Although is essential for growth of strain BY4741, the growth of a Δ haploid was partially restored by the addition of 0·6 M sorbitol to the growth medium. Rot1p is predicted to contain 256 amino acids, to have a molecular mass of 29 kDa, and to possess a transmembrane domain near its C-terminus. and have Rot1p homologues with high identity that also have predicted transmembrane domains. To explore the role of Rot1p, the phenotypes of the Δ haploid were analysed. Deletion of caused cell aggregation and an abnormal morphology. Analysis of the cell cycle showed that Δ cells are delayed at the G2/M phase. The Δ cells were resistant to K1 killer toxin and hypersensitive to SDS and hygromycin B, suggesting that they had cell wall defects. Indeed, greatly reduced levels of alkali-soluble and -insoluble 1,6--glucan, and increased levels of chitin and 1,3--glucan, were found in Δ cells. Furthermore, the phenotypes of Δ cells resemble those of disruption mutants of the and genes, which show greatly reduced levels of cell wall 1,6--glucan. Incorporation of glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Δ and Δ cells was examined using a GFP–Flo1 fusion protein. GFP fluorescence was detected both on the cell surface and in the culture medium, suggesting that, in these mutants, mannoproteins may become only weakly bound to the cell wall and some of these proteins are released into the medium. Electron microscopic analyses of Δ and Δ cells showed that the electron-dense mannoprotein rim staining was more diffuse and paler than that in the wild-type, and that the outer boundary of the cell wall was irregular. A ΔΔ double mutant had a growth rate similar to the corresponding single mutants, suggesting that Rot1p and Big1p have related functions in 1,6--glucan synthesis.


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