Rot1p of Saccharomyces cerevisiae is a putative membrane protein required for normal levels of the cell wall 1,6- -glucan

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Abstract
Although ROT1 is essential for growth of Saccharomyces cerevisiae strain BY4741, the growth of a rot1Δ 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. Candida albicans and Schizosaccharomyces pombe have Rot1p homologues with high identity that also have predicted transmembrane domains. To explore the role of Rot1p, the phenotypes of the rot1Δ haploid were analysed. Deletion of ROT1 caused cell aggregation and an abnormal morphology. Analysis of the cell cycle showed that rot1Δ cells are delayed at the G2/M phase. The rot1Δ 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-β-glucan, and increased levels of chitin and 1,3-β-glucan, were found in rot1Δ cells. Furthermore, the phenotypes of rot1Δ cells resemble those of disruption mutants of the KRE5 and BIG1 genes, which show greatly reduced levels of cell wall 1,6-β-glucan. Incorporation of glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in big1Δ and rot1Δ 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 rot1Δ and big1Δ 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 big1Δrot1Δ double mutant had a growth rate similar to the corresponding single mutants, suggesting that Rot1p and Big1p have related functions in 1,6-β-glucan synthesis.

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