Pradimicin resistance of yeast is caused by a mutation of the putative N-glycosylation sites of osmosensor protein Sln1.

Pradimicin, a mannose-binding antifungal antibiotic, induces apoptosis-like cell death in Saccharomyces cerevisiae. Previously we found that the substitution of the 74th amino acid from glycine to cysteine in Ypd1 yields a mutant resistant to pradimicin. In this study, the involvement of a membrane-spanning osomosensor, Sln1, which is located upstream of Ypd1, was investigated. A mutant, sln1 DeltaNG, that lacks the putative N-glycosylation sites in the extracellular domain became resistant to pradimicin. On the other hand, the null mutants of Ssk1, Pbs2, and Hog1, which are located downstream of the Sln1 cascade, were sensitive to pradimicin as well as the wild-type strain. In conclusion, pradimicin exerts its fungicidal action with the involvement of Sln1, but the downstream branch, Ssk1 and the HOG pathway, is not involved.

Apoptosis-like cell death of Saccharomyces cerevisiae induced by a mannose-binding antifungal antibiotic, pradimicin.

Pradimicin A (PRM), a mannose-binding antifungal antibiotic, induced the apotosis-like cell death in Saccharomyces cerevisiae. The nuclear breakage and DNA fragmentation were observed in yeast cells by DAPI and TUNEL staining after the treatment with PRM. Accumulation of reactive oxygen species (ROS) was also detected in PRM-treated yeast cells by staining with dichlorodihydrofluorescein diacetate. PRM-induced cell death and the accumulation of ROS were prevented by pretreating the yeast cells with a radical scavenger, N-acetylcysteine. These results indicate that PRM induces the apoptosis-like cell death in yeast through the generation of ROS.

Pradimicin-resistance of yeast is caused by a point mutation of the histidine-containing phosphotransfer protein Ypd1.

Pradimicin is an antifungal antibiotic which induces apoptosis like cell death in the yeast Saccharomyces cerevisiae. Pradimicin-resistant mutants were isolated from the S. cerevisiae and the mutation points were analyzed. A point mutation of YPD1 that led to a substitution of the 74th glycine (Gly74) to cysteine (Cys) was identified in a mutant strain NH1. In S. cerevisiae, Ypd1 transfers a phosphoryl group from the sensor kinase Slnl to the response regulator Sskl which regulates a downstream MAP kinase in response to hyperosmotic stress. Gly74 is located in a three-residue reverse turn domain that connects two alpha-helices, one of which contains a histidine residue which is phosphorylated. In the reverse turn, glycine (relative position +10 to the active-site histidine) is highly conserved in Ypd1 and other histidine-containing phosphotransfer proteins. It was therefore suggested that the substitution of Gly74 to Cys altered the Ypd1 structure, which resulted in the resistance to pradimicin.