THE CARBOXY TERMINI OF SIR4 AND RAP1 AFFECT SIR3 LOCALIZATION - EVIDENCE FOR A MULTICOMPONENT COMPLEX REQUIRED FOR YEAST TELOMERIC SILENCING

The Silent Information Regulatory proteins, Sir3 and Sir4, and the tel omeric repeat-binding protein RAP1 are required for the chromatin-medi ated gene repression observed at yeast telomeric regions. All three pr oteins are localized by immunofluorescence staining to foci near the n uclear periphery suggesting a relationship between subnuclear localiza tion and silencing. We present several lines of immunological and bioc hemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast ce lls. First, immunolocalization of Sir3 to foci at the yeast nuclear pe riphery is lost in rap1 mutants carrying deletions for either the term inal 28 or 165 amino acids of RAP1. Second, the perinuclear localizati on of both Sir3 and RAP1 is disrupted by overproduction of the COOH te rminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes u sing either anti-RAP1 or anti-Sir4 antibodies. We propose that the int egrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.