In this work we provide evidence that Ccw12p has a specific role in maintaining the stability of the newly synthesized cell wall especially during bud emergence and formation of the mating projection. Ccw12p localizes to areas of active cell wall synthesis such as forming buds (Figure 5B). In the absence of Ccw12p, buds are abnormally round  and cells tend to lyse during bud emergence and development (Figure 5A, panel b; 16% of ccw12Δ vs. 3% of wt cells). Moreover, when ccw12Δ cells are subjected to hypotonic shock buds lyse more frequently in early stages of development (Figure 5A, panel d; 41% of ccw12Δ cells vs. 8% of wt cells). Even after cytokinesis, daughter cells tend to lyse (Figure 5A, panel e). In agreement with the observed lysis phenotype, Ccw12p is specifically localized at the daughter side of the septum (Figure 5B, panel d). During mating, cell lysis is observed when the shmoo is forming (Figure 7A, panel c; 10% of ccw12Δ cells vs. 1% of wt cells) and Ccw12p is specifically localized to the mating projection (Figure 7B), further supporting a local function for cell wall stability during remodelling.
The CWI pathway plays a crucial role in cell wall stability especially during bud and shmoo formation. Mutants in this pathway show some of the phenotypic traits that are characteristic for ccw12Δ . We could demonstrate, that in ccw12Δ cells the pathway is hyper-activated (Figure 4) and that osmotic stabilization suppresses CWI pathway hyper-activation (Figure 4) as well as the growth defect of ccw12Δ . In addition, SGA and transcriptome analyses further demonstrate the interdependence between components of the CWI pathway and CCW12 (Tables 1 and 2). SGA revealed synthetic lethality of the CCW12 deletion in combination with mutants of the key regulatory kinases of the CWI pathway, BCK1 and SLT2 (Table 1). In addition, combinatory deletions with chitin synthesis genes (Table 1; ), and MID2, one of the main sensors of the CWI pathway, (Table 1; ) displayed severe synthetic growth phenotypes. Surprisingly, no direct involvement of the sensors WSC1-3  was found. Wsc1p normally localizes to sites of new cell surface growth as similarly observed for Ccw12p-GFP during the budding process. However, we could demonstrate an interaction with SLA1. It is known that Sla1p, a component of the actin- and clathrin-based endocytic machinery can serve as an NPFX(1,2)D-specific endocytic adaptor and that the NPFX(1,2)D-Sla1p system is responsible for directing Wsc1p into an endocytic and recycling pathway necessary to maintain yeast cell wall polarity . In turn, inhibition of Wsc1p endocytosis causes defects in polarized deposition of cell wall material and increased sensitivity to perturbation of cell wall synthesis. In our analysis, SLA1 deletion conferred synthetic lethality in combination with ccw12Δ (Table 1), similar to what was found in independent screens performed combining deletions of SLA1 and genes involved in glucan and chitin synthesis [60, 30]. Moreover, the SLA1 transcript is 2.69-fold increased when CCW12 is absent (Table 1). These results suggest that NPFX(1,2)D/Sla1p-mediated endocytosis and polarized localization of Wsc1p are important to ensure cell wall integrity in the developing daughter cell in the absence of Ccw12p. The lack of synthetic lethality of CCW12 in combination with WSC1 could be explained by a redundant function of one of the other WSC sensors. In contrast, deletion of SLA1 might not only affect WSC1 but other WSC-family members as well.
Transcriptional profiling of the ccw12Δ mutant revealed predominantly cell wall remodelling activities. Members of all categories of cell wall related genes are regulated: CWI pathway signalling, structural components, chitin/glucan synthesis and glycosylation with many of them under the control of PKC1-mediated signalling cascade. Interestingly, despite the high transcriptional activation of some of these genes, their deletion mostly generates mild or no cell wall phenotypes and only a few of them conferred synthetic lethality when deleted in combination with CCW12. This may either be explained by functional redundancy or the need to repress several cell wall functions and/or components in order to cause severe cell wall damage.
In addition to the CWI pathway, the high-osmolarity glycerol (HOG) and the SLN1-SKN7 pathway contribute to cell wall integrity, The HOG pathway is modulated by various plasma membrane sensors . One of which is the kinase Sln1p, the initiating member of a two-component type of phospho-relay cascade that negatively regulates the HOG pathway. In addition, Sln1p activates the transcription factor Skn7p which in turn triggers the transcription of specific target genes such as OCH1 and NCA3 . CWI and HOG pathway are closely interconnected suggesting a cooperative role for both pathways controlling cell integrity [15–17, 40, 41]. Likewise, it was suggested that the CWI and the SLN1-SKN7 pathway may function in parallel to protect cells from lysis . Interestingly, SGA revealed the interdependence between CCW12 and HOG1, the MAP kinase of the HOG pathway (Table 1). Further, transcription of OCH1 and NCA3 is significantly increased in the ccw12Δ mutant (2.69- and 3.36-fold increase respectively; Additional file 2) confirming previous data that suggested Ccw12p to be a modulator of Sln1p activity . Thus, different signalling pathways are involved to ensure cell wall integrity in the absence of Ccw12p. However, the CWI pathway and cell wall remodelling genes play a dominant role as demonstrated by the preponderance of CWI related genes identified in the SGA screen.
We further addressed the question whether different cell wall defects induce a similar response in yeast cells. Although there is some variation in the regulation of individual genes under different conditions, their functional classification indicates a common principle. We found significant overlap in the transcriptional response to CCW12 deletion as compared to that in other cell wall mutants and treatment with cell wall perturbing agents such as Zymolyase or CR [15, 16]. Notably, under all tested conditions we found 20 genes to be strongly induced, which could constitute the core response for cell wall stress (Figure 3, Cluster I). With the exception of SUR1, all the genes in this cluster are CWI pathway targets, reinforcing the notion that survival under cell wall stresses is mediated by different aspects regulated by CWI pathway.
In a previous work, a marked decrease in the mating and agglutination ability of ccw12Δ strain was detected . Here we demonstrate that ccw12Δ cells exhibit diminished α-factor sensitivity and earlier release from the pheromone-induced G1 arrest (Figure 6). The mating-related phenotypes could be due to a combination of different factors: i) during pheromone treatment, the absence of Ccw12p at the shmoo apex results in mating induced cell death of ~10% of the cells (Figure 7A). As a result, mating frequency is significantly reduced. ii) The ccw12Δ expression profile revealed down-regulation of genes involved in the mating process which is not detected in other cell wall mutants. iii) ccw12Δ cells show an early release from the pheromone-induced G1-arrest. The time cells display mating projections and the recovery time from pheromone arrest can greatly influence the mating efficiency. It is well known that in the absence of a successful mating event, G1-arrested cells re-enter the mitotic cycle through a recovery process that involves down-regulation of the mating mitogen-activated protein kinase cascade mainly through Msg5p phosphatase activity on the MAP kinase, Fus3p . Furthermore, recovery is regulated by POG1, coding for a putative transcriptional activator . Overexpression of POG1 inhibits α-factor-induced G1-arrest and transcriptional repression of the CLN1 and CLN2 genes. This loss of transcriptional repression occurs through SCB/MCB promoter elements and requires Bck1p, known to up-regulate Swi4-dependent cell-cycle box (SCB)/MluI cell-cycle box (MCB) promoter elements during vegetative growth . The G1-cyclin Cln2p, in addition to driving the G1- to S-phase transition , when over-expressed blocks the ability of cells to arrest in the presence of α-factor, primarily through an effect on Ste20p, an activator of the mating MAPK cascade . In agreement with these data, we detected a 3.14-fold increase for POG1 and a 2.18-fold increase for CLN2 in ccw12Δ cells, explaining the earlier G1-release.