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The β-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.

Ha CW, Kim K, Chang YJ, Kim B, Huh WK - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.

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The lack of Gas1 β-1,3-glucanosyltransferase activity promotes nuclear accumulation of Msn2/4 and enhances the association of Msn2/4 with the PNC1 promoter region. (A) The absence of Gas1 β-1,3-glucanosyltransferase activity induces nuclear accumulation of Msn2 (upper panels) and Msn4 (lower panels). Subcellular localization of Msn2- and Msn4-GFP was analyzed by fluorescence microscopy in wild-type (WT) and gas1Δ cells containing an empty vector and gas1Δ cells expressing WT GAS1 or gas1E161Q, E262Q on the pRS415 vector (left panels). The percentage of nuclear Msn2- and Msn4-GFP is shown in the right panels. Values represent the average of three independent experiments and at least 100 cells were counted for each determination. Error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (B) The association of Msn2 (left panel) and Msn4 (right panel) with the PNC1 promoter region is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. Shown is the degree of association of Msn2 and Msn4 with the PNC1 promoter region measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (C) The protein level of Pnc1 increases in the absence of Gas1 β-1,3-glucanosyltransferase activity. Total protein was extracted from the corresponding cells, and immunoblotting was performed using an HRP-conjugated anti-mouse IgG antibody for the detection of TAP-tagged protein. Actin was used as a loading control. The relative ratio of Pnc1 to actin, normalized against that of WT cells, is shown below each lane. Data are representative of at least three independent experiments. (D) The association of Sir2 with rDNA is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. The degree of Sir2 binding to four representative regions in the rDNA locus (25S, NTS1, NTS2/18S and 18S regions) was measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments, and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test).
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Figure 2: The lack of Gas1 β-1,3-glucanosyltransferase activity promotes nuclear accumulation of Msn2/4 and enhances the association of Msn2/4 with the PNC1 promoter region. (A) The absence of Gas1 β-1,3-glucanosyltransferase activity induces nuclear accumulation of Msn2 (upper panels) and Msn4 (lower panels). Subcellular localization of Msn2- and Msn4-GFP was analyzed by fluorescence microscopy in wild-type (WT) and gas1Δ cells containing an empty vector and gas1Δ cells expressing WT GAS1 or gas1E161Q, E262Q on the pRS415 vector (left panels). The percentage of nuclear Msn2- and Msn4-GFP is shown in the right panels. Values represent the average of three independent experiments and at least 100 cells were counted for each determination. Error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (B) The association of Msn2 (left panel) and Msn4 (right panel) with the PNC1 promoter region is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. Shown is the degree of association of Msn2 and Msn4 with the PNC1 promoter region measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (C) The protein level of Pnc1 increases in the absence of Gas1 β-1,3-glucanosyltransferase activity. Total protein was extracted from the corresponding cells, and immunoblotting was performed using an HRP-conjugated anti-mouse IgG antibody for the detection of TAP-tagged protein. Actin was used as a loading control. The relative ratio of Pnc1 to actin, normalized against that of WT cells, is shown below each lane. Data are representative of at least three independent experiments. (D) The association of Sir2 with rDNA is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. The degree of Sir2 binding to four representative regions in the rDNA locus (25S, NTS1, NTS2/18S and 18S regions) was measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments, and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test).

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.


The β-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.

Ha CW, Kim K, Chang YJ, Kim B, Huh WK - Nucleic Acids Res. (2014)

The lack of Gas1 β-1,3-glucanosyltransferase activity promotes nuclear accumulation of Msn2/4 and enhances the association of Msn2/4 with the PNC1 promoter region. (A) The absence of Gas1 β-1,3-glucanosyltransferase activity induces nuclear accumulation of Msn2 (upper panels) and Msn4 (lower panels). Subcellular localization of Msn2- and Msn4-GFP was analyzed by fluorescence microscopy in wild-type (WT) and gas1Δ cells containing an empty vector and gas1Δ cells expressing WT GAS1 or gas1E161Q, E262Q on the pRS415 vector (left panels). The percentage of nuclear Msn2- and Msn4-GFP is shown in the right panels. Values represent the average of three independent experiments and at least 100 cells were counted for each determination. Error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (B) The association of Msn2 (left panel) and Msn4 (right panel) with the PNC1 promoter region is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. Shown is the degree of association of Msn2 and Msn4 with the PNC1 promoter region measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (C) The protein level of Pnc1 increases in the absence of Gas1 β-1,3-glucanosyltransferase activity. Total protein was extracted from the corresponding cells, and immunoblotting was performed using an HRP-conjugated anti-mouse IgG antibody for the detection of TAP-tagged protein. Actin was used as a loading control. The relative ratio of Pnc1 to actin, normalized against that of WT cells, is shown below each lane. Data are representative of at least three independent experiments. (D) The association of Sir2 with rDNA is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. The degree of Sir2 binding to four representative regions in the rDNA locus (25S, NTS1, NTS2/18S and 18S regions) was measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments, and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test).
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Figure 2: The lack of Gas1 β-1,3-glucanosyltransferase activity promotes nuclear accumulation of Msn2/4 and enhances the association of Msn2/4 with the PNC1 promoter region. (A) The absence of Gas1 β-1,3-glucanosyltransferase activity induces nuclear accumulation of Msn2 (upper panels) and Msn4 (lower panels). Subcellular localization of Msn2- and Msn4-GFP was analyzed by fluorescence microscopy in wild-type (WT) and gas1Δ cells containing an empty vector and gas1Δ cells expressing WT GAS1 or gas1E161Q, E262Q on the pRS415 vector (left panels). The percentage of nuclear Msn2- and Msn4-GFP is shown in the right panels. Values represent the average of three independent experiments and at least 100 cells were counted for each determination. Error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (B) The association of Msn2 (left panel) and Msn4 (right panel) with the PNC1 promoter region is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. Shown is the degree of association of Msn2 and Msn4 with the PNC1 promoter region measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test). (C) The protein level of Pnc1 increases in the absence of Gas1 β-1,3-glucanosyltransferase activity. Total protein was extracted from the corresponding cells, and immunoblotting was performed using an HRP-conjugated anti-mouse IgG antibody for the detection of TAP-tagged protein. Actin was used as a loading control. The relative ratio of Pnc1 to actin, normalized against that of WT cells, is shown below each lane. Data are representative of at least three independent experiments. (D) The association of Sir2 with rDNA is enhanced in the absence of Gas1 β-1,3-glucanosyltransferase activity. The degree of Sir2 binding to four representative regions in the rDNA locus (25S, NTS1, NTS2/18S and 18S regions) was measured using a ChIP assay in the corresponding cells. Values represent the average of three independent experiments, and error bars indicate the standard deviation. Asterisks indicate P < 0.05, compared with WT cells with an empty vector (two-tailed Student's t-test).
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus