The β-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.
Bottom Line: The lack of enzymatic activity of Gas1 or treatment with a cell wall-damaging agent, Congo red, exhibits effects similar to those of the gas1Δ mutation.Collectively, our results suggest that the dysfunction of Gas1 plays a positive role in the maintenance of rDNA integrity by decreasing PKA activity and inducing the accumulation of Msn2/4 in the nucleus.It seems that nuclear-localized Msn2/4 stimulate the expression of Pnc1, thereby enhancing the association of Sir2 with rDNA and promoting rDNA stability.
Affiliation: Department of Biological Sciences and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.Show MeSH
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Mentions: The β-1,3-glucanosyltransferase activity of Gas1 plays a role in the formation of β-1,3-glucanosidic bonds and the crosslinking of cell wall components (20,35). Two amino acid residues, E161 and E262, are critical for the β-1,3-glucanosyltransferase activity of Gas1 (36,37). Koch and Pillus (21) demonstrated that cells carrying the GAS1 gene with E161Q and E262Q mutations (gas1E161Q, E262Q) are defective in telomeric silencing, indicating that the enzymatic activity of Gas1 is necessary for telomeric silencing. However, whether the β-1,3-glucanosyltransferase activity of Gas1 is linked to the Msn2/4–Pnc1–Sir2 pathway of rDNA silencing is unknown. To check this, we analyzed the subcellular localization of Msn2/4 in gas1E161Q, E262Q mutant cells. Like gas1Δ cells, gas1E161Q, E262Q cells also exhibited a significant increase in nuclear-localized Msn2/4 (Figure 2A). The protein level of Gas1 was little, if any, changed in cells carrying the GAS1 gene with E161Q and E262Q mutations (gas1E161Q, E262Q) compared with cells carrying wild-type GAS1 (Supplementary Figure S3). We next examined the degree of Msn2/4 binding to the PNC1 promoter in gas1E161Q, E262Q cells. As expected, the binding of Msn2/4 to the PNC1 promoter was significantly increased in gas1E161Q, E262Q cells (Figure 2B). Consistent with this result, cells with Gas1E161Q, E262Q exhibited increased expression of Pnc1 compared with cells with wild-type Gas1 (Figure 2C). Moreover, a ChIP assay to measure the association of Sir2 with the rDNA region revealed that cells with Gas1E161Q, E262Q exhibited significantly enhanced association of Sir2 with the rDNA region compared with cells with wild-type Gas1 (Figure 2D). These results suggest that the gas1E161Q, E262Q mutant defective in β-1,3-glucanosyltransferase activity and the gas1Δ knockout mutant exhibit similar effects on the nuclear localization of Msn2/4, the binding of Msn2/4 to the PNC1 promoter, the expression of Pnc1, and the association of Sir2 with rDNA.
Affiliation: Department of Biological Sciences and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.