Hsp82 Is Involved in Sir2p Abundance Figure 4. Western blot analysis shows over-expression of HSP82 down regulates Sir2p in a dose dependent manner. (A) Different lanes are marked with the respective genotypes. pHSP82 implies over-expression of HSP82 from a CEN plasmid. Total proteins from each lysates are probed with anti Hsp82 antibody, anti-Sir2 antibody and with anti-Actin antibody (as loading control). (B) Graphical representation of (A), where densitometric measurements of the bands from four experiments are plotted, after normalizing with Actin band intensities. Each bar represents mean density 6 SD. (C) Multiple levels of Hsp82 over-expression is shown using high (2 m) and low copy plasmids (CEN), to demonstrate dose response between Hsp82 and Sir2p levels. (D) The graphical representation of Western blot as in figure (C) shows over expression of Hsp82 from a high copy plasmid confers more reduction of Sir2p level. Each bar represents the mean density 6 SD from 4 independent experiments. The data are normalized with respect to Actin. doi:10.1371/journal.pone.0023406.g004 consequence as occasionally the amount of Hsp82 increases within the cell when they are exposed to heat shock or subjected to other stressed condition. Our study demonstrates, in a condition where Hsp82 is overexpressed, there occurs a gradual decrement of Sir2p which impairs its ability in transcriptional silencing at telomere, although silencing at HML/HMR locus remains unperturbed. This data suggests that under stressed condition, though Sir2p level is diminished, there are enough Sir2p still present to perform other cellular activities. We speculate that although Sir2p is the key protein to control both mating type silencing and telomere silencing, the threshold cellular concentration of Sir2p for these two different functions may be different. In case of silencing at the mating type loci, the reduced cellular level of Sir2p, which is the consequence of Hsp82 over-expression, may be sufficient to maintain the silence state, however, it may be limiting for silencing the stretch of subtelomeric regions spread over 32 chromosomal ends. The question remains that why at low concentration, Sir2p comes out of the subtelomeric region, but not from the mating type loci. Considering that telomere silencing is a reversible phenomenon, meaning that the silencing complex is in a dynamic equilibrium between DNA bound and unbound states; and mating type silencing is not reversible under normal conditions, provoke us to speculate that the binding affinity of silencing complex towards the silencer sequences at the cryptic mating type loci is much stronger than that for the subtelomeric regions. Thus, binding of Sir proteins at the silent mating loci may be achieved at much less cellular concentration. It is important to understand how deficiencies of Hsp82 as well as overexpression of Hsp82 both lead to derepression of TPE. Our results suggest that under overexpression condition the loss of TPE PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23406