INTRODUCTION

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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 -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 -amylase production in yeast cultured in a fed-batch fermentation (46).

In this study, we overexpressed mouse -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

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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 -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 -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 -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.

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 -amylase (1:1,000 dilution; Sigma). Immune complexes were detected by alkaline phosphatase-conjugated secondary antibodies and enhanced chemiluminescence.

Measurement of -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 -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 [-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

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Morphology of cells secreting high levels of -amylase. Transformed cells that expressed and secreted -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 -amylase (Fig. 2B), respectively, formed elongated buds (Fig. 1B and F). Secretion of -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 -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).

View larger version (113K): [in this window] [in a new window]   FIG. 1.   Phenotypes of yeast cells oversecreting mouse -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.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Relation between -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 -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 -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.

Cell cycle arrest of cells oversecreting -amylase. Morphology similar to that observed for strains oversecreting -amylase is often associated with a block in cell cycle progression. We examined the DNA content of cells expressing this mouse--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 -amylase, remained asynchronous during the 24-h culture period (Fig. 3B). In contrast, NI-D4-14 cells, which secrete a very high level of -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 -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 -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.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Flow cytometric analysis of the DNA content of yeast cells secreting -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.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Distributions of nuclei in buds of yeast cells overproducing -amylase. -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.

-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).

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Effects of high levels of -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 -amylase. Levels of Cdc28 protein were used as a protein loading control.

Effect of -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 -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 -amylase.

View larger version (46K): [in this window] [in a new window]   FIG. 6.   High levels of -amylase negatively regulate PP2A activity and cause defective MPF during mitosis. (A) Cdc55 abundance. Nontransformed (NI-D4) and -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.

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 -amylase-expressing wild type and cdc28 mutants (Fig. 7). The wild-type cells overproducing -amylase accumulated higher levels of Clb2 than cdc28 mutant cells producing -amylase (Fig. 7A). However, cells overproducing -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 -amylase (Fig. 7B).

View larger version (68K): [in this window] [in a new window]   FIG. 7.   Cdc28VF cured the defect in MPF activity and aberrant bud morphology in amylase-overproducing cells. (A) The -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 -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.

Influence of defective PP2A on cell mitosis and the cell integrity of cells overproducing -amylase. We examined wild-type and pph22 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, pph22 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.

View larger version (25K): [in this window] [in a new window]   FIG. 8.   High levels of amylase induced defective PP2A and triggered cell lysis. (A) Deletion of PPH22 suppresses the amylase-induced mitotic defect. The -amylase-overproducing wild-type strain (BY4741-14) and a pph22 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 -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.

	DISCUSSION

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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).

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 -amylase-induced cell lysis process provides another alternative for the efficient secretion and release of heterologous proteins by yeast.