RESUMO
BACKGROUND: The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Delta) causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Delta strains. RESULTS: Global mRNA expression profiles of myo1Delta strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p < or = 0.01) were identified with 263 up regulated and 284 down regulated genes in the myo1Delta strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Deltaslt2Delta double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Delta strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. CONCLUSION: Following this analysis of global mRNA expression in yeast myo1Delta strains, we conclude that 547 genes were differentially regulated in myo1Delta strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Delta strain viability, supporting that the up regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Delta arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Delta strains.
Assuntos
Fenômenos Fisiológicos Celulares , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Miosina Tipo II/deficiência , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Aminoglicosídeos/farmacologia , Animais , Western Blotting , Metabolismo dos Carboidratos/genética , Regulação para Baixo , Hipersensibilidade a Drogas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para CimaRESUMO
Myosin II is important for normal cytokinesis and cell wall maintenance in yeast cells. Myosin II-deficient (myo1) strains of the budding yeast Saccharomyces cerevisiae are hypersensitive to nikkomycin Z (NZ), a competitive inhibitor of chitin synthase III (Chs3p), a phenotype that is consistent with compromised cell wall integrity in this mutant. To explain this observation, we hypothesized that the absence of myosin type II will alter the normal levels of proteins that regulate cell wall integrity and that this deficiency can be overcome by the overexpression of their corresponding genes. We further hypothesized that such genes would restore normal (wild-type) NZ resistance. A haploid myo1 strain was transformed with a yeast pRS316-GAL1-cDNA expression library and the cells were positively selected with an inhibitory dose of NZ. We found that high expression of the ubiquitin-conjugating protein cDNA, UBC4, restores NZ resistance to myo1 cells. Downregulation of the cell wall stress pathway and changes in cell wall properties in these cells suggested that changes in cell wall architecture were induced by overexpression of UBC4. UBC4-dependent resistance to NZ in myo1 cells was not prevented by the proteasome inhibitor clasto-lactacystin-beta-lactone and required the expression of the vacuolar protein sorting gene VPS4, suggesting that rescue of cell wall integrity involves sorting of ubiquitinated proteins to the PVC/LE-vacuole pathway. These results point to Ubc4p as an important enzyme in the process of cell wall remodelling in myo1 cells.
