Fig. 1.

Not4p is required for cyclin C destruction. (A) Two-hybrid interactions between lexA–cyclin-C and two isolates of Ccr4–AD (activation domain) fusion proteins in EGy48 cells. Interactions are indicated by the ability to activate the lexAop-LEU2 growth on medium lacking leucine (–LEU). The positive control is lexA fused to the Gal4p activation domain. (B) An extract prepared from a mid-log-phase wild-type culture (RSY10) expressing myc–cyclin-C and FLAG–Not4p^I37A was incubated with (+) or without (−) anti-FLAG antibody. The presence of myc–cyclin-C in the immunoprecipitates was determined by western blot analysis probing for myc. (C) Wild-type (RSY10) and not4Δ mutant (RSY1579) strains expressing myc–cyclin-C (pKC337) were grown to mid-log phase (0 hour) then treated with H₂O₂ for the indicated times. Cyclin C levels were determined by western blotting of immunoprecipitates. Tub1p levels were used as a loading control. (D) The experiment described in C was repeated with the not4Δ strain RSY1579 expressing Not4p^ringΔ on plasmid pN287 or the ubc4Δ ubc5Δ double mutant (MHY508).