Western blotting provides a sensitive assay, whereas staining with Coomassie brilliant blue allows the simple detection of strong and efficient interactions. and H4 are required for silencing and interact with the SIR3 and SIR4 proteins (20C22). Consistent with this observation, SIR3 and SIR4 copurify with nucleosomal histones (23), although it is possible that some of these interactions are mediated by DNA. Based TAS-116 on these observations, Grunstein and colleagues have proposed that after associating with silencer-bound proteins SIR3 and SIR4 interact with the N-termini of histones H3 and H4 and that these interactions allow for the spreading of the SIR proteins along chromatin (23). Whether a complex containing all three SIR proteins exists and is recruited to the silencer, or whether each protein is recruited independently and assembled in stepwise fashion is not known. Furthermore, two-hybrid studies, either as general screens or as direct assays for proteinCprotein interactions, have not uncovered an interaction between SIR2 and other silencing proteins. SIR2-like sequences have been identified in both eukaryotes, including and human, and in (24). This evolutionary conservation suggests that interactions involving SIR2 are likely to play a central role in silencing. We previously have shown that the SIR2 and SIR3 proteins are specifically retained by an affinity column composed of the C-terminal half of the SIR4 protein, indicating that these proteins can physically associate (25). Here we report on studies involving the interactions of the SIR2, SIR3, and SIR4 proteins in yeast extracts and also examine these interactions using SIR proteins purified from promoter followed by the GST coding region in the polylinker of pRS316 (26). An as previously described (pDM118) (25). An open reading frame and 200 bp downstream of the stop codon was ligated into the fragment (encoding amino acids 1115C1358 of SIR4 and the 3 untranslated region) into the gene, pDM196 was constructed as described below. pDM196 contains 600 bp of DNA from the upstream and promoter region of the gene followed by 2.5 kb of DNA encoding TAS-116 the N-terminal half of the SIR4 protein; the N terminus of SIR4 in pDM196 is modified to insert six histidines followed by three hemagglutinin (HA) epitopes. The upstream region was PCR-amplified using an oligonucleotide to insert six histidine codons after the start codon followed by an region in plasmid pDM118a was then replaced with promoter region-ATG-6His fragment by digesting pDM118a with were previously described (25). pDM111 (GST-SIR2) was produced by ligation of TAS-116 an promoter). pDM120a (in JRY2334) and FM135 (for 2 min, resuspended in 5 ml of lysis buffer, transferred to a small column, and washed with 20 column volumes of wash buffer [50 mM Hepes, pH 7.6/10% glycerol/10 mM EDTA/0.5 M NaCl/5 mM Mg(OAc)2/1% Triton X-100/1 mM DTT/5 mM benzamidine hydrochloride, 1 mM phenylmethylsulfonyl fluoride/1 g/ml each leupeptin, bestatin and pepstatin] and with 4 column volumes of the same buffer without Triton X-100 or the protease inhibitors. Bound protein then was eluted with 10 mM glutathione in 50 mM Hepes, pH 7.8/10% glycerol/200 mM NaCl/1 mM DTT. Peak protein fractions (0.7 ml each) PIK3C3 were identified using the Bradford protein assay and pooled. Approximately equal amounts of each protein were loaded on SDS 8.5% polyacrylamide gels, and after electrophoresis and transfer to a poly(vinylidene difluoride) membrane (Millipore), probed sequentially or in parallel with appropriate antibodies. Proteins on Western blots were detected by using the Amersham Enhanced Chemiluminescence (ECL).