Abstract
BackgroundMannoproteins 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Mannoproteins construct the outer cover of the fungal cell wall
The 21 candidate genes were arranged into functional categories according to the Saccharomyces Genome Database (SGD): Cell wall synthesis and regulation Three of the identified genes are elements of the Cell Wall Integrity (CWI) pathway: the upstream sensor MID2, the mitogen-activated protein (MAP) kinase kinase kinase BCK1 and the MAP kinase SLT2
Downstream targets of CWI pathway involved in chitin deposition were found: CHS3, a N-acetylglucosamine transferase responsible for chitin synthesis at the bud neck and in the lateral cell wall as well as for chitin deposition in response to cell wall stress; CHS4, a direct activator of Chs3p activity [32] anchoring Chs3p to the septin ring via Bni4p [33]; CHS5, a component of the exomer complex involved in the polarized transport of Chs3p in specialized vesicles [34]; and CHS7, a membrane protein controlling Chs3p export from the ER [35,36,37]
Summary
The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. In Saccharomyces cerevisiae, the cell wall adds up to 30% of the total cell dry weight It is mainly composed of polysaccharides (85%) and highly glycosylated mannoproteins (15%) [1]. B-1,3glucan is synthesized at the plasma membrane and constitutes the principal cell wall polysaccharide to which other components are cross-linked [2,3]. Most of the chitin at the bud neck is linked to non-reducing ends of b-1,3-glucan, whereas the chitin of the lateral cell wall is mainly attached to b-1,6-glucan [4,5]. At the outer layer of the b-1,3-glucan meshwork highly branched b-1,6-glucan is present to which mannoproteins are linked via a remnant of their glycosylphosphatidylinositol (GPI) anchor (GPI-anchored covalently linked cell wall proteins; GPI-CWPs) [4]
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