The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections

Abstract

Background: Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency.

Results: In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection.

Conclusions: The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of CCW12. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.

Figure 1

Characterization of individual single mutants in genes identified by SGA. (A) Calcofluor white (CW) sensitivity. Indicated mutant strains were processed as described in Methods. 3 × 10⁵ cells, and 10-fold serial dilutions thereof were spotted on YPD plates with, and without 25 μg/ml of CW. Plates were incubated at 30°C for 2 days. (B and C) Activation of the CWI pathway. Cell extracts were analysed by Western blot using phospho-p44/p42 MAPK antibodies to detect the dually phosphorylated form of Slt2p, the MAP kinase of the CWI pathway (lower panel, PP-Slt2p). Phosphofructokinase 1 (Pfk1p) was used as a loading control (upper panel, Pfk1p antibodies are directed against both Pfk1p subunits). (B) To analyse constitutive Slt2p phosphorylation, wt and indicated mutant strains were grown at 30°C. (C) To analyse induction of Slt2p phosphorylation in response to different stresses, cells were incubated with CW or at elevated temperatures as described in Methods. For unstressed cells, one representative extract is shown. PP-Slt2p and Pfk1p protein levels were quantified using the ScionImage™ software, and the amount of PP-Slt2p was normalized to the amount of the larger Pfk1p subunit. Values of the mutants were referred to the wt value (set to 1), and are shown below the figure.

Figure 2

Functional categories of the differentially expressed genes. Black bars represent the functional catalogue of 6200 genes according to the MIPS classification. White bars represent the distribution of the 472 differentially expressed genes from ccw12Δ mutant according to the MIPS functional catalogue. The percentage of entries is given by the ratio of the number of regulated genes in each category and the total number of differentially expressed genes. Asterisks highlight categories with p-values < 0.05. P-values were obtained using hypergeometric distribution statistics (default setting in FunCatDB tools, MIPS). Note that the sum of entries is more than 100% since some genes are annotated in more than one functional category in the FunCat database.

Figure 3

Hierarchical clustering of genes differentially expressed in ccw12Δ. The transcriptional response of the ccw12Δ mutant was compared to transcriptomes of other cell wall mutants (gas1Δ, fks1Δ, mnn9Δ and kre6Δ) and cell wall stress conditions (Zymolyase and Congo Red) [15,16]. Each column represents a different condition. Each row represents the ratio of expression for each gene as it is indicated in the colour scale. The clustering tree was built using MeV software from TIGR.

Figure 4

Activation of the CWI pathway in mutant ccw12Δ. Mutant ccw12Δ, slt2Δ, gas1Δ and BY4741 (isogenic wt) cells, were grown to mid-exponential phase, and cell extracts were analysed by Western blot using phospho-p44/p42 MAPK antibodies to detect dually phosphorylated Slt2p (mid and low panel, PP-Slt2p). Pfk1p was used as a loading control (upper panel, Pfk1p). PP-Slt2p and Pfk1p protein levels were quantified as described in Figure 1

Figure 5

Ccw12p localizes to areas of active cell wall synthesis and ccw12Δ cells display bud lysis. (A) Cell lysis phenotypes. Wt (SEY6211) and ccw12Δ mutant (MEY12B) strains, exponentially growing in YPD (a, b) or YPD supplemented with 1 M sorbitol (c, d), were stained with the vital dye methylene blue to identify dead cells (details are described in Methods): (a) wt cells display a typical ellipsoidal shape; (b) mutant cells show a pronounced round morphology and lyse as small budded cells (16% of ccw12Δ cells vs. 3% of wt cells); (c) mutant cells round morphology is partially reverted in presence of osmotic stabilization; (d) After hypotonic shock (transfer to YPD) lysis as small budded cell is observed(41% of ccw12Δ cells vs. 8% of wt cells) and (e-f) cell lysis occurs after completion of cytokinesis as shown by DAPI staining (panel e and f represent the same cells that have been stained with methylene blue and DAPI). (B) Localization of Ccw12p during vegetative growth. To exclude artefacts due to over-expression of CCW12-GFP, Ccw12p-GFP is expressed from plasmid pCCW12-GFP in mutant MEY12B. (a) Ccw12p-GFP is enriched at sites of emerging buds. The arrow marks the site of bud emergence. When cells proceed in the cell cycle, Ccw12p-GFP is specifically enriched in small (b) and medium-sized (c) buds. (d) After cytokinesis Ccw12p-GFP marks the septum.

Figure 6

Budding and formation of the mating projection are affected in ccw12Δ cells. Wt (SEY6211) and ccw12Δ mutant (MEY12B) strains were treated with the mating pheromone α-factor as detailed in Methods. At time zero 20 μg/ml of α-factor were added. Budding index (A) and shmoo formation (B) was monitored over the time. Mean values of four independent experiments are shown. At least 200 cells were analysed at the indicated times.

Figure 7

Ccw12p localizes to the shmoo tip and ccw12Δ cells display pheromone induced cell lysis. (A) Wt (SEY6211) and ccw12Δ mutant (MEY12B) strains were treated with α-factor (20 μg/ml) for two hours. Cells were stained with the vital dye methylene blue. (a) Wt cells display a typical elongated shmoo. (b) Shmoos of mutant cells are rounder and less polarized. (c) Mutant cells dye during shmoo formation (10% of ccw12Δ vs. 1% of wt cells). (d, e) Buds of mutants cells released from G1-arrest undergo cell lysis after cytokinesis is completed; a representative cell stained with methylene blue (d) and DAPI (e) is shown. (f) Mutant cells tend to lyse as small budded cells after re-entry of the mitotic cell cycle (23% of ccw12Δ vs. 5% of wt cells). (B) Localization of Ccw12p-GFP to the shmoo during pheromone treatment. Yeast strain described in figure 7B was treated with α-factor as indicated in 7A.