. Research Article Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway Helene Martin-Yken 1 , Adilia Dagkessamanskaia 1 , Piet De Groot 2 , Arthur Ram 2 , Frans Klis 2 and Jean Franc ¸ois 1 * 1 Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, UMR-INRA 792, F-31077, Toulouse, France 2 Biocentrum Amsterdam, University of Amsterdam, Swammerdam Institute for Life Sciences, 1098 SM Amsterdam, The Netherlands * Correspondence to: J. M. Franc ¸ois, De ´partement de Ge ´nie Biochimique et Alimentaire, INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 04, France. E-mail: fran_jm@insa-tlse.fr Received: 21 November 2000 Accepted: 5 February 2001 Abstract The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1–MPK1 pathway, including a growth defect and increased release of b-1,6-glucan and b- glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14–16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanoga- ster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43–GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele- specific phenotype appears to be due to a G–R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway. However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G 1 , no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: cell wall; Calcofluor white hypersensitivity; MAP kinase; PKC1; cell integrity; cell cycle Introduction The yeast cell wall is an essential outer organelle that confers cell shape, mechanical strength and osmotic protection and acts as a selective barrier for external compounds. The cell wall is mainly composed of carbohydrates and accounts for 20–30% of the cell dry mass (Klis, 1994; Dallies Yeast Yeast 2001; 18: 827–840. DOI: 10.1002/yea.731 Copyright # 2001 John Wiley & Sons, Ltd.