ARV1 deficiency induces lipid bilayer stress and enhances rDNA stability by activating the unfolded protein response in Saccharomyces cerevisiae

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the replicative lifespan of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner.


Table of Contents
Table S1.Yeast strains used in this study.Table S2.Oligonucleotide primers used in this study.

Measurement of Cki1 phosphorylation
Total protein extraction and western blot analysis was performed as described previously (1).Myc-tagged Cki1 proteins are detected using a HRP-conjugated mouse anti-Myc antibody (sc-40 HRP, Santa Cruz Biotechnology).Images were captured Using a luminescent image analyzer AE-9150 Ez-Capture II (ATTO) and CS analyzer version 3.0 software (ATTO).Densitometry determinations was performed using ImageJ software.

Figure S1 .
Figure S1.Loss of Arv1 enhances Sir2-mediated rDNA silencing and rDNA stability in a Slt2independent manner.

Figure S4 .
Figure S4.Overexpression of Arv1 does not induce UPR and does not increase rDNA stability Figure S5.Loss of Arv1 does not inhibit PKA or TORC1.

Figure S6 .
Figure S6.Loss of Alg12 does not affect rDNA silencing and rDNA stability.

Figure S1 .
Figure S1.Loss of Arv1 enhances Sir2-mediated rDNA silencing and rDNA stability in a Slt2independent manner.A, rDNA silencing assay performed with indicated cells.WT + ARV1 and arv1 + ARV1 indicate wild-type (WT) and arv1 cells expressing ARV1-TAP under its native promoter.Silencing at the rDNA region was assessed by monitoring the growth of 10-fold serial dilutions of cells on SC media lacking uracil.SC medium was used as a plating control.B, Relative mURA3 transcript levels in the indicated cells.Total RNA was extracted and analyzed by quantitative real-time reverse transcription-PCR.The relative transcript levels of the mURA3 gene were calculated as the ratio of the normalized transcript levels of the mURA3 gene inside the rDNA array (NTS1::mURA3) to those outside the rDNA array (leu2::mURA3).C, rDNA recombination assay performed with the indicated cells.rDNA recombination is represented by the frequency of loss of the ADE2 marker gene integrated at the rDNA locus in the corresponding cells.For B and C, values represent the average of three independent experiments, and error bars indicate the standard deviation.Asterisks indicate significant differences (paired two-tailed Student's t-test): *P < 0.05; **P < 0.01; ns, not significant.

Figure S2 .
Figure S2.Various ER stresses enhance rDNA silencing.A, rDNA silencing assay performed with wildtype (WT) and hog1 cells the indicated cells under 0.05 g/ml Tm treatment or inositol depletion.B, Relative mURA3 transcript levels in the indicated cells.C, rDNA silencing assay performed with WT, pmt1, bst1, hog1, pmt1 hog1, and bst1 hog1 cells.D, Relative mURA3 transcript levels in the indicated cells.For A and C, silencing at the rDNA region was assessed by monitoring the growth of 10fold serial dilutions of cells on SC media lacking uracil.SC medium was used as a plating control.For B and D, Total RNA was extracted and analyzed by quantitative real-time reverse transcription-PCR.The relative transcript levels of the mURA3 gene were calculated as the ratio of the normalized transcript levels of the mURA3 gene inside the rDNA array (NTS1::mURA3) to those outside the rDNA array (leu2::mURA3).Values represent the average of three independent experiments, and error bars indicate the standard deviation.Asterisks indicate significant differences (paired two-tailed Student's t-test): *P < 0.05; **P < 0.01; ns, not significant.

Figure S3 .
Figure S3.Various ER stresses enhance rDNA stability.The rDNA recombination assay was performed with the indicated cells treated with or without 0.05 μg/ml tunicamycin (Tm) or inositol for 3 h.rDNA recombination is represented by the frequency of loss of the ADE2 marker gene integrated at the rDNA locus in the corresponding cells.Values represent the average of three independent experiments, and error bars indicate the standard deviation.Asterisks indicate significant differences (paired two-tailed Student's t-test): *P < 0.05; **P < 0.01; ns, not significant.

Figure S4 .
Figure S4.Overexpression of Arv1 does not induce UPR and does not increase rDNA stability.A, Analysis of HAC1 mRNA splicing in wild-type cells harboring p413ADH or p413ADH-ARV1-TAP vector.B, Relative KAR2 transcript levels in the indicated cells Total RNA was extracted and analyzed by quantitative real-time reverse transcription-PCR.The relative KAR2 transcript level was normalized against TAF10 and calculated using the 2 -Ct method.C, rDNA recombination assay performed with the indicated cells.rDNA recombination is represented by the frequency of loss of the ADE2 marker gene integrated at the rDNA locus in the corresponding cells.For C and D, values represent the average of three independent experiments, and error bars indicate the standard deviation.

Figure S5 .
Figure S5.Loss of Arv1 does not inhibit PKA or TORC1.A, Activity of PKA was examined by monitoring phosphorylation of Cki1.B, Activity of TORC1 was examined by monitoring phosphorylation of Sch9.In wild-type (WT) and arv1 cells, total protein was extracted and immunoblotting was performed using a mouse anti-Myc antibody for detection of Myc-tagged Cki1 or a mouse anti-HA antibody for detection of HA-tagged Sch9.Act1 was used as a loading control.The relative ratio of phosphorylated protein to total protein, normalized against that of WT cells, is shown below each lane.

Figure S6 .
Figure S6.Loss of Alg12 does not affect rDNA silencing.A, rDNA silencing assay performed with wildtype (WT) and alg12 cells.Silencing at the rDNA region was assessed by monitoring the growth of 10fold serial dilutions of cells on SC media lacking uracil.SC medium was used as a plating control.B, Relative mURA3 transcript levels in WT and alg12 cells.Total RNA was extracted and analyzed by quantitative real-time reverse transcription-PCR.The relative transcript levels of the mURA3 gene were calculated as the ratio of the normalized transcript levels of the mURA3 gene inside the rDNA array (NTS1::mURA3) to those outside the rDNA array (leu2::mURA3).C, rDNA recombination assay performed with WT and alg12 cells.rDNA recombination is represented by the frequency of loss of the ADE2 marker gene integrated at the rDNA locus in the corresponding cells.Values represent the average of three independent experiments, and error bars indicate the standard deviation.Statistical analysis was performed using a two-tailed Student's t-test.ns, not significant.