Figure 2: Characterization of the FRQ–FRH interaction.

(a) Western blot analysis of the indicated FLAG-tagged FRH versions expressed in a frh⁺ background. WT: _FLAGFRH; K208A and AAA: Walker A box mutants; AEIH and DAIH: DEAD box mutants. FRH antibody and FLAG antibody was used for decoration in the left and right panel, respectively. Asterisks indicate FLAG-tagged FRH. (b) FRQ does not stably accumulate in complex with ATPase and DEAD box mutants of FRH. Co-IP of FRQ with _FLAGFRH versions pulled down with M2 FLAG Sepharose is shown. Protein extracts of light-grown cultures expressing WT and mutant versions of FLAG-tagged FRH were used. Western blots of the total protein extract (T), the immunoprecipitate (IP) and the supernatant (S) were decorated with FRQ antibody (upper panels) and FLAG antibody (lower panels). (c) Turnover kinetics of FRQ in complex with FLAG–FRH versions. Light-grown cultures were incubated with cycloheximide (CHX). Samples were harvested at the indicated time points and the indicated _FLAGFRH versions were immunoprecipitated (FLAG-IP). Immunoprecipitates of mutant FRH versions are five-fold overrepresented. Co-IP of FRQ was detected by western blotting (left panel) and quantified by densitometry (right panel). FRQ levels at time 0 were set equal to 1 (a.u., arbitrary units). Error bars indicate s.d. (n=3).