Fig. 1.

Detection of F-box protein-substrate interactions by the refined two-hybrid system. (A) The refined two-hybrid system allows the detection of Cdc4-dF-Sic1 interaction. By using the filter assay, we examined the expression of reporter LacZ in wild-type (WT) and cdc4–1 mutants harboring the indicated combinations of bait and prey constructs. (B) The refined two-hybrid system identified interactions of Cdc4-dF with Gcn4, Swi5, Rcn1, and Spo74. (C) Stability of AD- and HA-tagged proteins expressed from the GAL1 promoter after the addition of cycloheximide was compared in the indicated cells. Aliquots were taken at 10 min intervals and immunoblotted with anti-HA (12CA5) and anti-PSTAIRE antibodies. Cdc28 is shown as a loading control. (*, Nonspecific signals detected in this experiment.)