Silent information regulator protein complexes in Saccharomyces cerevisiae : A SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3

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Abstract
The SIR2, SIR3, and SIR4 silent information regulator proteins are involved in the assembly of silent chromatin domains in the budding yeast Saccharomyces cerevisiae. Using a series of biochemical experiments, we have studied protein-protein interactions involving these proteins. We found that yeast extracts contained a SIR2/SIR4 complex that was associated with little or no SIR3. However, truncations of the N-terminal two-thirds of the SIR4 protein allowed it to efficiently associate with SIR3, suggesting that the N-terminal domain of SIR4 inhibited its interaction with SIR3. We propose that the SIR3 and SIR4 proteins interact only during the assembly of the SIR protein complex at the silencer and that an early step in assembly unmasks the SIR4 protein to allow its association with SIR3. To test whether the interactions observed in yeast extracts were direct, we tested these SIR-SIR interactions using bacterially expressed SIR proteins. We observed direct interactions between SIR4 and SIR2, SIR4 and SIR3, SIR2 and SIR3, SIR2 and SIR2, and SIR4 and SIR4, indicating that the associations observed in yeast extracts were direct.