Figure 3: Expression and analysis of Neurospora clock proteins in yeast.

(a) FRH stabilizes FRQ and attenuates its CK1a-dependent hyperphosphorylation. FRQ, FRH and CK1a were expressed in S. cerevisiae and expression levels and phosphorylation state were analysed by western blot. (b) The degradation kinetics of FRQ is dependent on FRH and CK1a. S. cerevisiae strains expressing FRQ under control of the GAL1 promoter together with CK1a and FRH when indicated were shifted from galactose to glucose containing medium. Samples were harvested at the indicated time points and analysed by immunoblotting with FRQ antibodies (upper panel). FRQ levels were quantified by densitometry (lower panel). Error bars indicate s.d. (n=3). It should be noted that the apparent degradation kinetics includes dilution of FRQ because of growth of the yeast cells. (c) Expression levels in yeast and phosphorylation state of FRQ6B2 (ref. 14) are independent of FRH. FRQ6B2 is a mutant that does not interact with FRH. (d) CK1a-dependent phosphorylation of FRQ is dependent on the CK1a interaction domains FCD1 and FCD2 of FRQ. Western blot analysis of the phosphorylation state of indicated FRQ versions expressed in yeast. Co-expression of CK1a is indicated. (e) The phosphorylation state of FRQ is dependent on expression of catalytically active CK1a. FRQ was co-expressed in yeast with active CK1a and with the catalytically inactive versions D131N and K41R as indicated. (f) ATP binding and hydrolysis by FRH regulates CK1a-dependent hyperphosphorylation of FRQ but the ATPase activity of FRH is not required for the stabilization of FRQ. FRQ was expressed without and with FRH versions as indicated. Upper panel: CK1a was co-expressed. Lower panel: No co-expression of CK1a. FRQ expression level and phosphorylation state were analysed by western blotting. Hyper- and hypophosphorylated FRQ accumulated in the presence (upper panel) and absence (lower panel) of CK1a, respectively. Hyperphosphorylation was attenuated by WT FRH but not by the FRH mutants.