Role of the α-Glucanase Agn1p in Fission-Yeast Cell Separation

Strains and Culture Media

Mutant strains were derived from S. pombe wild type, 972 (Table 1). Cells were grown in YEA medium (Hochstenbach et al., 1998) or in EMM2 medium (Moreno et al., 1991) with addition of 250 mg/l adenine sulfate (EMMA). GEMMA was EMMA with addition of 2% (wt/vol) sodium d-gluconic acid instead of d-glucose, adjusted to pH 4.0 by using 2 M phosphoric acid. In a conjugation and sporulation assay, cells were crossed on agar plates containing ME medium at 25°C for 2 d and treated with iodine vapor to test for ascospore formation. Diploids were selected on EMM2 medium (Moreno et al., 1991).

DNA Sequencing

For agn1⁺ and agn2⁺ sequencing (GenBank accession nos. AY626901 and AY626902, respectively), total RNA was isolated from vegetatively grown 972 cells or sporulating ND080 cells, respectively (Koerkamp et al., 2002). cDNA of a part of the agn1⁺ 5′-untranslated region, the open reading frame (ORF), and 3′-untranslated region was synthesized by polymerase chain reaction (PCR), by using pfu polymerase (Stratagene, La Jolla, CA). For agn2⁺ sequencing, cDNA of the agn2⁺ 5′-untranslated region, ORF, and 3′-untranslated region was synthesized in three separate PCR amplification reactions. These PCR amplification products were used for direct DNA sequencing (BDT sequencing kit; Applied Biosystems, Foster City, CA). The sequence of the 3′-end of the agn2 ORF also was confirmed by direct DNA sequencing of several PCR amplification products obtained for the region of interest using genomic DNA from strain 972 as template (our unpublished data).

Disruption and Tagging

Using a PCR-mediated strategy (Bähler et al., 1998), ORFs of agn1⁺ (systematic gene name, SPAC14C4.09), agn2⁺ (SPBC646.06c), eng1⁺ (SPAC821.09), ace2⁺ (SPAC6G10.12C), and sep1⁺ (SPBC4C3.12), were replaced by a loxP-kanMX4-loxP cassette (pUG6; Güldener et al., 1996). agn1⁺ and eng1⁺ ORFs were tagged at their 3′ ends with the 13myc-kanMX6 cassette (Bähler et al., 1998). Hot-start PCR amplification products were obtained in 28 cycles by using Taq polymerase (TaKaRa). Transformations were performed using a lithium-acetate method (Bähler et al., 1998), and cells were allowed to recover for 30 h at 28°C, followed by replica plating onto YEA plates containing 200 μg/ml Geneticin (Invitrogen, Carlsbad, CA). PCR analyses were used to verify correct integration, and tetrad analyses were used to confirm single integration events. Double and triple mutants were generated using tetrad dissection, and genotypes were rechecked by PCR analyses.

Cloning and Overexpression of agn1⁺

For overexpression of agn1⁺ from its chromosomal locus, a kanMX6–3nmt1 cassette was integrated in front of the ORF by homologous recombination (Bähler et al., 1998). For agn1p-his overexpression from a plasmid, the agn1 ORF was amplified from genomic DNA of strain 972 and cloned into plasmid pSE01, a pREP4-based plasmid (Maundrell, 1993) containing sequences encoding a protease X cleavage site followed by six histidines. After verifying the agn1 ORF sequence, the resulting plasmid, pND02, was transformed into a strain with genotype h^- ura4-D18 by using a lithium acetate method (Akada et al., 2000).

Synchronization and Immunoblotting

For synchronization, cdc25-22 strains were grown at 25°C to an optical density at 595-nm wavelength (OD₅₉₅) of 0.3 and shifted to 37°C for 4 h. Then, cells were released into the cell cycle by quick transfer to 25°C. Septation index was determined by calculating the percentage of cells with a septum after calcofluor-white staining (Fluostain I; F-0386; Sigma-Aldrich, St. Louis, MO). Cells were taken up in 100 mM Tris-HCl, pH 7.6, containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and broken with washed glass beads by using a FastPrep 120 (Bio 101, Vista, CA) at speed 5.5 for two intervals of 10 s. Protein concentrations were measured using the Bradford method with bovine serum albumin as a standard. Total cell lysates were resolved at 6 μg of protein per lane on a SDS/8% polyacrylamide gel under reducing conditions, blotted onto nitrocellulose membranes (0.45 μm; Schleicher & Schuell, Keene, NH), and probed with anti-myc monoclonal antibody (mAb) 9E10 (Evan et al., 1985) and an anti-α-tubulin antibody (T-5168; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad, Hercules, CA) and developed by chemiluminescence (ECL kit; Amersham Biosciences, Piscataway, NJ).

Microscopy

For light microscopy, cells were stained with calcofluor white and embedded in 2% (wt/vol) low-melting agarose. Micrographs were obtained using an Axiophot 2 microscope (Carl Zeiss, Thornwood, NY) equipped with a Coolsnap HQ digital camera (Photometrics, Tucson, AZ). For transmission electron microscopy, cells were fixed with potassium permanganate (Hochstenbach et al., 1998). Thin sections were stained with lead citrate (Reynolds, 1963) and uranyl acetate, and micrographs were obtained using a model 420 transmission electron microscope (Philips, Eindhoven, The Netherlands) operating at 80 kV.

Sedimentation Assay

Cells were grown in YEA medium in an orbital shaker at 200 rpm at 28°C to an OD₅₉₅ of 1. Subsequently, 1 ml of culture was transferred into a cuvette (Sarstedt 67.742), gently mixed, and placed without further disturbance in an Ultrospec III spectrophotometer (Pharmacia AB, Uppsala, Sweden). OD₅₉₅ was registered at 2.5-min intervals over a period of 30 min.

Purification of Agn1p-his

Agn1p-his expression was induced in GEMMA for 24 h. Culture medium was concentrated 10-fold under N₂ pressure at 4°C by using a polyethersulfone filter (10 kDa cut-off; Millipore, Billerica, MA), and its pH was changed to 8.0 by addition of 100 mM MOPS buffer. Then, supernatant was incubated at 4°C for 1 h with nickel-nitrilotriacetic acid beads (QIAGEN, Valencia, CA). Beads were washed with 300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0, buffer (buffer A), containing 15 mM imidazole. Agn1p-his was eluted with buffer A containing 250 mM imidazole, and fractions with (1,3)-α-glucanase activity were pooled. After exchanging buffer A for 20 mM sodium acetate, pH 5.0, by using a gel-filtration column (PD-10; Amersham Biosciences), the eluate was loaded onto an anion-exchange column (Mono-Q; Amersham Biosciences). Agn1p-his was eluted using a linear gradient of KCl to 250 mM, and fractions with highest (1,3)-α-glucanase activity were used for further study. Size exclusion chromatography was performed at a flow rate of 0.5 ml/min, by using a Superdex-75 column (Amersham Biosciences) equilibrated with 50 mM sodium acetate, pH 6.0, and calibrated with low-molecular-mass markers (Amersham Biosciences) and blue dextran.

Purification of MutAp

Fifty mg of Novozym (L-2265: Sigma-Aldrich) was dissolved in 50 mM Tris-HCl, pH 7.6, containing 1 mM PMSF and 10 μl of protease inhibitor cocktail (P-8215; Sigma-Aldrich) and run over an anion-exchange column (DEAE; Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.6. Flow-through fractions containing (1,3)-α-glucanase activity were pooled. For adsorption chromatography, buffer was exchanged for 5 mM sodium azide, 50 mM sodium acetate, pH 5.6 (buffer B), and 0.85 mM PMSF and 10 μl of protease inhibitor cocktail were freshly added. Twenty mg of (1,3)-α-glucan (Pleurotus ostreatus) was dissolved in 10 M HCl, incubated at 37°C for 15 min, and reprecipitated overnight by addition of ice-cold 1.25 M sodium acetate and neutralization with 10 M NaOH. Then, MutAp was bound to (1,3)-α-glucan in buffer B containing 10% (vol/vol) ethylene glycol and 250 mM NaCl at 4°C for 45 min, washed, and eluted by a 5-min incubation in 80% (vol/vol) ethylene glycol in buffer B at room temperature. Finally, ethylene glycol was removed using a PD-10 column equilibrated with buffer B.

Isolation of (1,3)-α-Glucan

Fruiting bodies of P. ostreatus or Laetiporus sulfureus were minced in a blender and boiled in 2% (wt/vol) SDS, 40 mM 2-mercaptoethanol for 15 min to extract cytosolic contaminants. To precipitate the alkali-soluble, water-insoluble fraction, cell wall material was dissolved in 2 mM NaBH₄, 2 M KOH for 30 min on ice and reprecipitated overnight at 4°C by adjusting the pH to 6 with acetic acid. Then, the pellet was resuspended in 5 mM sodium azide, 40 mM 2-mercaptoethanol, 50 mM citrate-phosphate buffer, pH 5.3, containing 45 mg of Zymolyase-100T (Seikagaku America, Rockville, MD) and incubated overnight at 37°C. After collection, the insoluble fraction was reextracted as described above and washed in 5 mM sodium azide.

Mass Spectrometry

For matrix-assisted laser desorption ionization analysis, protein spots were excised from a Coomassie-stained SDS/8% polyacrylamide gel and treated as described previously (Shevchenko et al., 1996). Dried peptides, cleaned with ZipTip_C18 (Millipore), were resuspended in 5 μl 60% (vol/vol) acetonitrile, 1% (vol/vol) formic acid. Approximately 4 μg of protein also was digested directly with 2 μg of endoproteinase Glu-C (V8 protease) (Roche Diagnostics, Indianapolis, IN) overnight in 50 mM sodium phosphate, pH 7.9, with 5% (vol/vol) acetonitrile at 25°C. Subsequently, the peptide solution was alkylated (Shevchenko et al., 1996), cleaned, and concentrated on a ZipTip_C18 (Millipore), and eluted in 10 μl 60% (vol/vol) acetonitrile, 1% (vol/vol) formic acid. Peptide solutions were mixed with an equal volume of 52 mM (aceton-prewashed) α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in 50% (vol/vol) ethanol, 48% (vol/vol) acetonitrile, 2% (vol/vol) trifluoroacetic acid, 1 mM ammonium acetate; spotted on a target plate; and dried at room temperature. Mass spectrometry spectra were collected on a MALDI R (Micromass, Wythenshawe, United Kingdom). For tandem mass spectrometry analysis, V8-protease digests were analyzed on a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass). Low-energy collision-induced dissociation was performed using argon as a collision gas.

Enzyme Reactions

Substrate specificity was determined by an incubation at 37°C for 20 min of ∼2 pmol of Agn1p-his with substrates at a final concentration of 4 mg/ml in 5 mM sodium azide, 50 mM sodium acetate, pH 5.6. Reduced and carboxymethylated (1,3)-α-glucan (L. sulfureus), (1,4)(1,6)-α-glucan (starch; 85642; Fluka, Buchs, Switzerland), reduced (1,4)(1,6)-α-glucan (glycogen; G-8751; Sigma-Aldrich), reduced (1,4)(1,6)-α-glucan (pullulan; P-4516; Sigma-Aldrich), reduced (1,6)-α-glucan (dextran; D-9260; Sigma-Aldrich), reduced (1,3)-β-glucan (laminarin; 61430; Fluka), carboxymethylated (1,4)-β-glucan (cellulose; C-5678; Sigma-Aldrich), or reduced (1,3)-α-mannan (mannan; M-7504; Sigma-Aldrich) were used as substrates. O-(Carboxy)methylation and polysaccharide reduction were performed as described previously (Takasaki and Kobata, 1978; Kiho et al., 1989). Reaction products were analyzed using a colorimetric assay (Lever, 1972).

For high-performance anion-exchange chromatography (HPAEC), (1,3)-α-glucan (0.5 mg) purified from S. pombe cell walls was incubated with Agn1p-his or MutAp in 5 mM sodium azide, 50 mM sodium acetate, pH 5.6, at 37°C for 5 h. As standards, oligosaccharides from S. pombe (1,3)-α-glucan were prepared as described previously (Koizumi et al., 1989). HPAEC was performed using a CarboPac PA-1 column on a DX 500 system (Dionex, Sunnyvale, CA) by using a linear gradient from 0 to 500 mM sodium acetate in 0.1 M NaOH at a flow rate of 1.0 ml/min.

Agn1p Contains a Signal Sequence for Secretion, whereas Agn2p Does Not

Annotation of the S. pombe genome sequence suggested that the agn1⁺ and agn2⁺ genes each contain one continuous ORF (Wood., 2002). This prediction was confirmed by our analysis of the transcripts. agn1⁺ cDNA contained a 5′-untranslated region of at least 136 base pairs and a 3′-untranslated region of 422 base pairs, whereas agn2⁺ cDNA contained a 5′-untranslated region of 157 base pairs and a 3′-untranslated region of 122 base pairs. Analysis of their ORFs showed that agn1⁺ and agn2⁺ encode polypeptides of 424 and 433 amino acids, respectively, with calculated molecular masses of 46.9 and 48.0 kDa (Figure 1). These predicted polypeptides contain 22 additional amino acids at the amino terminus of Agn1p and 26 additional amino acids at the carboxy terminus of Agn2p beyond those predicted by the original genome-project annotation. These polypeptides share 39% amino acid sequence identity, suggesting that they are paralogs. Furthermore, Agn1p and Agn2p show 41 and 34% amino acid sequence identity, respectively, to the catalytic domain (residues 38–461) of the known Trichoderma harzianum (1,3)-α-glucanase MutAp, the founding member of glycoside hydrolase family 71 (Fuglsang et al., 2000), identifying Agn1p and Agn2p as putative (1,3)-α-glucanases. This prediction is consistent with the absence of Agn homologues in S. cerevisiae and Candida albicans (our unpublished data), both of which lack cell wall (1,3)-α-glucan. However, Agn1p and Agn2p lack sequences corresponding to the carboxy-terminal Pro-Ser-Thr-rich linker (residues 462–550) and polysaccharide-binding domain (residues 551–634) present in MutAp (Figure 1).

A hydropathy profile of the Agn1 polypeptide reveals a hydrophobic region at its amino terminus (residues 1–20) that is predicted to function as a cleavable signal sequence (Kyte and Doolittle, 1982; Nielsen et al., 1997). However, a signal sequence was found to be absent from the Agn2 polypeptide. To study secretion of Agn1p and Agn2p directly, we tagged these proteins at their carboxy terminus and performed an immunoblot analysis on exponentially growing agn1-myc and agn2-myc cells. In total cell lysates, Agn1-myc and Agn2-myc protein bands resolved at apparent molecular masses of ∼78 and ∼76 kDa, respectively (Figure 2, lanes 2 and 3), which are in agreement with the calculated molecular masses of 67.4 and 68.5 kDa. Immunoblot analysis on corresponding culture media revealed the presence of Agn1-myc protein in culture medium of agn1-myc cells, whereas no Agn2-myc protein was observed in culture medium of agn2-myc cells (Figure 2, compare lanes 5 and 6). Comparison of the cell-bound fraction with the culture-medium fraction revealed that the majority of steadystate Agn1-myc protein was bound to cells (Figure 2, compare lanes 2 and 5). Our data demonstrate that, unlike Agn2p, Agn1p shares a functional signal sequence with characterized MutA proteins of T. harzianum and Penicillium purpurogenum, both of which also are secreted (Fuglsang et al., 2000).

Deletion of agn1⁺ Causes Cells to Clump

To study the cellular function of Agn1p, we deleted the complete agn1⁺ ORF from the genome of wild type, 972, and analyzed the phenotype of the resulting deletion mutant. agn1Δ cells were viable and displayed a typical cylindrical rod morphology (Figure 3, compare agn1Δ with wild type). However, rather than single cells, the agn1Δ mutant often formed small clumps of cells attached to each other via an area located near their tips (Figure 3, arrows). These data indicate that agn1Δ cells fail to separate effectively at the end of the cell cycle.

Cell clumping has been observed not only with agn1Δ cells but also with cells deleted for eng1⁺ or ace2⁺ (Martín-Cuadrado et al., 2003). To compare these clumping phenotypes, we deleted the eng1⁺ and ace2⁺ ORFs individually and verified the phenotypes of the resulting strains (Figure 3). Although eng1Δ and ace2Δ cells, like agn1Δ cells, formed clumps, the extent of cell clumping seemed to be most severe for the ace2Δ mutant in light microscopy. To compare the extents of clumping more quantitatively, we set up a simple sedimentation assay (see MATERIALS AND METHODS). Wild-type cells hardly sedimented during a 30-min time period, whereas ace2Δ cells sedimented rapidly, thereby clearing the culture medium to a large extent (Table 2). agn1Δ and eng1Δ cells, on the other hand, sedimented with intermediate velocities, indicating that they each formed clumps that were generally smaller than those formed by the ace2Δ mutant (Table 2). Together, these data indicate that loss of agn1⁺ function causes S. pombe cells to clump to a similar extent as loss of eng1⁺ function.

For a complementation analysis, we expressed the agn1-his gene from a multicopy plasmid in the agn1Δ strain. Induction of agn1-his resulted in a sedimentation index comparable with that of wild-type cells, whereas repression of agn1-his gene expression showed a clumping phenotype typical for the agn1Δ strain (Table 2, compare induction of pND02 with its repression). The observation that during agn1-his repression clumping was not completely identical to that of agn1Δ cells is explained by the fact that repression of the nmt1 promotor is not complete. Despite continuous overexpression of the agn1 ORF under the strong nmt1 promotor, cells showed a wild-type morphology and did not lyse, indicating that cells are resistant to agn1⁺ overexpression (our unpublished data). We confirmed this conclusion by demonstrating that cells overexpressing agn1⁺ are as resistant to an incubation with (1,3)-β-glucanase as wild-type cells, providing no evidence for a change in cell wall rigidity (our unpublished data). Together, these data show that Agn1p-his restores Agn1p function in agn1Δ cells, but that Agn1p-his, upon overexpression, is not cytolytic.

Agn1p Is an endo-(1,3)-α-Glucanase

To characterize the role of Agn1p in cell separation, we studied its specific enzymatic activity. To obtain recombinant Agn1-his protein, we purified it from culture medium by immobilized metal-ion affinity chromatography and anion-exchange chromatography (Figure 4A, lanes 1 and 2), and assessed its purity as follows. First, SDS-PAGE followed by silver staining resolved only a single band with an apparent molecular mass of ∼47 kDa (Figure 4A, lane 2), in close agreement with its calculated molecular mass of 48.4 kDa. In size exclusion chromatography, Agn1p-his eluted as a single peak with an elution volume corresponding to a molecular mass of ∼50 kDa, indicating that it is a monomer (Figure 4B). Although mature Agn1p-his possesses one potential N-glycan attachment site at Asn-384, treatment with trifluoromethanesulfonic acid to eliminate glycosyl residues did not change its mobility in SDS-PAGE (our unpublished data), indicating that recombinant Agn1p-his was not glycosylated. Second, the 47-kDa protein band was subjected to peptide mass fingerprinting by matrix-assisted laser desorption ionization time-of-flight by using trypsin or V8 protease. All major nonprotease peaks in the obtained spectra were accounted for by Agn1p-his, providing no evidence for the presence of contaminating proteins or for posttranslational modification of Agn1-his protein. Electrospray mass spectrometry analysis of Agn1p-his yielded a major peak of molecular mass 45,980.1 ± 1.2 Da and a minor peak of mass 46,117.4 ± 1.8 Da. The major peak corresponds to an Agn1-his polypeptide without posttranslational modifications that starts, as predicted above, at Asp-21 and terminates with four His residues, indicating that two carboxy-terminal His residues were removed, presumably by proteases. The minor peak corresponds to an Agn1 polypeptide that terminates with five His residues. In conclusion, we purified Agn1-his protein to homogeneity and showed that its signal sequence was cleaved off to generate mature protein.

Substrate specificity of recombinant Agn1p-his was assayed with different glucan or mannan substrates. In the presence of (1,3)-α-glucan, we detected the release of digestion products by using a colorimetric assay (Figure 4C). No activity was detected using (1,3)-β-glucan, (1,3)-α-mannan, (1,4)-α-glucan, or (1,6)-α-glucan as substrates, demonstrating a specificity for (1,3)-α-glucosidic linkages (Figure 4C). Agn1p-his shows a broad pH optimum, with a maximum activity ranging from pH 3.5–5.5 (our unpublished data). Furthermore, 4-nitrophenyl-α-glucose, a synthetic compound that is cleaved only by glucanases with an exo-type mechanism of hydrolysis, was not hydrolyzed (our unpublished data). Also, Agn1p-his activity was not inhibited by 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin, castanospermin, or d-glucono-1,5-lactone, all inhibitors of exo-type enzymes (our unpublished data).

The observation that S. pombe Agn1p and T. harzianum MutAp share a putative catalytic domain but not a Pro-Ser-Thr-rich linker or polysaccharide-binding domain (see above) prompted us to compare the enzyme characteristics of both (1,3)-α-glucanases. For this purpose, we purified MutA protein by (1,3)-α-glucan adsorption chromatography, yielding a single band in SDS-PAGE analysis that resolved at a molecular mass of ∼75 kDa (Figure 4A, lane 4), consistent with previous results (Fuglsang et al., 2000). To compare the reaction products of both enzymes, insoluble (1,3)-α-glucan purified from S. pombe was incubated with Agn1p-his or MutAp for 5 h. A high-performance anion-exchange chromatography profile of MutAp reaction products showed the presence of mostly glucose, with small amounts of (1,3)-α-glucan trisaccharide (Figure 4D, bottom). Importantly, the reaction product of Agn1p-his lacked glucose, but instead contained (1,3)-α-glucan oligosaccharides with a degree of polymerization of 2–7, with (1,3)-α-glucan pentasaccharide as the predominant reaction product (Figure 4D, top). Together, these results demonstrate that Agn1p is an endo-(1,3)-α-glucanase that releases predominantly (1,3)-α-glucan pentasaccharides.

Agn1p Is Involved in Hydrolysis of the Septum Edging but Not the Primary Septum

Given that Agn1p and Eng1p are both glucanases, we investigated whether their functions are redundant. First, we compared the cellular morphologies of mutants with single or double deletions for agn1⁺ and eng1⁺. Although the morphology of agn1Δ eng1Δ cells resembled that of eng1Δ cells, the double mutant often contained more cells per chain of cells (Figure 3). Frequently, these chains of cells contained branches (Figure 3, arrowheads), a morphology hardly ever observed with either single deletion mutant. Second, we compared the extent of clumping of the single and double mutants in our sedimentation assay. Interestingly, agn1Δ eng1Δ cells sedimented significantly faster than agn1Δ or eng1Δ cells (Table 2), indicating that clumps of the double mutant were on average significantly larger than those of either single mutant. Together, these data suggest that agn1⁺ and eng1⁺ fulfill nonoverlapping functions.

To gain insight into the precise roles of Agn1p and Eng1p in cell separation, we analyzed the ultrastructures of agn1Δ and eng1Δ cells. Electron microscopy analysis of agn1Δ cells showed that septum assembly (and thus cytokinesis) and dissolution of the primary septum progressed normally in this mutant (Figure 5A). These results were corroborated by microscopy analysis on agn1Δ cells stained with calcofluor white, a dye that specifically stains the primary septum and remnants thereof. Staining of the septa in agn1Δ cells was intense during cytokinesis, but diminished in intensity during cell separation (Figure 3). By contrast, a defect in dissolution of the septum edging was evident in agn1Δ cells (Figure 5A). The septum edging in agn1Δ cells was broken down only partially, causing daughter cells to remain attached via a small stretch of septum-edging material (Figure 5A, solid arrow).

The ultrastructure of the septal region in agn1Δ cells is clearly distinct from that in eng1Δ cells. In eng1Δ cells, we observed that the primary septum often fails to break down (Figure 5B), exposing primary-septum material to the extracellular space (Figure 5D, open arrow). This defect causes eng1Δ cells to remain attached via the primary septum, which is visualized clearly by staining with calcofluor white (Figure 3). The septum edging, on the other hand, seems to be broken down normally in eng1Δ cells (Figure 5B), resulting in invaginations at the septa and in exposure of fission scars (Figure 5B, asterisks). We conclude that eng1⁺ and agn1⁺ fulfill distinct roles in cell separation: eng1⁺ is involved in dissolution of the primary septum, whereas agn1⁺ is involved in dissolution of the septum edging.

Agn1p Protein Expression Oscillates during the Cell Cycle

The observation that Agn1p acts during cell separation prompted us to examine whether Agn1 protein expression was up-regulated during this stage of the cell cycle. To this end, we arrested temperature-sensitive (ts) cdc25-22 agn1-myc cells at the G2-M transition and then released them synchronously into the cell cycle, while monitoring cell synchrony by septation-index analysis and Agn1-myc protein levels by immunoblot analysis. Agn1-myc protein levels oscillated considerably as cells traversed the first cell cycle, peaking concomitantly with septation and cell separation (Figure 6A). During the following cell cycle, the oscillation was less dramatic, because Agn1-myc protein is a stable protein with a half-life of >2 h (our unpublished data), and its levels did not decrease to their initial levels observed at arrest. In an arrest-release experiment on cdc25-22 eng1-myc cells, Eng1-myc protein levels oscillated in a manner similar to the oscillation of Agn1-myc protein levels (Figure 6, compare B and A). This oscillation in Eng1 protein levels is in good agreement with the oscillation reported for eng1⁺ transcript levels (Martín-Cuadrado et al., 2003).

To compare more precisely Agn1 protein levels at specific stages of the cell cycle, we used, along with the cdc25^ts mutant to block entry into mitosis, a cps1^ts mutant to block septum assembly and a cdc10^ts mutant to block in late G1 phase. Also, we treated agn1-myc cells with hydroxyurea for arrest in S phase. Immunoblot analysis showed that during the G2 and C phases Agn1-myc protein levels are low, whereas during the G1 and S phases they increased (Figure 6D). These data not only confirm the results of the cdc25-22 arrest-release experiment, they also narrow down the broad period of septation and cell separation to the G1 and S phases. In summary, we conclude that Agn1 and Eng1 protein levels oscillate during the cell cycle and that Agn1 protein levels peak during G1 and S phases.

agn1⁺ and eng1⁺ Expression Depend on Transcription Factors Sep1p and Ace2p

To investigate whether transcription factors Sep1p and Ace2p are involved in regulating Agn1 protein levels, we crossed agn1-myc into ace2Δ and sep1Δ backgrounds. Immunoblot analysis showed that the Agn1-myc protein level had decreased dramatically in both these mutants (Figure 7, compare lanes 2 and 3 to lane 1). To exclude the possibility that the observed lack of Agn1-myc protein expression was the result of a change in the frequency of cells in G1 and S phases, we performed an arrest-release experiment on cdc25-22 agn1-myc cells deleted for ace2⁺. At no stage of the cell cycle were we able to detect Agn1-myc protein in the ace2Δ background (Figure 6, compare A and C). These data show that ace2⁺ and sep1⁺ each are required for Agn1 protein expression.

Not only did Agn1-myc protein levels decrease considerably in both the sep1Δ and ace2Δ backgrounds but also Eng1-myc protein levels (Figure 7, compare lanes 5 and 6 to lane 4). Simultaneous deletion of sep1⁺ and ace2⁺ did not further decrease Eng1-myc protein levels, suggesting that their effects are not additive (Figure 7, lane 7). This observation was substantiated by the finding that the ace2Δ and sep1Δ single and double mutants formed clumps to a similar extent, although the double mutant sedimented a little faster than either single mutant (Table 2).

To study the effect of a simultaneous loss of agn1⁺ and eng1⁺ expression in the ace2Δ mutant (see above), we studied its ultrastructure. We observed that in ace2Δ cells both the septum edging and the primary septum remained intact (Figure 5C). Also, in this mutant, apical growth was redirected toward the side of the septum, causing branching (Figure 5E, solid arrowheads). This phenotype was also observed for the agn1Δ eng1Δ double mutant (Figure 3, arrowheads). From these experiments we conclude that both Sep1p and Ace2p are required for periodic expression of both Agn1 and Eng1 proteins.

Agn2p Is Essential for Endolysis of the Ascus Wall during Sporulation

To investigate whether the cellular function of agn2⁺ is similar to that of agn1⁺, we deleted the agn2⁺ ORF and analyzed the resulting deletion phenotype. agn2Δ cells were viable and grew in YEA culture medium at 28°C with a generation time similar to that of wild-type cells (our unpublished data). agn2Δ cells also displayed a cylindrical rod morphology indistinguishable from that of wild-type cells (Figure 3), and they did not form clumps (Table 2). Furthermore, northern blot analysis indicated that agn2 transcript levels did not fluctuate during the cell cycle (our unpublished data). We conclude that, unlike agn1⁺, agn2⁺ does not seem to have a role during vegetative growth.

agn2⁺ expression is up-regulated temporarily during sporulation (Mata et al., 2002). To localize Agn2p in sporulating cells, we crossed haploid agn2-gfp cells with haploid wild-type cells of opposite mating type and analyzed Agn2p-GFP fluorescence in living mating products. Although Agn2p-GFP fluorescence in the haploid agn2-gfp cells was weak and diffuse, it was more intense in sporulating diploid cells. Agn2p-GFP was excluded from ascospores, but instead localized to the residual cytoplasm within the ascus wall (Figure 8A), known as the epiplasm (Yoo et al., 1973). To assess Agn2p function, we analyzed agn2Δ cells in a simple mating assay. No differences in conjugation and sporulation efficiency were observed with agn2Δ cells compared with wild-type cells (our unpublished data). To determine whether agn2⁺ plays a role in the subsequent step of endolysis of the ascus wall, during which the ascospores are released, we transferred agn2Δ/agn2Δ diploid cells to sporulation medium. Like the majority of matching wild-type diploids, the majority of agn2Δ/agn2Δ cells developed into asci containing four ascospores, called tetrads, within 48 h after start of induction, confirming that sporulation was not affected by agn2⁺ deletion (our unpublished data). However, whereas the ascus walls lysed in a majority of wild-type tetrads, releasing individual ascospores within the following 48 h, the ascus walls remained intact in a majority of agn2Δ/agn2Δ tetrads, even after 5 d after tetrad formation (Figure 8B). Small numbers of free ascospores were observed, but they represented only ∼10% of the tetrads. We conclude from these experiments that Agn2p is involved in endolysis of the ascus wall.