The effect of Tunicamycin on Wortmannin resistant Schizosaccharomyces pombe mutants

Tunicamycin causes accumulation of incorrectly-folded proteins by inhibiting N-glycosylation of asparagine groups, and is an antibiotic used extensively in cell biology studies because it increases endoplasmic reticulum (ER) stress. Tunicamycin causes ER stress by blocking N-glycosylation vice inhibiting of N-acetylglucosaminphosphotransferase, the enzyme that transfers N-acetylglucosamin-1-phosphate to phosphorylated dolichol. In this study, tunicamycin, known to cause ER stress, was applied to the collection known to be resistant to Wortmannin and signaling pathway, mechanism of action and functions of related genes with single gene deletions were investigated. 111 haploid strains with different mutations in each, which were found Wortmannin-resistant in our previous study, were treated by a semi-lethal dose of tunicamycin (0.5 µg/mL). Twenty mutant strains were identified as tunicamycin-resistant or sensitive. The differences in intracellular oxidation (ROT levels, reactive oxygen species) of the control groups and tunicamycin-treated cells, the changes in nucleus with DAPI method, the viability and apoptosis levels in the cells with acridine orange/ethidium bromide (AO/EtBr) technique were measured. In addition, the viability and resistance levels of strains with the spot assay method and the changes occurring in proteosomal activities with 20S proteasome activity measurement were shown in comparison with both their control groups and parental strain. When comparing the groups grown in the media with or without tunicamycin, it was observed that the cells grow fewer in tunicamycin-containing media and were morphologically larger size, nucleus structures changed significantly, intracellular oxidation levels increased, proteosomal activities changed significantly and the cells were directed to apoptosis. The results will help to clarify the molecular mechanism of some human diseases.