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

Piet W J De Groot,Luis J García,Frans M Klis,Annemiek Andel,Concha Gil,Pilar Pérez,Víctor J Cid,César Nombela,Javier Arroyo,Juan C García,Carlos R Vázquez De Aldana,Julio Caubín,María Molina,Encarnación Duenas,José M Rodríquez-Peña,Cristina Ruiz,Francisco Del Rey

doi:10.1002/cfg.85

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.

Highlights

The cell wall of S. cerevisiae is an essential organelle whose rigid structure determines cell shape, enables cells to withstand internal turgor pressure and protects cells against environmental stresses
For primary identification of potential cell wallrelated genes, the 620 EUROFAN mutants, each deleted in an individual ORF, were analyzed using phenotypic assays which are indicative of mutations leading to a defective cell wall
The cell wall is responsible for maintaining cell shape and, morphological abnormalities may be indicative of alterations in cell wall dynamics during morphogenesis

Summary

Introduction

The cell wall of S. cerevisiae is an essential organelle whose rigid structure determines cell shape, enables cells to withstand internal turgor pressure and protects cells against environmental stresses. The four major components of the cell wall are the polysaccharides b1,3-glucan, b1,6-glucan and chitin, and various mannoproteins. These may be covalently linked to each other to form macromolecular complexes (Kollar et al, 1995, 1997; Kapteyn et al, 1999; Smits et al, 1999). The cell walls contain more Cwp and more Pir proteins, and transcription of FKS2, which encodes a catalytic subunit of the b1,3-glucan synthase complex, is upregulated (Kapteyn et al, 1999; Ram et al, 1998)

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Results

Discussion

Conclusion