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Molecular and Cellular Biology, August 2004, p. 6931-6946, Vol. 24, No. 16
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.16.6931-6946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

Jason C. Tanny,1 Donald S. Kirkpatrick,1,2 Scott A. Gerber,1,2 Steven P. Gygi,1,2 and Danesh Moazed1*

Department of Cell Biology,1 Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Boston, Massachusetts 021152

Received 13 February 2004/ Returned for modification 19 March 2004/ Accepted 18 May 2004

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.

* Corresponding author. Mailing address: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1258. Fax: (617) 432-1144. E-mail: danesh{at}hms.harvard.edu.

Molecular and Cellular Biology, August 2004, p. 6931-6946, Vol. 24, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.16.6931-6946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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