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. 2001 Aug;67(8):3693–3701. doi: 10.1128/AEM.67.8.3693-3701.2001

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

Bi-Dar Wang 1, Tsong-Teh Kuo 1,2,*
PMCID: PMC93073  PMID: 11472949

Abstract

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase 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 the cyclin-dependent kinase (Cdk) protein family (30). The Cdk Cdc28 plays an important role in the initiation of mitosis in Saccharomyces cerevisiae (34, 35, 41), and its association with B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 is required for entry into mitosis (15, 16, 22, 37, 41). Inactivation of 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 exist and can affect cell mitosis. For example, Cdc55 is a regulatory subunit of protein phosphatase 2A (PP2A) and has been implicated in a variety of cell procresses, including exit from mitosis (17, 19). The Cdk inhibitor Sic1 may also play a role in mitotic exit (38, 50).

Cell cycle progression may be correlated with protein production in yeast or other eukaryotic cells. For example, antibody synthesis and the secretion rate in murine hybridoma cells are regulated during the cell cycle (1, 9, 25, 28). With respect to cell cycle dependency and foreign protein production, most of the work has focused on yeast as a model system. For example, Uchiyama et al. (45) reported that the specific secretion rate of rice α-amylase fluctuated during the cell cycle and reached a maximum during the M phase, although the basis of the cell cycle dependency was unknown. They also developed a mathematical model describing the cell cycle dependency of rice α-amylase production in yeast cultured in a fed-batch fermentation (46).

In this study, we overexpressed mouse α-amylase in S. cerevisiae to determine if high levels of foreign proteins affect cell mitosis or cell integrity. We examined the levels of PP2A, Cdc55, and MPF in M-phase cells to determine if they were influencing the timing of mitosis. Our experiments tested the effects of the synthesis of foreign proteins on the mechanism of the cell cycle perturbation and checkpoint response in yeast.

MATERIALS AND METHODS

Yeast strains, plasmids, and cell growth.

The yeast strains used in this study were TL154, 20B12, NI-C, NI-D4, and W303-derived strains (Table 1). TL154 is a 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 strain 20B12 (6) that were used to express and secrete high levels of α-amylase. Cells were 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 ammonium sulfate, 0.5% ammonium sulfate, 2% dextrose) supplemented with uracil and leucine, and YPDS agar (1% yeast extract, 2% peptone, 2% dextrose, 2% soluble starch, 2% agar). Plasmid pMS12 (23) contains the mouse salivary α-amylase cDNA under the control of the ADH1 promoter and was transformed into 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 excreted high levels of α-amylase. These transformants had clear zones around the colonies as a result of the degradation of starch in the medium (8). Transformants grown in YNBD-uracil-leucine were also transferred to YPD broth and cultivated for 4 to 6 days at 28°C for determination of growth curves; cell number was estimated by direct counts in a hemacytometer chamber or by measurement of optical density at 600 nm.

TABLE 1.

Yeast strains used

Strain Genotype Reference or source
TL154 α trp1 leu2 6
20B12 α trp1 pep4 6
NI-C α trp1 pep4 6
NI-D4 α trp1 ura3 pep4 This study
W303 α trp1-1 leu2-3 ura3-1 his3-11 ade2-1 can1-100 A. W. Murray
ADR508 W303 CDC28-HA-URA3 A. W. Murray
ADR640 W303 cdc28-Y19F-HA-URA3 A. W. Murray
BY4741 a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 This study
Y03386 BY4741 pph22::kanMX4 This study
TL154-14 TL154/pMS12 (2μ ADHI-AMY TRP1) This study
20B12-14 20B12/pMS12 (2μ ADHI-AMY TRP1) This study
NI-C-14 NI-C/pMS12 (2μ ADHI-AMY TRP1) This study
NI-D4-14 NI-D4/pMS12 (2μ ADHI-AMY TRP1) This study
ADR508-14 ADR508/pMS12 (2μ ADHI-AMY TRP1) This study
ADR640-14 ADR640/pMS12 (2μ ADHI-AMY TRP1) This study
BY4741-14 BY4741/pAMY (2μ ADHI-AMY URA3) This study
Y03386-14 BY4741/pAMY (2μ ADHI-AMY URA3) This study
DXN-8 NI-D4/pXYN (2μ PGK1-XYN2 URA3) This study
AST-3 TL154-14/pGAM (2μ PGK1-GAM1 LEU2) This study
A18ST-3 TL154-14/pGAM (2μ PGK1-GAM1 LEU2) pMS12 (2μ ADHI-AMY TRP1) This study
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Scanning electron microscopy.

Yeast cells grown in YPD broth were transferred to 0.22-μm-pore-size filters and fixed for 1 to 2 h at room temperature with 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 h at room temperature to 1% (wt/vol) osmium tetroxide in phosphate buffer, and then dehydrated in a graded series of ethanol solutions. After being dried with liquid CO2 and coated with gold and palladium, the cells were examined with a scanning electron microscope (model JSM T330A; JEOL, Tokyo, Japan).

DAPI staining and flow cytometry.

For DAPI (4′,6′-diamidino-2-phenylindole) staining, cells were harvested by centrifugation, fixed with 95% (vol/vol) ethanol, and exposed to DAPI (1 μg/ml) as described previously (44). Stained cells were examined with a microscope (Nikon, Tokyo, Japan) equipped with epifluorescence illumination at 340 to 365 nm. Flow cytometry was performed as previously described (49); cells (107) were harvested at various times, fixed with ethanol, and stained with propidium iodide (16 μg/ml).

Cell cycle synchronization.

To arrest yeast cells in S phase, cultures were grown at 28°C, diluted to an optical density at 600 nm of 0.2, and cultured for 3 h at 24°C in the presence of 0.2 M hydroxyurea (Sigma, St. Louis, Mo.). For M-phase arrest, cultures were grown, diluted, and cultured for 3 h at 24°C in the presence of 15 μg of nocodazole (Sigma) per ml. The S- and M-phase-arrested cells were filtered, washed, suspended in fresh YPD medium, and cultured at 28°C.

Preparation of cell extracts and immunoblot analysis.

Cells were harvested by centrifugation (4,000 × g for 5 min), washed with 10 mM Tris-HCl (pH 7.5), and resuspended in 200 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 50 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 μg of aprotinin per ml, 1 μg of leupeptin per ml, and 2 μg of pepstatin per ml). After addition of an equal volume of glass beads, the cells were broken by vigorous vortexing for 3 min at 4°C. A portion (10 μl) of the resulting cell lysate was removed for assay of protein concentration, and after the addition of 100 μl of 3× sodium dodecyl sulfate (SDS) sample buffer to the remainder, the resulting mixture was boiled for 3 min. The glass beads and cell debris were removed by centrifugation (12,000 × g for 30 min at 4°C), and a portion of the remaining cell extract (50 μg of total protein) was fractionated by SDS-polyacrylamide gel electrophoresis on a 10% gel. The gel was soaked in transfer buffer containing 10% (vol/vol) methanol before transfer of proteins to a polyvinylidene difluoride membrane with the use of an Electroblotter (Novex, San Diego, Calif.). The membrane was incubated for 1 h with 5% (wt/vol) nonfat dried milk in Tris-buffered saline (pH 7.5) containing 0.05% (wt/vol) Tween 20 and then incubated overnight at 4°C with monoclonal antibodies to Clb2 (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif.), to Cdc28 (1:500 dilution; Calbiochem, San Diego, Calif.), to Cdc55 (1:200 dilution; Santa Cruz Biotechnology), or to human α-amylase (1:1,000 dilution; Sigma). Immune complexes were detected by alkaline phosphatase-conjugated secondary antibodies and enhanced chemiluminescence.

Measurement of α-amylase activity.

Cells from YPD cultures (1 ml) were harvested by centrifugation (4,000 × g for 5 min). The resulting pellet was collected for preparation of a cell extract as previously described, and the supernatant was buffered with 15 mM HEPES-NaOH (pH 7.0). Portions (20 μl) of the cell extract and buffered supernatant were used for determination of intracellular and secreted α-amylase activities (7), respectively, with Sigma diagnostic kit 577-3.

Histone H1 kinase assay.

Clb2 was immunoprecipitated from yeast lysates (50 μg of total protein) using 10 μl of protein A (Sigma). For histone H1 kinase (Clb2-Cdc28 kinase) analysis, the Clb2 immunoprecipitates were preincubated at 37°C for 5 min. Subsequently, 8 μl of a solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 750 μM ATP, 2 μg of bovine histone H1 (Sigma), and 10 μCi of [γ-32P]ATP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) was added. The reaction was incubated at 37°C for 10 min and was stopped by adding 30 μl of 2× loading buffer, and the mixture was heated at 95°C for 5 min and loaded onto a 10% SDS–polyacrylamide gel. The gel was fixed and dried, and the phosphorylated H1 was visualized by autoradiography.

Assay of protein phosphatase activity.

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

RESULTS

Morphology of cells secreting high levels of α-amylase.

Transformed cells that expressed and secreted α-amylase had an abnormal morphology (Fig. 1A, C, E, and G). Transformed TL154-14 and NI-C-14 cells that secreted moderate (∼500 U of activity per liter of culture medium) and high (1,500 U/liter) levels of α-amylase (Fig. 2B), respectively, formed elongated buds (Fig. 1B and F). Secretion of α-amylase at even higher levels (3,500 U/liter) by transformed NI-D4-14 cells (Fig. 2B) resulted in the formation of highly elongated buds (Fig. 1H). In contrast, a low level of α-amylase secretion (∼20 U/liter) by 20B12-14 cells (Fig. 2B), from which the plasmid was lost after 10 generations (data not shown), did not result in morphologic changes (Fig. 1D).

FIG. 1.

FIG. 1

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Phenotypes of yeast cells oversecreting mouse α-amylase. Nontransformed and transformed S. cerevisiae strains were grown in YPD medium at 28°C for 4 days and then examined by scanning electron microscopy. The phenotypes of nontransformed TL154 (A), 20B12 (C), NI-C (E), and NI-D4 (G) cells and of their respective pMS12-transformed TL154-14 (B), 20B12-14 (D), NI-C-14 (F), and NI-D4-14 (H) cells are shown. Scale bar, 5 μm.

FIG. 2.

FIG. 2

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Relation between α-amylase production and altered bud morphology 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 the indicated times, portions of the culture were removed for analysis of cell morphology and α-amylase secretion. Cell morphology was examined by phase-contrast microscopy, and the average percentage of cells with elongated buds (•, those more than twice as long as normal buds) was calculated. The average activity of α-amylase (○) in the culture medium was determined after removal of cells by centrifugation. Data are means of values from nine experiments with three cultures (TL154-14, NI-C14, and NI-D4-14), with each culture being repeated three times with similar results.

We also determined the percentages of cells exhibiting elongated buds and the extents of α-amylase secretion at various times. Secretion of α-amylase by transformed NI-C-14 and NI-D4-14 cells was first detected after culture for 8 h, at which time ∼10% of the cells exhibited elongated buds (data not shown). Both the average amount of α-amylase activity in the culture medium and the average percentage of budded cells increased over similar time courses in TL154-14, NI-C-14, and NI-D4-14 cultures (Fig. 2). Moderate, high, and very high levels of α-amylase secretion by TL154-14, NI-C-14, and NI-D4-14 strains, respectively, resulted in the formation of elongated buds in 7.6, 12, and 27% of cells, respectively, after cultivation for 95 h.

We saw no morphologic abnormalities following phase-contrast microscopy of TL154, 20B12, NI-C, or NI-D4 cells harboring the vector pMA56, which does not contain α-amylase cDNA (data not shown). Thus, high-level secretion of α-amylase (rather than transformation per se) affects bud morphogenesis in S. cerevisiae.

Cell cycle arrest of cells oversecreting α-amylase.

Morphology similar to that observed for strains oversecreting α-amylase is often associated with a block in cell cycle progression. We examined the DNA content of cells expressing this mouse-α-amylase protein by flow cytometry. Cultures of all transformed strains were asynchronous and had a bimodel distribution of DNA content at the zero time point, with peaks at 1 and 2N (Fig. 3). 20B12-14 cells, which secrete only a very low level of α-amylase, remained asynchronous during the 24-h culture period (Fig. 3B). In contrast, NI-D4-14 cells, which secrete a very high level of α-amylase, began to accumulate cells with a G2/M DNA content after culture for 4 h (Fig. 3D). TL154-14 (Fig. 3A) and NI-C-14 (Fig. 3C) cells, which secrete moderate and high levels of α-amylase, respectively, showed accumulation of G2/M cells after 8 h. After 24 h, TL154-14 and NI-C-14 cells 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 the cell cycle. The correlation of high-level secretion of α-amylase from yeast cells with a DNA content typical of G2/M suggests that the checkpoint that governs the transition between G2 and M or M and G1 is impaired in these cells.

FIG. 3.

FIG. 3

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

The elongated buds of TL154-14, NI-C-14, and NI-D4-14 carrying pMS12 each contained two nuclei (Fig. 4A, 4E and 4G), and about 5% of NI-C-14 (Fig. 4E) and NI-D4-14 (Fig. 4G) cells were segmented and contained multiple nuclei (three to five nuclei). Cells secreting α-amylase at high or very high levels thus appeared to be impaired in cell division, with incomplete separation between newly budding cells and the mother cell.

FIG. 4.

FIG. 4

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Distributions of nuclei in buds of yeast cells overproducing α-amylase. α-Amylase-overproducing yeast strains TL154-14 (A and B), 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) or dark-field (B, D, F, and H) microscopy. The positions of nuclei are indicated by arrowheads.

α-Amylase secretion and the accumulation of Clb2.

Levels of CLB1 and CLB2 transcripts and of the encoded G2 cyclins exhibit marked periodicity in S. cerevisiae, peaking about 10 min before anaphase (15, 16, 37, 42). The kinase activity of the Clb-Cdc28 complex shows a similar periodicity (16, 21). Eighty to 85% of nontransformed NI-C and NI-D4 cells had a DNA content of 1N after release from S arrest and again 120 min later (Fig. 5A, left panels), whereas 65 to 70% of transformed NI-C-14 and NI-D4-14 cells had a DNA content of 2N 120 min after release from S arrest (Fig. 5A, right panels).

FIG. 5.

FIG. 5

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Effects of high levels of α-amylase on DNA content and Clb2 abundance during cell cycle progression. (A) S-phase cells of nontransformed (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 by treatment with hydroxyurea (0.2 M). Cells subjected to S-phase arrest were analyzed for DNA content by flow cytometry at 0 and 120 min after release from S-phase arrest. (B) Effect on Clb2 abundance. Cell lysates were prepared at the indicated times after release from S-phase arrest as for the experiment whose results are shown in panel A and subjected to immunoblot analysis with antibodies to Clb2, to Cdc28, or to α-amylase. Levels of Cdc28 protein were used as a protein loading control.

The level of Clb2 protein in α-amylase-overproducing cells did not exhibit the periodicity seen in nontransformed cells (Fig. 5B, left panels). In AMY cells (NI-D4-14), which had the highest level of α-amylase secretion, the amount of Clb2 increased 40 min after release from S arrest. Clb2 protein then accumulated for 100 min (40 to 140 min after S release) and finally decreased again 160 min after S release (Fig. 5B). The synthesis of the mouse α-amylase in transformed yeast cells appeared to be highly periodic (Fig. 5B), peaking in the G2 and M phases, similar to the periodicity of Clb2. These results suggest that high-level secretion of heterologous α-amylase in yeast is correlated with a G2-M delay and an associated defect in the regulation of Clb2 protein levels.

Effect of α-amylase secretion on PP2A and MPF (Clb-Cdc28 kinase) activities during mitosis.

In NI-D4 cells, levels of Cdc55, a regulatory subunit of PP2A, appeared relatively stable for 2 h after release from nocodazole-induced arrest, whereas the level of this protein in cells overproducing α-amylase (NI-D4-14) began to decrease 90 min after release from nocodazole arrest (Fig. 6A). MPF (histone H1 kinase) activity decreased after the amylase-oversecreting cells were released from nocodazole arrest (Fig. 6A, right panel), compared with the level in nontransformed cells (Fig. 6A, left panel). Thus, MPF (Clb-Cdc28 kinase) activity appears to be defective in cells secreting high levels of α-amylase.

FIG. 6.

FIG. 6

FIG. 6

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High levels of α-amylase negatively regulate PP2A activity and cause defective MPF during mitosis. (A) Cdc55 abundance. Nontransformed (NI-D4) and α-amylase-overproducing cells (NI-D4-14) were subjected to growth arrest with nocodazole, released into YPD medium at 28°C, and at the indicated times thereafter lysed and subjected to immunoblot analysis with antibodies to the regulatory B subunit (Cdc55) of PP2A. A protein sample withdrawn at each time point was examined for histone H1 kinase activity as described in Materials and Methods. Protein levels of Cdc28 were used as a protein control. (B) PP2A activity. Portions of the cell extracts analyzed for panel A were assayed for phosphatase activity in the absence or presence of the type 1 phosphatase inhibitor I-2 (0.1 μM) or the type 2 phosphatase inhibitor okadaic acid (OA) (2 nM). The reaction was performed for 30 min at room temperature and at an extract protein concentration of 200 μg/ml in the absence of divalent cations and free phosphate. Data are expressed as nanomoles of phosphate generated per minute per milligram of extract protein and are means of values obtained from two independent experiments. Left panel, −AMY1; right panel, +AMY1.

We also measured PP2A activity with a synthetic phosphopeptide substrate in extracts of cells released from nocodazole arrest. The chosen phosphopeptide is a poor substrate for protein phosphatase type 1, and we measured phosphatase activity in the absence and presence of specific inhibitors of type 1 (I-2) and type 2 (okadaic acid) phosphatase activity. Phosphatase activity in the presence of I-2 increased after release from nocodazole arrest in both nontransformed and α-amylase-overproducing cells; however, the increase was more marked in the nontransformed cells and the activity subsequently decreased in the α-amylase-overproducing cells to ∼50% of the initial value (Fig. 6B). These results suggest that PP2A activity is negatively regulated in α-amylase-overproducing cells.

Cdc28VF prevents MPF inactivation and aberrant buds in amylase-overproducing cells treated with nocodazole.

Phosphorylation of Tyr19 inhibits Cdc28 H1 kinase activity in S. cerevisiae (4). We transformed the cdc28VF mutant (in which Thr18 was changed to Val and Tyr19 was changed to Phe) with pMS12 to overproduce amylase. The amounts of Cdc28 remained relatively constant and did not differ markedly between the α-amylase-expressing wild type and cdc28 mutants (Fig. 7). The wild-type cells overproducing α-amylase accumulated higher levels of Clb2 than cdc28 mutant cells producing α-amylase (Fig. 7A). However, cells overproducing α-amylase had a lower level of Clb2-Cdc28 kinase (Fig. 7A, left panel) than that of wild-type cells (Fig. 6A), whereas the cdc28VF mutation prevented the inactivation of Clb2-Cdc28 kinase (Fig. 7A, right panel). The cdc28VF mutation also suppressed the defective bud morphology exhibited by wild-type cells overproducing α-amylase (Fig. 7B).

FIG. 7.

FIG. 7

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Cdc28VF cured the defect in MPF activity and aberrant bud morphology in amylase-overproducing cells. (A) The α-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 were lysed at the indicated times thereafter and subjected to immunoblot analysis with antibodies to Cdc55, Clb2, and hemagglutinin (HA) (against Cdc28-HA or Cdc28VF-HA). (B) Photographs of the wild-type strain (ADR508-14) and a cdc28 mutant overproducing α-amylase (ADR640). These cells were grown to stationary phase in YPD medium. (C) Photographs of wild-type strains overproducing heterologous glucoamylase (+ GAM1) and xylanase (+ XYN2). Cell overproducing glucoamylase (AST-3 and A18ST-3) or xylanase (DXN-8) were grown to stationary phase in YPD medium at 28°C.

We also examined yeast strains that express and secrete high levels of heterologous proteins, including mouse α-amylase, glucoamylase from Rhizopus orizae, or xylanase from Trichoderma reesei (Fig. 7C). Overproduction of glucoamylase results in cells with large buds. Overproduction of glucoamylase and either amylase or xylanase resulted in extremely elongated budded cells. These data suggest that the mitosis delay may not be specific to α-amylase but that it instead is a response to the abnormally high levels of secretory proteins.

Influence of defective PP2A on cell mitosis and the cell integrity of cells overproducing α-amylase.

We examined wild-type and pph22Δ cells overproducing amylase for their Clb2 levels after cells were released from nocodazole arrest. Wild-type cells that overproduce amylase accumulated Clb2 protein for 150 min after M-phase release (Fig. 8A). However, pph22Δ cells that overproduce amylase began to degrade Clb2 protein 90 min after release from nocodazole arrest and Clb2 protein appeared again 150 min after release from M phase, suggesting that the lack of PPH22 suppresses the amylase-induced mitosis delay. These data also provided direct evidence that the PPH22-encoding subunit of PP2A was affected by high levels of amylase.

FIG. 8.

FIG. 8

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High levels of amylase induced defective PP2A and triggered cell lysis. (A) Deletion of PPH22 suppresses the amylase-induced mitotic defect. The α-amylase-overproducing wild-type strain (BY4741-14) and a pph22Δ mutant (Y03386-14) were arrested with nocodazole and released into fresh YPD. Samples were taken every 30 min to analyze the amounts of Clb2 and Cdc28. Cells were lysed at the indicated times thereafter and subjected to immunoblot analysis with antibodies to Clb2 and Cdc28. (B) Phenotype of α-amylase-overproducing cells. Cultures were grown to a density of 5 × 107 to 8 × 107 cells/ml in YPD medium containing 1 M sorbitol at 28°C, subcultured in YPD medium containing or lacking 1 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.

pph21Δ pph22Δ cells have a double deletion that causes slow growth at 24°C and temperature-sensitive growth at 37°C (14, 27). To test whether the amylase-induced PP2A defect can cause a growth defect similar to that of pph21Δ pph22Δ cells, we examined the effect of high osmolarity on growth in nontransformed cells and α-amylase-overproducing cells. Cells overproducing α-amylase displayed a partial arrest of proliferation 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 cells were viable after 8 h at 37°C), whereas in high-osmolarity medium containing 1 M sorbitol (Fig. 8C), the cells died at a lower rate (47% of cells were viable after 8 h at 37°C). In contrast, nontransformed cells remained viable under all the conditions tested (Fig. 8C), indicating that a defect in cell wall integrity was induced when α-amylase was overproduced.

DISCUSSION

High levels of amylase, PP2A, cell integrity, and nuclear division.

A variety of serine/threonine protein phosphatases have been implicated in mitosis in various organisms, but the underlying mechanisms through which they operate are not known (10, 27, 29, 54). PP2A is a heterotrimeric protein that consists of a catalytic subunit and two regulatory subunits (A and B), the latter of which confers substrate specificity to the catalytic subunit (54). In S. cerevisiae, the regulatory B subunit of 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 PPH22 genes (21, 27).

Genetic analysis implicates PP2A in mitosis and cellular morphogenesis in yeast (19, 29, 53, 55). Lin and Arndt (27) showed that defects in PP2A induced S. cerevisiae cells to arrest with small or aberrant buds, at which sites the actin cytoskeleton is disorganized and chitin deposition is delocalized. When released from hydroxyurea treatment (S-phase arrest), pph21 mutants have a reduced MPF activity but accumulate Clb2 at levels similar to those of wild-type cells (27). Minshull et al. (31) showed that MPF inactivation occurs without cyclin degradation in cdc55 mutant cells; cells released into nocodazole contain little MPF activity but accumulate mitotic cyclins in a manner similar to that of wild-type cells. In contrast, cdc55Δ cells, which carried the cdc28 mutation, retain high MPF activity in the presence of nocodazole (31).

From our results, PP2A activity was negatively regulated during high-level secretion of α-amylase and consequently caused a decrease in MPF activity (Fig. 6 and 8A). We also found that the dephosphorylation of tyrosine-phosphorylated Cdc28, a critical step for MPF activation, was perturbed by high levels of α-amylase (Fig. 7). The net effect was a delay in mitosis.

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

There is evidence that, in both mammalian cells and yeast cells, Rho-like GTPases are key regulators of signaling pathways that link extracellular growth signals or intracellular stimuli 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 pathway is required for cell wall integrity (12). A direct role for PP2A in controlling cell integrity has been suggested (3). For example, BEM2 encodes GTPase-activating protein for small G protein encoded by RHO1 (24, 33) and bem2 mutations can suppress cdc55-1 (19). Moreover, bem2 mutants and temperature-sensitivity-negative pph22 strains display many common phenotypic features, including temperature-dependent disruption of the actin cytoskeleton, a bud growth defect, and a sorbitol-remediated temperature-sensitivity-negative cell lysis defect (14, 52). Our data also support the hypothesis that defective PP2A, induced by α-amylase overproduction, leads to defects in cell integrity. Such a defect can be rescued by treatment with 1 M sorbitol (Fig. 8).

Our data also show that the amylase-induced PP2A defect causes a mitotic delay and the accumulation of cells with replicated DNA and separated nuclei (Fig. 4). Cells with divided nuclei also resulted from the defect in bud growth observed in pph22 cells at 37°C (14). Together, these data suggest that PP2A is required for maintenance of polarized growth, cell integrity, and nuclear division.

Cell lysis system

The cell wall of S. cerevisiae is a tough, rigid structure which presents a significant barrier to the release of native or recombinant proteins. Lysis mutants provide one route to mechanical or chemical disruption of the cell wall that precedes the recovery of yeast contents. Alvarez et al. (2) reported on the release of intracellular proteins, including virus-like particles, from an slT2 mutant by osmotic shock. The use of an srb1-1 mutant for similar purposes has also been documented (40). Zhang et al. (56) developed an approach to trigger cell lysis by a genetic switch of three genes involved in cell wall biogenesis: PDE2, SRB1 (also called PSA1), and PKC1. The α-amylase-induced cell lysis process provides another alternative for the efficient secretion and release of heterologous proteins by yeast.

In summary we hypothesize that high levels of α-amylase induce a checkpoint response, mediated by a cell integrity signaling pathway, and cause a negative regulation of PP2A, which in turn causes mitosis delay and cell lysis. The α-amylase-induced mitotic block we observed supports the hypothesis that PP2A has a role in the maintenance of bud morphology, nuclear division, and cell integrity. In addition, pph22-induced cell lysis and mitotic delay suggest an alternative approach to the production of M-phase-dependent foreign proteins by this lysis system.

ACKNOWLEDGMENTS

We thank A. W. Murray and A. D. Ruder for providing cdc28VF mutants and D.-C. Chen and H. J. Huang for valuable suggestions and discussions.

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

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