Isolation and characterization of the aureobasidin A-resistant gene, aur1 R , on Schizosaccharomyces pombe : roles of Aur1p + in cell morphogenesis

Abstract

To study the mechanism of action of the antibiotic aureobasidin A (AbA) on yeasts, we isolated a dominant mutant of Schizosaccharomyces pombe which gave high resistance to AbA. From a genomic library of the mutant, an aur1R mutant gene conferring AbA resistance was isolated. One amino-acid mutation, a substitution of glycine with cysteine at residue 240, was responsible for the acquisition of AbA resistance. The wild-type aur1+ gene was essential for viability, and its over-expression enhanced significant resistance to AbA. The predicted protein of S. pombe aur1R was highly homologous in primary structure and hydropathy profile with that of Saccharomyces cerevisiae AUR1R isolated as an AbA-resistance gene. To analyze a role in cell growth of S. pombe aur1+, temperature-sensitive mutants (aur1ts) were obtained by random mutagenesis procedures using a modified PCR. The aur1ts mutation caused a defect in cell elongation at the non-permissive temperature and finally led to cell death. These results suggest that Aur1p was a target of the antibiotic AbA and was required in the cell elongation of cell-end tips and in the viability of S. pombe.

Chromosomes Rein Back the Spindle Pole Body during Horsetail Movement in Fission Yeast Meiosis

“…The resulting plasmid was integrated into the chromosome at the lys1 gene locus. The mCherry-Bqt4 fusion constructs were made as follows: the coding sequence of the mCherry gene was ligated in-frame between the bqt4 promoter sequence and the bqt4 coding sequence with the nmt1 terminator sequence, and it was integrated into the chromosome at the aur1 gene locus using the aur1R allele, which dominantly confers resistance to Aureobasidin A (Takara) (Hashida-Okado et al, 1998). Strains carrying Sid4-GFP, Sid4-mRFP, Taz1-mCherry and Hht1-mRFP were constructed by replacing each wild-type gene with the selection marker kan r by a PCR-based gene targeting method (Bähler et al, 1998).…”

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

“…A particularly interesting target is the fungal inositol phosphorylceramide (IPC) synthase (10), inhibitors of which are highly toxic to many mycopathogens but exhibit low toxicity in mammals (11)(12)(13). IPC synthase, generally believed to be encoded by orthologous AUR1/IPC1 genes discovered in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida species, Aspergillus species, and Cryptococcus neoformans (10,(14)(15)(16)(17)(18), catalyzes the transfer of myo-inositol-1-phosphate from phosphatidylinositol to ceramide. Neither IPC, IPC synthase, nor any orthologous AUR1/IPC1 genes have been found in mammals, consistent with the lack of susceptibility of mammalian species to inhibitors of this enzyme.…”

Glycosphingolipids of the model fungus Aspergillus nidulans

“…On the other hand, it is already well appreciated that disruption of the GIPC pathway, by either inhibition or deletion of IPC synthase activity, is fatal for fungi (24-27, 29, 30), except for some yeast strains with compensating mutations (58)(59)(60). Some fungal strains selected for resistance to the IPC synthase inhibitor Aureobasidin A were found to have a mutation in the AUR1 gene that affects the interaction of the AUR1 protein with the drug but not catalytic activity (25)(26)(27)29). Overexpression of the AUR1 gene in S. pombe conferred significant resistance to Aureobasidin A (25); in addition, wild-type strains of some fungal species, such as the mycopathogen A. fumigatus, are relatively resistant by virtue of highly efficient transporter-mediated drug efflux (61).…”

“…Synthesis of inositol phosphorylceramide (IPC), an obligate intermediate in GIPC catabolism, appears to be required for normal growth of S. cerevisiae (23). The IPC synthases are believed to be coded by orthologous AUR1 / IPC1 genes discovered in S. cerevisiae , Schizosaccharomyces pombe , Candida spp., Aspergillus spp., and C. neoformans (24)(25)(26)(27)(28)(29), and a number of inhibitors of this reaction have been discovered (30)(31)(32). These appear to be very useful tools for studying GIPC biosynthesis and function, as well as offering promise as antifungal agents, insofar as they are highly toxic to many fungi but exhibit low toxicity in mammals (33).…”

“…These appear to be very useful tools for studying GIPC biosynthesis and function, as well as offering promise as antifungal agents, insofar as they are highly toxic to many fungi but exhibit low toxicity in mammals (33). Disruption of the AUR1 gene in S. cerevisiae and S. pombe results in morphological changes, disappearance of microtubules, abnormal deposition of chitin, and cessation of growth (25,29). More recently, Luberto et al (34) observed that downregulation of the IPC1 -encoded IPC synthase in C. neoformans significantly lowered the expression of certain virulence traits, as well as impairing intracellular growth in an in vitro murine macrophage model and pathogenicity in an established rabbit in vivo model.…”