Hsp82 Is Involved in Sir2p Abundance Figure 2. Over-expression of HSP82 negatively regulates TPE but not mating type silencing. (Ai) ADE2 color phenotype of various strains i.e., HSP82 (wild type), Dhsp82, Dhsc82, Dsba1, Dsir2, over-expressing HSP82 (HSP82+pHSP82) and over-expressing HSP82 in Dsba1 background. Pink colored colony represents telomere silencing and white colored colony represents de-repression of telomere silencing. (Aii) YFR057w is silenced in all single mutants as well as in wild type strain. (Bi) Over-expression of HSP82 de-represses the expression of YFR057w. ACT1 serves as loading control. (Bii) Quantification of YFR057w transcript is done from 4 independent experiments in HSP82 overexpressed cells. The band intensity values are normalized against ACT1. Each bar represents mean density 6 SD. doi:10.1371/journal.pone.0023406.g002 dependent on Hsp82 in a dose dependent manner or not. In order to address this question, we generated another strain SLY32, which over-expresses Hsp82 from a multicopy 2 m plasmid (pTA/ hsp82). We compared the level of Hsp82 and Sir2 among SLY20 (endogenous HSP82), SLY13 (endogenous plus one copy from a CEN plasmid) and SLY32 (multiple copies of HSP82) strains by Western blot analysis. Our result showed a gradual increment in Hsp82 protein levels from SLY20 to SLY13 to SLY32, which was in accordance with the copy number. However, the level of Sir2p showed a reverse trend. The most dramatic reduction in Sir2p level was observed in SLY32 cells, while SLY13 exhibited a moderate decrement compared to the wild type cells (SLY20) (Figure 4C). The quantification of the bands, taking the average from independent three sets of experiment, showed that while the increase in Hsp82 level is 4 times in SLY32 strain compared to the wild type, the decrease in Sir2 level was at least by 75% compared to the wild type and by 50% compared to the SLY13 strain (Figure 4D). Thus, it appears that under stressed condition the increased level of Hsp82 results in concomitant reduction of the total cellular pool of Sir2p, which begs us to speculate that such adaptation in Sir2p abundance in response to Hsp82 homeostasis could be responsible for the reversible nature of transcriptional silencing at telomere. Heat shock induces derepression of telomere silencing in yeast Our findings propose that the Sir2p level and/or their function is controlled by Hsp82 homeostasis, which may be responsible for reversible nature of telomere silencing in yeast. This observation led us to think whether heat shock induced elevated level of Hsp82p is sufficient to alter the Sir2p level and consequently its function. To achieve this we exposed wild type cells (SLY20) to 39uC heat shock for 40 minutes. This is the optimum condition for induction of heat shock gene HSP82 [35]. After heat shock we harvested the cell and isolated protein from them. The Western blot analysis showed (Figure 5Ai) similar kind of reduction in Sir2p level in heat stressed condition compared to cells growing at 30uC. The experimental data were plotted (Figure 5Aii) which showed heat shock induced the expression of Hsp82p which in turn reduced about 50% of the total cellular pool of Sir2p. In order to find out the effect of heat shock on telomere silencing we aimed to monitor the transcript level of the PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23406