Dosage rescue by UBC4 restores cell wall integrity in Saccharomyces cerevisiae lacking the myosin type II gene MYO1

Abstract

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-β-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.

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

“…The RPS31 gene product constitutes a fusion of a non-essential small ribosomal (40S) subunit protein with ubiquitin, which facilitates the assembly of ribosomal proteins into ribosomes and is required for the proper translation of GCN4 , a transcription factor that regulates amino acid biosynthesis [ 22 ]. A previous screen for suppressors of Nikkomycin Z (NZ) hypersensitivity in myo1Δ strains identified the cDNAs encoding RPL30 and RPS31 among others [ 24 ]. Nikkomycin Z is a competitive inhibitor of Chs3p and our previous studies established the hypersensitivity of myo1Δ strains to this antibiotic which inhibits cell growth and affects cell integrity by blocking chitin synthesis [ 8 ].…”

“…We previously demonstrated that overexpression of the cDNA for ubiquitin conjugating enzyme UBC4 lowered the p-Slt2p levels and conferred resistance to NZ in myo1Δ strains, suggesting that cell integrity was restored [ 24 ]. We have now discovered that overexpression of RPL30 and RPS31 can also up regulate the cell integrity pathway to achieve a similar effect.…”

“…The screening for Nikkomycin Z sensitivity suppression in myo1Δ was performed as described in [ 24 ]. Briefly, 1 × 10 5 cells/ml of WT, myo1Δ , myo1Δ p RPL30 and myo1Δ p RPS31 cells were grown in ura - glu - or ura - gal - in the presence or absence of 6.25 μM Nikkomycin Z at 26°C and 225 rpm.…”

“…Wild type, myo1Δ , wild type pRS316- RPL30 , wild type pRS316- RPS31 , myo1Δ pRS316- RPL30 , and myo1Δ pRS316- RPS31 yeast strains were grown in selective medium in 2% glucose or 2% galactose to an OD 600 0.5–0.8 at 26°C, overnight. Cells were centrifuged for 5 minutes, washed in ice cold medium, and resuspended in lysis buffer as described in [ 24 ]. Protein extract were centrifuged at 13,000 for 10 minutes at 4°C, the supernatant was removed and quantified using the DC Protein Assay method (BioRad).…”

Differential gene expression signatures for cell wall integrity found in chitin synthase II (chs2Δ) and myosin II (myo1Δ) deficient cytokinesis mutants of Saccharomyces cerevisiae

“…Yeast strains were grown in selective medium between 0.5–0.8 OD 600 at 26°C. Cells treatment, protein extraction and quantification methods were performed as described [ 6 , 9 , 10 , 17 ]. Total protein extracts (75 μg) were separated in a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane at 70 V for 2 h at 4°C.…”