Hsp82 Is Involved in Sir2p Abundance Figure 3. Single mutants as well as overexpression of HSP82 do not alter mating type silencing. (A) Mating type silencing phenotype of different strains (as indicated) when mated with the tester strain YDS32 (MATa). Dsir2 acts as a negative control. (Bi) RT-PCR analysis of HMLa2 transcripts in different strain backgrounds (as indicated) shows that HMLa2 transcript levels are similar in HSP82 overexpressing cells and wild type cells, ACT1 serves as loading control. (Bii) Quantification of HMLa2 transcript from three independent experiments are normalized with ACT1 and plotted. Each bar represents mean density 6 SD. doi:10.1371/journal.pone.0023406.g003 subtelomeric gene YFR057w. For that we isolated the total RNA from the wild type cells grown under normal (30uC) as well as stressed condition (exposing at 39uC for 40 min). The semi- quantitative RTPCR showed the abundance of YFR057w transcript as a result of heat shock, indicating loss of telomere silencing (Figure 5B). These data strengthen our model that Hsp82 homeostasis is indeed responsible for the reversible nature of transcriptional silencing at telomere. Though the mechanism of this is not clear at present, but it is evident that Hsp82 is directly or indirectly controlling the cellular abundance of Sir2p. Discussion Several important findings came out through this study. Firstly, our study shows for the first time that Sir2p function is directly or indirectly dependent on Hsp82, and its steady state level in the cell is also controlled by Hsp82. We observe that in iGD170hsp82 temperature sensitive mutant, where Hsp82 is nonfunctional at restrictive temperature, there is significant reduction of Sir2p which is associated with loss of mating type silencing as well transcriptional silencing at telomere. As Hsp82 controls phero- mone signaling pathway in yeast, we wanted to decipher whether the loss of mating type silencing at restrictive temperature is also due to derepression at the HMLa locus in MATa strain. Our experimental result shows significant amount of MATa2 transcript at 37uC, which is well correlated with the sterile nature of iG170Dhsp82 (MATa). The Western blot shows that considerable amount of Sir2p is still present at the restrictive temperature (albeit at 50% reduced level); however functional studies at hidden mating locus demonstrated that these Sir2 proteins are inadequate for its functional activity. It has been reported earlier that silencing in yeast is dependent on temperature [34]. Increase in the temperature is associated with stronger TPE and mating type silencing in yeast. As an explanation of this effect it has been shown that at higher temperature Sir2-Net1 complex is destabi- lized whereas Sir2-Sir4 complex is having no effect on temper- ature [36]. Thus at higher temperature more Sir2 is available to bind with Sir4 leading to an increase in telomere and mating type silencing at the expense of ribosomal silencing in yeast. In our system the derepression of transcriptional silencing both at telomere and MAT locus at higher temperature is directly linked with the lack of Hsp82/Hsc82 activity. Secondly, over-expression of Hsp82 in the cell causes reduction of Sir2p level in a dose dependent manner. This has important PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23406