis and the secretion rate in murine hybridomacells are regulated during the cell cycle (1, 9, 25, 28). Withrespect to cell cycle dependency and foreign protein produc-tion, most of the work has focused on yeast as a model system.For example, Uchiyama et al. (45) reported that the specificsecretion rate of rice a-amylase fluctuated during the cell cycleand reached a maximum during the M phase, although thebasis of the cell cycle dependency was unknown. They alsodeveloped a mathematical model describing the cell cycle de-pendency of rice a-amylase production in yeast cultured in afed-batch fermentation (46).

In this study, we overexpressed mouse a-amylase in S. cer-evisiae to determine if high levels of foreign proteins affect cellmitosis or cell integrity. We examined the levels of PP2A,Cdc55, and MPF in M-phase cells to determine if they wereinfluencing the timing of mitosis. Our experiments tested the

effects of the synthesis of foreign proteins on the mechanism ofthe cell cycle perturbation and checkpoint response in yeast.

MATERIALS AND METHODS

Yeast strains, plasmids, and cell growth. The yeast strains used in this studywere TL154, 20B12, NI-C, NI-D4, and W303-derived strains (Table 1). TL154 isa moderate-level-secretion strain, and 20B12 is a low-level-secretion strain. NI-C(7) and NI-D4 (51) are oversecreting strains derived from the parental strain20B12 (6) that were used to express and secrete high levels of a-amylase. Cellswere grown in the following media (all percentages reflect weights per volume):YP (1% yeast extract, 2% peptone), YPD (1% yeast extract, 2% peptone, 2%dextrose), YNBD (0.17% yeast nitrogen base without amino acids and ammo-nium sulfate, 0.5% ammonium sulfate, 2% dextrose) supplemented with uraciland leucine, and YPDS agar (1% yeast extract, 2% peptone, 2% dextrose, 2%soluble starch, 2% agar). Plasmid pMS12 (23) contains the mouse salivarya-amylase cDNA under the control of the ADH1 promoter and was transformedinto yeast strains (5). The transformed strains were cultivated in YNBD-uracil(0.002%)-leucine (0.003%) for 2 to 3 days. Colonies formed on YNBD-uracil-leucine agar were transferred to YPDS agar to identify transformants that ex-creted high levels of a-amylase. These transformants had clear zones around thecolonies as a result of the degradation of starch in the medium (8). Transfor-mants grown in YNBD-uracil-leucine were also transferred to YPD broth andcultivated for 4 to 6 days at 28°C for determination of growth curves; cell numberwas estimated by direct counts in a hemacytometer chamber or by measurementof optical density at 600 nm.

Scanning electron microscopy. Yeast cells grown in YPD broth were trans-ferred to 0.22-mm-pore-size filters and fixed for 1 to 2 h at room temperaturewith 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0).Fixed cells were washed three times with phosphate buffer, exposed for 1 to 2 hat room temperature to 1% (wt/vol) osmium tetroxide in phosphate buffer, andthen dehydrated in a graded series of ethanol solutions. After being dried withliquid CO2 and coated with gold and palladium, the cells were examined with ascanning electron microscope (model JSM T330A; JEOL, Tokyo, Japan).

DAPI staining and flow cytometry. For DAPI (49,69-diamidino-2-phenylin-dole) staining, cells were harvested by centrifugation, fixed with 95% (vol/vol)ethanol, and exposed to DAPI (1 mg/ml) as described previously (44). Stainedcells were examined with a microscope (Nikon, Tokyo, Japan) equipped withepifluorescence illumination at 340 to 365 nm. Flow cytometry was performed aspreviously described (49); cells (107) were harvested at various times, fixed withethanol, and stained with propidium iodide (16 mg/ml).

Cell cycle synchronization. To arrest yeast cells in S phase, cultures weregrown at 28°C, diluted to an optical density at 600 nm of 0.2, and cultured for 3 hat 24°C in the presence of 0.2 M hydroxyurea (Sigma, St. Louis, Mo.). For

* Corresponding author. Mailing address: Institute of MolecularBiology, Academia Sinica, Nankang, Taipei 11529, Taiwan. Phone: 8862 27899213. Fax: 886 2 27826085. E-mail: [email protected].

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M-phase arrest, cultures were grown, diluted, and cultured for 3 h at 24°C in thepresence of 15 mg of nocodazole (Sigma) per ml. The S- and M-phase-arrestedcells were filtered, washed, suspended in fresh YPD medium, and cultured at28°C.

Preparation of cell extracts and immunoblot analysis. Cells were harvested bycentrifugation (4,000 3 g for 5 min), washed with 10 mM Tris-HCl (pH 7.5), andresuspended in 200 ml of lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA,50 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mg of aprotinin perml, 1 mg of leupeptin per ml, and 2 mg of pepstatin per ml). After addition of anequal volume of glass beads, the cells were broken by vigorous vortexing for 3min at 4°C. A portion (10 ml) of the resulting cell lysate was removed for assayof protein concentration, and after the addition of 100 ml of 33 sodium dodecylsulfate (SDS) sample buffer to the remainder, the resulting mixture was boiledfor 3 min. The glass beads and cell debris were removed by centrifugation(12,000 3 g for 30 min at 4°C), and a portion of the remaining cell extract (50 mgof total protein) was fractionated by SDS-polyacrylamide gel electrophoresis ona 10% gel. The gel was soaked in transfer buffer containing 10% (vol/vol)methanol before transfer of proteins to a polyvinylidene difluoride membranewith the use of an Electroblotter (Novex, San Diego, Calif.). The membrane wasincubated for 1 h with 5% (wt/vol) nonfat dried milk in Tris-buffered saline (pH7.5) containing 0.05% (wt/vol) Tween 20 and then incubated overnight at 4°Cwith monoclonal antibodies to Clb2 (1:300 dilution; Santa Cruz Biotechnology,Santa Cruz, Calif.), to Cdc28 (1:500 dilution; Calbiochem, San Diego, Calif.), toCdc55 (1:200 dilution; Santa Cruz Biotechnology), or to human a-amylase (1:1,000 dilution; Sigma). Immune complexes were detected by alkaline phos-phatase-conjugated secondary antibodies and enhanced chemiluminescence.

Measurement of a-amylase activity. Cells from YPD cultures (1 ml) wereharvested by centrifugation (4,000 3 g for 5 min). The resulting pellet wascollected for preparation of a cell extract as previously described, and the su-pernatant was buffered with 15 mM HEPES-NaOH (pH 7.0). Portions (20 ml) ofthe cell extract and buffered supernatant were used for determination of intra-cellular and secreted a-amylase activities (7), respectively, with Sigma diagnostickit 577-3.

Histone H1 kinase assay. Clb2 was immunoprecipitated from yeast lysates (50mg of total protein) using 10 ml of protein A (Sigma). For histone H1 kinase(Clb2-Cdc28 kinase) analysis, the Clb2 immunoprecipitates were preincubated at37°C for 5 min. Subsequently, 8 ml of a solution containing 50 mM Tris-HCl (pH7.5), 10 mM MgCl2, 750 mM ATP, 2 mg of bovine histone H1 (Sigma), and 10mCi of [g-32P]ATP (Amersham, Little Chalfont, Buckinghamshire, United King-dom) was added. The reaction was incubated at 37°C for 10 min and was stoppedby adding 30 ml of 23 loading buffer, and the mixture was heated at 95°C for5 min and loaded onto a 10% SDS–polyacrylamide gel. The gel was fixed anddried, and the phosphorylated H1 was visualized by autoradiography.

Assay of protein phosphatase activity. The activity of PP2A in cell extracts wasmeasured with a nonradioactive serine/threonine protein phosphatase assay sys-

tem (Promega, Madison, Wis.). The synthetic phosphopeptide RRA(pT)VA wasused as the substrate; this peptide is a good substrate for PP2A but a poorsubstrate for protein phosphatase 1. Cell extracts were applied to a spin columnpacked with Sephadex G-25 (Promega) in order to remove free phosphate. Assayof phosphatase activity was initiated by mixing 40 ml of phosphate-free extractswith 360 ml of a premixed reaction solution (100 mM phosphopeptide, 50 mMimidazole [pH 7.2], 0.2 mM EGTA, 0.02% [vol/vol] 2-mercaptoethanol, and 0.1mg of bovine serum albumin/ml). Bacterially expressed human inhibitor 2 (I-2;Sigma) and okadaic acid (Sigma) were included in the assay mixture to inhibitthe activities of type 1 and type 2 phosphatases, respectively. The reaction wasterminated by addition of an equal volume of Molybdate Dye-additive Mixture(Promega). The resulting molybdate-malachite green-phosphate complex wasquantitated by measurement of absorbance at 630 nm with a spectrophotometer(Beckman model DU-68).

RESULTS

Morphology of cells secreting high levels of a-amylase.Transformed cells that expressed and secreted a-amylase hadan abnormal morphology (Fig. 1A, C, E, and G). TransformedTL154-14 and NI-C-14 cells that secreted moderate (;500 Uof activity per liter of culture medium) and high (1,500 U/liter)levels of a-amylase (Fig. 2B), respectively, formed elongated

FIG. 1. Phenotypes of yeast cells oversecreting mouse a-amylase.Nontransformed and transformed S. cerevisiae strains were grown inYPD medium at 28°C for 4 days and then examined by scanningelectron microscopy. The phenotypes of nontransformed TL154 (A),20B12 (C), NI-C (E), and NI-D4 (G) cells and of their respectivepMS12-transformed TL154-14 (B), 20B12-14 (D), NI-C-14 (F), andNI-D4-14 (H) cells are shown. Scale bar, 5 mm.

TABLE 1. Yeast strains used

Strain Genotype Referenceor source

TL154 a trp1 leu2 620B12 a trp1 pep4 6NI-C a trp1 pep4 6NI-D4 a trp1 ura3 pep4 This studyW303 a trp1-1 leu2-3 ura3-1 his3-11 ade2-1

can1-100A. W. Murray

ADR508 W303 CDC28-HA-URA3 A. W. MurrayADR640 W303 cdc28-Y19F-HA-URA3 A. W. MurrayBY4741 a his3D1 leu2D0 met15D0 ura3D0 This studyY03386 BY4741 pph22::kanMX4 This studyTL154-14 TL154/pMS12 (2m ADHI-AMY TRP1) This study20B12-14 20B12/pMS12 (2m ADHI-AMY TRP1) This studyNI-C-14 NI-C/pMS12 (2m ADHI-AMY TRP1) This studyNI-D4-14 NI-D4/pMS12 (2m ADHI-AMY TRP1) This studyADR508-14 ADR508/pMS12 (2m ADHI-AMY TRP1) This studyADR640-14 ADR640/pMS12 (2m ADHI-AMY TRP1) This studyBY4741-14 BY4741/pAMY (2m ADHI-AMY URA3) This studyY03386-14 BY4741/pAMY (2m ADHI-AMY URA3) This studyDXN-8 NI-D4/pXYN (2m PGK1-XYN2 URA3) This studyAST-3 TL154-14/pGAM (2m PGK1-GAM1 LEU2) This studyA18ST-3 TL154-14/pGAM (2m PGK1-GAM1 LEU2)

pMS12 (2m ADHI-AMY TRP1)This study

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buds (Fig. 1B and F). Secretion of a-amylase at even higherlevels (3,500 U/liter) by transformed NI-D4-14 cells (Fig. 2B)resulted in the formation of highly elongated buds (Fig. 1H). Incontrast, a low level of a-amylase secretion (;20 U/liter) by20B12-14 cells (Fig. 2B), from which the plasmid was lost after10 generations (data not shown), did not result in morphologicchanges (Fig. 1D).

We also determined the percentages of cells exhibiting elon-gated buds and the extents of a-amylase secretion at varioustimes. Secretion of a-amylase by transformed NI-C-14 andNI-D4-14 cells was first detected after culture for 8 h, at whichtime ;10% of the cells exhibited elongated buds (data notshown). Both the average amount of a-amylase activity in theculture medium and the average percentage of budded cellsincreased over similar time courses in TL154-14, NI-C-14, andNI-D4-14 cultures (Fig. 2). Moderate, high, and very highlevels of a-amylase secretion by TL154-14, NI-C-14, and NI-D4-14 strains, respectively, resulted in the formation of elon-gated buds in 7.6, 12, and 27% of cells, respectively, aftercultivation for 95 h.

We saw no morphologic abnormalities following phase-con-trast microscopy of TL154, 20B12, NI-C, or NI-D4 cells har-boring the vector pMA56, which does not contain a-amylasecDNA (data not shown). Thus, high-level secretion of a-amy-lase (rather than transformation per se) affects bud morpho-genesis in S. cerevisiae.

Cell cycle arrest of cells oversecreting a-amylase. Morphol-ogy similar to that observed for strains oversecreting a-amylase

is often associated with a block in cell cycle progression. Weexamined the DNA content of cells expressing this mouse-a-amylase protein by flow cytometry. Cultures of all transformedstrains were asynchronous and had a bimodel distribution ofDNA content at the zero time point, with peaks at 1 and 2N(Fig. 3). 20B12-14 cells, which secrete only a very low level ofa-amylase, remained asynchronous during the 24-h cultureperiod (Fig. 3B). In contrast, NI-D4-14 cells, which secrete avery high level of a-amylase, began to accumulate cells with aG2/M DNA content after culture for 4 h (Fig. 3D). TL154-14(Fig. 3A) and NI-C-14 (Fig. 3C) cells, which secrete moderateand high levels of a-amylase, respectively, showed accumula-tion of G2/M cells after 8 h. After 24 h, TL154-14 and NI-C-14cells exhibited partial arrests in G2/M whereas .80% of NI-D4-14 cells carrying pMS12 exhibited a DNA content of 2N,indicating a delay in transit through the G2 or M phase of thecell cycle. The correlation of high-level secretion of a-amylasefrom yeast cells with a DNA content typical of G2/M suggeststhat the checkpoint that governs the transition between G2 andM or M and G1 is impaired in these cells.

The elongated buds of TL154-14, NI-C-14, and NI-D4-14carrying pMS12 each contained two nuclei (Fig. 4A, 4E and4G), and about 5% of NI-C-14 (Fig. 4E) and NI-D4-14 (Fig.4G) cells were segmented and contained multiple nuclei (threeto five nuclei). Cells secreting a-amylase at high or very highlevels thus appeared to be impaired in cell division, with in-

FIG. 2. Relation between a-amylase production and altered budmorphology in yeast cells. Yeast cells (TL154-14, NI-C-14, and NI-D4-14) carrying pMS12 were cultivated in YPD broth at 28°C, and at theindicated times, portions of the culture were removed for analysis ofcell morphology and a-amylase secretion. Cell morphology was exam-ined by phase-contrast microscopy, and the average percentage of cellswith elongated buds (●, those more than twice as long as normal buds)was calculated. The average activity of a-amylase (E) in the culturemedium was determined after removal of cells by centrifugation. Dataare means of values from nine experiments with three cultures (TL154-14, NI-C14, and NI-D4-14), with each culture being repeated threetimes with similar results.

FIG. 3. Flow cytometric analysis of the DNA content of yeast cellssecreting a-amylase. TL154-14 (A), 20B12-14 (B), NI-C-14 (C), andNI-D4-14 (D) yeast cells harboring pMS12 were grown for 12 h at 28°Cin YNBD plus uracil-leucine and then transferred to fresh YPD. Cellswere harvested at zero time as well as at early log phase (4 h), mid-logphase (8 h), and stationary phase (24 h) for analysis of DNA contentby staining with propidium iodide and flow cytometry. Peaks corre-sponding to DNA contents of 1 and 2N are indicated by arrows.

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complete separation between newly budding cells and themother cell.

a-Amylase secretion and the accumulation of Clb2. Levelsof CLB1 and CLB2 transcripts and of the encoded G2 cyclinsexhibit marked periodicity in S. cerevisiae, peaking about 10min before anaphase (15, 16, 37, 42). The kinase activity of theClb-Cdc28 complex shows a similar periodicity (16, 21). Eightyto 85% of nontransformed NI-C and NI-D4 cells had a DNAcontent of 1N after release from S arrest and again 120 minlater (Fig. 5A, left panels), whereas 65 to 70% of transformedNI-C-14 and NI-D4-14 cells had a DNA content of 2N 120 minafter release from S arrest (Fig. 5A, right panels).

The level of Clb2 protein in a-amylase-overproducing cellsdid not exhibit the periodicity seen in nontransformed cells(Fig. 5B, left panels). In AMY cells (NI-D4-14), which had the

highest level of a-amylase secretion, the amount of Clb2 in-creased 40 min after release from S arrest. Clb2 protein thenaccumulated for 100 min (40 to 140 min after S release) andfinally decreased again 160 min after S release (Fig. 5B). Thesynthesis of the mouse a-amylase in transformed yeast cellsappeared to be highly periodic (Fig. 5B), peaking in the G2 andM phases, similar to the periodicity of Clb2. These resultssuggest that high-level secretion of heterologous a-amylase inyeast is correlated with a G2-M delay and an associated defectin the regulation of Clb2 protein levels.

Effect of a-amylase secretion on PP2A and MPF (Clb-Cdc28kinase) activities during mitosis. In NI-D4 cells, levels ofCdc55, a regulatory subunit of PP2A, appeared relatively sta-ble for 2 h after release from nocodazole-induced arrest,whereas the level of this protein in cells overproducing a-amy-lase (NI-D4-14) began to decrease 90 min after release fromnocodazole arrest (Fig. 6A). MPF (histone H1 kinase) activitydecreased after the amylase-oversecreting cells were releasedfrom nocodazole arrest (Fig. 6A, right panel), compared withthe level in nontransformed cells (Fig. 6A, left panel). Thus,MPF (Clb-Cdc28 kinase) activity appears to be defective incells secreting high levels of a-amylase.

We also measured PP2A activity with a synthetic phos-phopeptide substrate in extracts of cells released from nocoda-zole arrest. The chosen phosphopeptide is a poor substrate forprotein phosphatase type 1, and we measured phosphataseactivity in the absence and presence of specific inhibitors oftype 1 (I-2) and type 2 (okadaic acid) phosphatase activity.Phosphatase activity in the presence of I-2 increased afterrelease from nocodazole arrest in both nontransformed and a-amylase-overproducing cells; however, the increase was moremarked in the nontransformed cells and the activity subse-quently decreased in the a-amylase-overproducing cells to;50% of the initial value (Fig. 6B). These results suggest thatPP2A activity is negatively regulated in a-amylase-overproduc-ing cells.

Cdc28VF prevents MPF inactivation and aberrant buds inamylase-overproducing cells treated with nocodazole. Phos-phorylation of Tyr19 inhibits Cdc28 H1 kinase activity in S.cerevisiae (4). We transformed the cdc28VF mutant (in whichThr18 was changed to Val and Tyr19 was changed to Phe) withpMS12 to overproduce amylase. The amounts of Cdc28 re-mained relatively constant and did not differ markedly betweenthe a-amylase-expressing wild type and cdc28 mutants (Fig. 7).The wild-type cells overproducing a-amylase accumulatedhigher levels of Clb2 than cdc28 mutant cells producing a-amy-lase (Fig. 7A). However, cells overproducing a-amylase had alower level of Clb2-Cdc28 kinase (Fig. 7A, left panel) than thatof wild-type cells (Fig. 6A), whereas the cdc28VF mutationprevented the inactivation of Clb2-Cdc28 kinase (Fig. 7A, rightpanel). The cdc28VF mutation also suppressed the defectivebud morphology exhibited by wild-type cells overproducinga-amylase (Fig. 7B).

We also examined yeast strains that express and secrete highlevels of heterologous proteins, including mouse a-amylase,glucoamylase from Rhizopus orizae, or xylanase from Tricho-derma reesei (Fig. 7C). Overproduction of glucoamylase resultsin cells with large buds. Overproduction of glucoamylase andeither amylase or xylanase resulted in extremely elongatedbudded cells. These data suggest that the mitosis delay may not

FIG. 4. Distributions of nuclei in buds of yeast cells overproducinga-amylase. a-Amylase-overproducing yeast strains TL154-14 (A andB), 20B12-14 (C and D), NI-C-14 (E and F), and NI-D4-14 (G and H)harboring pMS12 were cultured for 20 h in YPD, fixed with ethanol,stained with DAPI, and visualized by fluorescence (A, C, E, and G) ordark-field (B, D, F, and H) microscopy. The positions of nuclei areindicated by arrowheads.



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be specific to a-amylase but that it instead is a response to theabnormally high levels of secretory proteins.

Influence of defective PP2A on cell mitosis and the cellintegrity of cells overproducing a-amylase. We examined wild-

type and pph22D cells overproducing amylase for their Clb2levels after cells were released from nocodazole arrest. Wild-type cells that overproduce amylase accumulated Clb2 proteinfor 150 min after M-phase release (Fig. 8A). However, pph22D

FIG. 5. Effects of high levels of a-amylase on DNA content and Clb2 abundance during cell cycle progression. (A) S-phase cells of nontrans-formed (left panels, NI-C or NI-D4) and pMS12-transformed (right panels, NI-C-14 or NI-D4-14) yeast strains were arrested in S phase bytreatment with hydroxyurea (0.2 M). Cells subjected to S-phase arrest were analyzed for DNA content by flow cytometry at 0 and 120 min afterrelease from S-phase arrest. (B) Effect on Clb2 abundance. Cell lysates were prepared at the indicated times after release from S-phase arrest asfor the experiment whose results are shown in panel A and subjected to immunoblot analysis with antibodies to Clb2, to Cdc28, or to a-amylase.Levels of Cdc28 protein were used as a protein loading control.

FIG. 6. High levels of a-amylase negatively regulate PP2A activityand cause defective MPF during mitosis. (A) Cdc55 abundance. Non-transformed (NI-D4) and a-amylase-overproducing cells (NI-D4-14)were subjected to growth arrest with nocodazole, released into YPDmedium at 28°C, and at the indicated times thereafter lysed and sub-jected to immunoblot analysis with antibodies to the regulatory B sub-unit (Cdc55) of PP2A. A protein sample withdrawn at each time pointwas examined for histone H1 kinase activity as described in Materialsand Methods. Protein levels of Cdc28 were used as a protein control.(B) PP2A activity. Portions of the cell extracts analyzed for panel Awere assayed for phosphatase activity in the absence or presence of thetype 1 phosphatase inhibitor I-2 (0.1 mM) or the type 2 phosphataseinhibitor okadaic acid (OA) (2 nM). The reaction was performed for30 min at room temperature and at an extract protein concentration of200 mg/ml in the absence of divalent cations and free phosphate. Dataare expressed as nanomoles of phosphate generated per minute permilligram of extract protein and are means of values obtained from twoindependent experiments. Left panel, 2AMY1; right panel, 1AMY1.



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cells that overproduce amylase began to degrade Clb2 protein90 min after release from nocodazole arrest and Clb2 proteinappeared again 150 min after release from M phase, suggestingthat the lack of PPH22 suppresses the amylase-induced mitosisdelay. These data also provided direct evidence that the PPH22-encoding subunit of PP2A was affected by high levels of amylase.

pph21D pph22D cells have a double deletion that causes slowgrowth at 24°C and temperature-sensitive growth at 37°C (14,27). To test whether the amylase-induced PP2A defect cancause a growth defect similar to that of pph21D pph22D cells,we examined the effect of high osmolarity on growth in non-transformed cells and a-amylase-overproducing cells. Cellsoverproducing a-amylase displayed a partial arrest of prolifer-ation that was not suppressed by the osmotic stabilizer sorbitol(1 M) (Fig. 8B). In medium lacking 1 M sorbitol (Fig. 8C),amylase-overproducing cells died rapidly at 37°C (21% of cellswere viable after 8 h at 37°C), whereas in high-osmolaritymedium containing 1 M sorbitol (Fig. 8C), the cells died at alower rate (47% of cells were viable after 8 h at 37°C). Incontrast, nontransformed cells remained viable under all theconditions tested (Fig. 8C), indicating that a defect in cell wallintegrity was induced when a-amylase was overproduced.

DISCUSSION

High levels of amylase, PP2A, cell integrity, and nucleardivision. A variety of serine/threonine protein phosphataseshave been implicated in mitosis in various organisms, but theunderlying mechanisms through which they operate are notknown (10, 27, 29, 54). PP2A is a heterotrimeric protein thatconsists of a catalytic subunit and two regulatory subunits (Aand B), the latter of which confers substrate specificity to thecatalytic subunit (54). In S. cerevisiae, the regulatory B subunitof PP2A is encoded by the RTS1 and CDC55 genes (13, 19),the regulatory A subunit is encoded by the TPD3 gene (48),and the catalytic subunit is encoded by the PPH21 and PPH22genes (21, 27).

Genetic analysis implicates PP2A in mitosis and cellularmorphogenesis in yeast (19, 29, 53, 55). Lin and Arndt (27)showed that defects in PP2A induced S. cerevisiae cells toarrest with small or aberrant buds, at which sites the actincytoskeleton is disorganized and chitin deposition is delocal-ized. When released from hydroxyurea treatment (S-phase ar-rest), pph21 mutants have a reduced MPF activity but accu-mulate Clb2 at levels similar to those of wild-type cells (27).Minshull et al. (31) showed that MPF inactivation occurs with-

FIG. 7. Cdc28VF cured the defect in MPF activity and aberrant bud morphology in amylase-overproducing cells. (A) The a-amylase-overproducing wild-type strain (ADR508-14) and a cdc28YF mutant (ADR640-14) were arrested with nocodazole and released into fresh YPD.Samples were taken every 30 min to analyze the amounts of Clb2, the amounts of Cdc28, and Clb2-associated histone H1 kinase activity. Cells werelysed at the indicated times thereafter and subjected to immunoblot analysis with antibodies to Cdc55, Clb2, and hemagglutinin (HA) (againstCdc28-HA or Cdc28VF-HA). (B) Photographs of the wild-type strain (ADR508-14) and a cdc28 mutant overproducing a-amylase (ADR640).These cells were grown to stationary phase in YPD medium. (C) Photographs of wild-type strains overproducing heterologous glucoamylase (1GAM1) and xylanase (1 XYN2). Cell overproducing glucoamylase (AST-3 and A18ST-3) or xylanase (DXN-8) were grown to stationary phase inYPD medium at 28°C.



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out cyclin degradation in cdc55 mutant cells; cells released intonocodazole contain little MPF activity but accumulate mitoticcyclins in a manner similar to that of wild-type cells. In con-trast, cdc55D cells, which carried the cdc28 mutation, retainhigh MPF activity in the presence of nocodazole (31).

From our results, PP2A activity was negatively regulated dur-ing high-level secretion of a-amylase and consequently causeda decrease in MPF activity (Fig. 6 and 8A). We also found thatthe dephosphorylation of tyrosine-phosphorylated Cdc28, acritical step for MPF activation, was perturbed by high levels ofa-amylase (Fig. 7). The net effect was a delay in mitosis.

It is possible that high levels of amylase induce intracellu-lar stimuli or some sort of damage, either to the cell wall orto polysaccharide-decorated membrane proteins, that is per-ceived by a mitotic checkpoint response that halts cytokinesis.This hypothesis is supported by the production of large orelongated buds by recombinant yeast strains that produce highlevels of heterologous amylase, glucoamylase, or xylanase (Fig.7C). Thus, we hypothesize that high levels of heterologousproteins in yeast induce a checkpoint response to perturb post-mitotic events through negative regulation of PP2A, whichthen causes a defect in MPF and delays cytokinesis.

There is evidence that, in both mammalian cells and yeastcells, Rho-like GTPases are key regulators of signaling path-ways that link extracellular growth signals or intracellular stim-uli to organization of the actin cytoskeleton (18, 20, 26, 32, 43,47). Rho1 regulates cell wall biosynthesis (11, 36), and the

mitogen-activated protein kinase signaling transduction path-way is required for cell wall integrity (12). A direct role forPP2A in controlling cell integrity has been suggested (3). Forexample, BEM2 encodes GTPase-activating protein for smallG protein encoded by RHO1 (24, 33) and bem2 mutations cansuppress cdc55-1 (19). Moreover, bem2 mutants and temper-ature-sensitivity-negative pph22 strains display many commonphenotypic features, including temperature-dependent disrup-tion of the actin cytoskeleton, a bud growth defect, and asorbitol-remediated temperature-sensitivity-negative cell lysisdefect (14, 52). Our data also support the hypothesis that de-fective PP2A, induced by a-amylase overproduction, leads todefects in cell integrity. Such a defect can be rescued by treat-ment with 1 M sorbitol (Fig. 8).

Our data also show that the amylase-induced PP2A defectcauses a mitotic delay and the accumulation of cells with rep-licated DNA and separated nuclei (Fig. 4). Cells with dividednuclei also resulted from the defect in bud growth observed inpph22 cells at 37°C (14). Together, these data suggest thatPP2A is required for maintenance of polarized growth, cellintegrity, and nuclear division.

Cell lysis system. The cell wall of S. cerevisiae is a tough,rigid structure which presents a significant barrier to the re-lease of native or recombinant proteins. Lysis mutants provideone route to mechanical or chemical disruption of the cell wallthat precedes the recovery of yeast contents. Alvarez et al. (2)reported on the release of intracellular proteins, including

FIG. 8. High levels of amylase induced defective PP2A and triggered cell lysis. (A) Deletion of PPH22 suppresses the amylase-induced mitoticdefect. The a-amylase-overproducing wild-type strain (BY4741-14) and a pph22D mutant (Y03386-14) were arrested with nocodazole and releasedinto fresh YPD. Samples were taken every 30 min to analyze the amounts of Clb2 and Cdc28. Cells were lysed at the indicated times thereafterand subjected to immunoblot analysis with antibodies to Clb2 and Cdc28. (B) Phenotype of a-amylase-overproducing cells. Cultures were grownto a density of 5 3 107 to 8 3 107 cells/ml in YPD medium containing 1 M sorbitol at 28°C, subcultured in YPD medium containing or lacking1 M sorbitol, and grown for several generations overnight at 28°C. The activated cells were transferred to 37°C and monitored for their cell density(B) and cell viability (C). Serial dilutions for the determination of cell viability (7) were performed with 1 M sorbitol.



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virus-like particles, from an slT2 mutant by osmotic shock. Theuse of an srb1-1 mutant for similar purposes has also beendocumented (40). Zhang et al. (56) developed an approach totrigger cell lysis by a genetic switch of three genes involvedin cell wall biogenesis: PDE2, SRB1 (also called PSA1), andPKC1. The a-amylase-induced cell lysis process provides an-other alternative for the efficient secretion and release of het-erologous proteins by yeast.

In summary we hypothesize that high levels of a-amylaseinduce a checkpoint response, mediated by a cell integrity sig-naling pathway, and cause a negative regulation of PP2A, whichin turn causes mitosis delay and cell lysis. The a-amylase-in-duced mitotic block we observed supports the hypothesis thatPP2A has a role in the maintenance of bud morphology, nu-clear division, and cell integrity. In addition, pph22-inducedcell lysis and mitotic delay suggest an alternative approach tothe production of M-phase-dependent foreign proteins by thislysis system.

ACKNOWLEDGMENTS

We thank A. W. Murray and A. D. Ruder for providing cdc28VFmutants and D.-C. Chen and H. J. Huang for valuable suggestions anddiscussions.

This work was supported by biotechnology grant BT-89-01 from theAcademia Sinica. B.-D. Wang was supported by a postdoctoral fellow-ship from the Academia Sinica.

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