Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions

Abstract

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.

Figure 1

Histograms derived from Gene Set Enrichment Analysis for categories with a corrected p-value ≤ 0.0004. A) Density versus t-value plots for protein biogenesis, stress response, unknown, carbohydrate metabolism, and RNA processing categories. Red lines represent the distribution of genes of a specific category in the array. Black lines represent the distribution of all genes in the array. B) t-value versus A-value plots for protein biogenesis, stress response, unknown, carbohydrate metabolism, and RNA processing categories. Red dots represent the genes of a specific category in the array. Black dots represent the distribution of all genes in the array. The cutoff for a significant category is based on a p-value calculated after 10,000 permutations and then a corrected p-value was calculated using the Bonferroni correction. See Methods section for details.

Figure 2

(A) Genetic disruption of the SLT2/MPK1 gene induces lethality in a myo1Δ strain. Wild type, myo1Δ, slt2Δ and myo1Δ slt2ΔpRS316-MYO1 strains were grown in CSM (1 mg/ml) 5-FOA for three days at 26°C. (B)Suppression of Nikkomycin Z hypersensitivity in myo1Δ strains overexpressing the ribosomal protein genes RPL30 and RPS31. Wt, myo1Δ, myo1ΔpRS316-RPL30 and myo1ΔpRS316-RPS31 strains were grown CSM or CSM URA^- in presence or absence of 6.25 μM Nikkomycin Z in 2% glucose or 2% galactose for 48 hours at 26°C. Percent of Nikkomycin Z resistance was calculated as: (OD_{600 nm treated}/OD_{600nm untreated}) × (100). Dark gray histograms represent cells where expression of the plasmid is repressed with 2% glucose and light gray histograms represent cells where expression of the plasmid gene is induced with 2% galactose. (C) Determination of the activation of the cell integrity pathway in myo1Δ strains overexpressing the ribosomal protein genes RPL30 and RPS31. Wild type, myo1Δ, wildtype pRS316-RPL30, wild type pRS316-RPS31, myo1ΔpRS316-RPL30 and myo1ΔpRS316-RPS31 strains were grown in CSM or CSM URA^- in the presence of 2%glucose or 2% galactose. Equal amounts of protein (75 μg) were analyzed by Western blot as described in the Methods section. The phosphorylated levels of Slt2p were observed using a mouse monoclonal antibody against phospho-p42/p44 Slt2p (p-Slt2p). The membrane was stripped and reprobed with a monoclonal antibody against Pgk1p as a loading control.