Figure 4.

Rap1 binds Sir4 in vitro. In vitro binding of Rap1 and GST–Sir fusion proteins. GST-fusion protein experiments were performed as described in Materials and Methods. Full-length Rap1 was labeled in vitro with ³⁵S-Methionine and incubated with GST (lane 1), GST–Sir2 (amino acids 275–562, lane 2), GST–Sir3 (amino acids 503–970, lane 3), GST–Sir4N (amino acids 142–591, lane 4), and GST–Sir4C (amino acids 1144–1358, lane 5). Interacting proteins were analyzed by SDS-PAGE and fluorography.