Hsp82 Is Involved in Sir2p Abundance Table 1. Yeast strains used in this study. Strains SLY20 YDS31 YDS32 iG170Dhsp82 YSC1021-551520 YSC1021-551935 YSC1021-552834 SLY4 SLY5 SLY6 SLY12 SLY13 SLY10 SLY31 SLY32 SLY46 Genotype MATa his12 MATa his12 MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 MATa can1-100 ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 HSP82::LEU2 HSC82::LEU2 HIS3::HSP82G170D MATa HSC82::KANr MATa HSP82::KANr MATa SBA1::KANr MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 HSC82::KANr MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 HSP82::KANr MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 SBA1::KANr MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 SIR2::KANr MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 pHCA/HSP82 MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 HSP82::KANr pHCA/HSP82 MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 SBA1::KANr pHCA/HSP82 MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 pTA/HSP82 MATa 15ade2-1, ura3-1, 112 his 3-11, trp1, leu2-3, VIIL::ADE2 pHCA doi:10.1371/journal.pone.0023406.t001 was then transferred to a polyvinylidene difluoride (PVDF) membrane and immuno blotted for Hsp82, Sir2 and Actin. The anti Hsp90 antibody (Calbiochem), the anti Sir2 (Santa Cruz Biotechnology Inc., CA) and anti Actin antibody (Abcam) were used at 1:5000 dilution. Horseradish peroxide-conjugated rabbit IgG (Santa Cruz Biotechnology Inc., CA) was used as a secondary antibody for Sir2 at 1:10000 dilution and HRP conjugated mouse IgG (Promega) was used as a secondary antibody for Hsp82 and Actin blot at the same dilution. The Western blots were developed using chemiluminescent detection system (Pierce). The bands on the gel were quantified using Gene Tools, Syngene and the relative densities, thus obtained, were plotted using GraphPad Prism 5 software. The mean value from four independent experiments was plotted with standard deviations (6SD). All blots were normalized against Actin. RNA isolation Total RNA was isolated from iG170Dhsp82 after growing them at 25uC and 37uC, as well as from SLY20, SLY12 and SLY13 strains by using acid phenol method as described [28] with some modifications. 10 ml culture of cells were grown up to mid log phase (OD600<1), centrifuged and the pellet was suspended in 400 ml TES buffer (10 mM Tris HCl, [pH = 7.5], 10 mM EDTA, 0.5% SDS). 400 ml phenol (pre equilibrated with water) was added and the mixture was incubated at 650C for 1 hour, with intermittent vortexing. The mixture was rapidly chilled on ice for 5 min, and centrifuged at 14000 rpm for 10 min at 40C. The aqueous layer was mixed with 400 ml chloroform, vortexed and centrifuged again for the same time at the same speed and temperature mentioned above. The extracted aqueous phase was precipitated by adding 1/10th volume of 3 M sodium acetate [pH 5.2] and 2.2 volume of chilled ethanol. The precipitate containing RNA was washed with 70% ethanol and the pellet was dissolved in 30 ml DEPC treated water. Equal amount of RNA measured by spectroscopic analysis (JASCO spectrophotometer EMC-709) was then subjected to DNase I (Fermentas) digestion for 15 min at room temperature. DNase I was finally inactivated by incubating with 25 mM EDTA at 650C for 10 min. About 10 mg of total RNA from each sample was then reverse transcribed with oligo dT primer (Sigma Aldrich) using reverse transcriptase (Omniscript kit, Qiagen). Resulting cDNA was first quantified, diluted appropriately to normalize and then subjected to PCR amplification (27 cycles) using gene specific primers. The PCR products were run on 1.4% agarose gel and stained with ethidium bromide. We used OSB16 (59TGA CCA AAC TAC TTA CAA CTC C39) and OSB14 (59TTA GAA ACA CTT GTG GTG AAC G39) for amplifying 307 base pair at the 39 end of ACT1 transcript. OSB19 (59 ATC ACG AGT AAG GAT CAA AG 39) and OSB20 (59 TTA TGG CTT TGT TAC GCT TG 39) were used to amplify YFR057w, which is located in the subtelomeric region on chromosome VIR. We used OSB62 (59 AAT CGG CGG ATG GGT TGG 39) and OSB63 (59 TCA TTC TTT CTT CTT TGC CAG 39) for amplifying 308 base pair at the 39end of the HMLa2 transcript. Results Sir2p function is dependent on Hsp82 Previously, yeast two hybrid screen, using Hsp82 as bait, has identified Sir2p interaction [16]. To explore whether Sir2p function is dependent on Hsp82, we examined both the mating type silencing as well as telomere position effect in a conditional mutant, where Hsp82 is functionally defective. We performed the assay using a temperature sensitive iG170Dhsp82 strain. In this strain both HSP82 and HSC82 genes are deleted and the strain behaves as wild type when grown at permissive temperature 25uC, but as mutant when grown at restrictive temperature at 37uC [24]. This particular strain harbors mutant G170Dhsp82 which is integrated into its chromosome. It tolerates little bit higher temperature than its counterpart where G170Dhsp82 is maintained PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23406