Hsp82 Is Involved in Sir2p Abundance episomaly in the cell [29]. Previous work by Nathan and Lindquist showed that the mutant G170Dhsp82 when present as an episomal copy within the cell, its growth is significantly diminished above 34uC. However if it is integrated within the chromosome it can tolerate (2–3)uC higher temperature [24,30]. We allowed to mate iG170Dhsp82 (MATa) with tester strain YDS32 (MATa) at 25uC and 37uC and compared its mating behavior with wild type (HSP82) and Dsir2 strains. We found that loss of function of HSP82 at 37uC impaired the mating ability of the strain and it behaved like Dsir2 (Figure 1Ai). Conversely, at 25uC, the strain showed mating ability comparable to wild type cells. In order to rule out the possibility that the lack of silencing at restrictive temperature is not due to any lack of growth, we tested the viability of both temperature sensitive mutant and that of the tester strains at 37uC, and found that all the strains were viable at 37uC (Figure S1). It was earlier reported that Hsp90 controls the pheromone signaling in yeast [25]. Therefore, it is important to understand whether the loss of mating type silencing of G170Dhsp82 at restrictive temperature is due to the defect in pheromone signaling pathway alone, or also due to de-repression of HMLa locus. To address this question the semi quantitative RT-PCR was done to measure the a2 transcript level from G170Dhsp82 (MATa) after growing them at 25uC as well as 37uC (Figure 1Aii). The quantification RT-PCR data showed negligible amounts of a2 transcript at 25uC, whereas at 37uC significant amount of a2 transcript was visible. This result was well corroborated with the sterility of iG170Dhsp82 at 37uC, since in order for a MATa strain to behave as a mating compatible haploid strain, a2 factor should remain repressed [31,32]. As a control, we had taken Dsir2 strain where the RT-PCR result showed the presence of considerable amount of a2 transcript, which made it sterile. Thus from this experiment it can be concluded that in hsp82 deficient condition, there is a loss of silencing at HML, which is not only due to defect in pheromone signaling pathway [25] but also due to derepression of MATa2 transcript in iGD170hsp82 (MATa). We also explored whether Hsp82 has any role in silencing of subtelomeric genes. For that purpose we monitored the mRNA level of YFR057w gene, which is located adjacent to chromosome Figure 1. Sir2p function is dependent on Hsp82. (Ai) The temperature sensitive strain iGD170hsp82 causes disruption of mating type silencing at restrictive temperature (37uC), where Hsp82 is non functional. (Aii) iG170Dhsp82 at 37uC shows considerable amount of HMLa2 transcript comparable to that present in Dsir2 cells; whereas at 25uC negligible amount of HMLa2 is seen. (Bi) Semi quantitative RT-PCR shows increase in YFR057w transcript in iG170Dhsp82 at 37uC compared to 25uC, indicating that the strain has defect in telomere position effect, whereas wild type strain (HSP82) shows silencing at higher temperature (37uC). (Bii) Graphical representation of four independent experiments of (Bi) is done after normalization with ACT1 control. (C) Western blot shows the relative abundance of Hsp82 and Sir2p at two temperatures. Actin is the loading control. (D) The quantification of Western blot (from 4 independent experiments) shows more than 50% reduction in steady state level of Sir2p at higher temperature. The data are normalized with respect to the loading control Actin. Each bar represents mean density 6 SD. doi:10.1371/journal.pone.0023406.g001 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23406