A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Abstract

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.

Cell-type-dependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p–Itc1p chromatin remodelling complex

“…As shown in Fig. 1, Slt2p was found to be activated in both itc1 strains, thus indicating that the previously reported activation of this MAPK (de Groot et al, 2001) is not mating type-specific. These results suggest that the aberrant morphology and the constitutive activation of the pheromone pathway, detected only in the MATa itc1 mutant cells, are not related to the cell wall phenotype.…”

“…itc1 mutants show mating type-independent activation of the cell integrity pathway ITC1 has been identified among a group of genes whose deletion results in defects in cell wall integrity (de Groot et al, 2001). The systematic screening of 620 MATa mutants for different cell wall-related phenotypes revealed the itc1 mutant to be sensitive to the presence of calcofluor white or caffeine in the growth medium, and to show constitutive activation of Slt2p/Mpk1p, the MAPK of the cell wall integrity signalling pathway.…”

“…No clues as to the biological function of the Itc1 protein were known when these phenotypes were reported. In a subsequent systematic screen for mutants displaying cell-wall-related phenotypes, also in the frame of EUROFAN, itc1 was identified among a group of mutants showing increased sensitivity to calcofluor white and constitutive activation of Slt2/Mpk1, the MAP kinase of the cell integrity signalling pathway (de Groot et al, 2001).…”

Identification of phosphatase 2A‐like Sit4‐mediated signalling and ubiquitin‐dependent protein sorting as modulators of caffeine sensitivity in S. cerevisiae

“…These results indicate that the suppressor genes work in parallel to set the normal level of caffeine resistance in wild-type cells. The fact that none of the suppressor genes was previously identified in largescale screens examining caffeine sensitivity (de Groot et al, 2001;Dudley et al, 2005) highlights the importance of multiple genetic approaches to studying a given cellular process.…”

“…Furthermore, the widespread human consumption of caffeine-containing beverages and foods makes understanding the molecular details of its effects on cells a medically relevant endeavour (Bode and Dong, 2007;Sabisz and Skladanowski, 2008). Although there are numerous reports of budding yeast mutations that confer caffeine sensitivity (de Groot et al, 2001;Dudley et al, 2005;SGD Project, 2009), relatively few mutations have been shown to cause caffeine resistance. Notably, certain mutations in the kinase domain and FK506 binding protein-interacting domain (FRB) of TOR confer synergistic caffeine resistance (Reinke et al, 2006), and screens for caffeine-resistant mutants in Schizosaccharomyces pombe have yielded strains with mutations in genes that function in oxidative stress response mediated by the transcription factor Pap1 (Benko et al, 1997(Benko et al, , 1998(Benko et al, , 2004Kumada et al, 1996).…”