FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation

Abstract

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.

Fig. 1. (A) Schematic depiction of the domain structure of FWD1. (B and C) Amino acid sequence alignment of the F-box domains (B) and the WD-40 repeat regions (C) from the Neurospora FWD1, C.elegans Lin23, Drosophila Slimb and human β-TRCP1. The alignments were based on the structural-based alignments (Orlicky et al., 2003). The seven WD40 repeats are labeled above.

Fig. 2. In the fwd1^RIP strains, FRQ is hyperphosphorylated, and the FRQ protein levels are higher due to increased protein stability. (A) Western blot analysis showing that, in the fwd1^RIP strains, FRQ was mostly hyperphosphorylated and its levels were higher than those of the wild-type strains. Cultures harvested in LL or DD29 were used. The arrows indicate the hyperphosphorylated FRQ species observed only in the fwd1^RIP strains. (B and C) Western blot analyses showing that FRQ was more stable in the fwd1^RIP strain after LD transition or cycloheximide (CHX) treatment (10 µg/ml). Cultures were first grown in LL for 1 day prior to the addition of CHX or were transferred into constant darkness and harvested at the indicated time. Densitometric analysis of the western blot results of (B) is shown in (C).

Fig. 3. Loss of circadian rhythms of gene expression in the fwd1^RIP strain. (A) Cultures were harvested at the indicated time in constant darkness. The representative results of the western blot analysis of FRQ and northern blot analyses of frq and ccg-1 mRNA in both the wild-type and the fwd1^RIP strains are shown. Densitometric analyses of the western blot results in (A) are shown in (B) and (C).

Fig. 4. Loss of circadian conidiation rhythms in the fwd1^RIP strains. (A and B) Race tube analysis results in light/dark cycles (A) or in constant dark after a LD transition (B). Severe independent fwd1^RIP strains were examined, and the race tubes shown are representative samples from more than six replicate tubes for each strain.

Fig. 5. FRQ and FWD1 interact physically in vivo. (A) Western blot analyses of the expression of FRQ and the Myc-FWD1 or Myc-FWD1ΔF proteins in fwd1^RIP strains. QA was added into all cultures grown in LL. Note that the FRQ expression profile in the fwd1^RIP,qa-Myc-FWD1 strain was similar to that of the wild-type strain (WT), while in the fwd1^RIP,qa-Myc-FWD1ΔF strain, the FRQ profile was similar to that of the fwd1^RIP mutants. The arrow indicates the hyperphosphorylated FRQ species observed in the fwd1^RIP,qa-Myc-FWD1ΔF strain. (B) Growth phenotype of different strains in slants showing the rescue of the fwd1^RIP phenotypes by the expression of the Myc-FWD1 protein, but not Myc-FWD1ΔF. (C) Circadian conidiation rhythm in the fwd1^RIP,qa-Myc-FWD1 strain in the presence of QA (10^–3 M). (D) Immunoprecipitation assay showing that FRQ and Myc-FWD1ΔF form a complex in vivo. The wild-type strain (WT) was used as the negative control. Cultures were grown in LL in the presence of QA (10^–2 M). The total lysates or the immunoprecipitates were analyzed by immunoblotting using FRQ or c-Myc antibodies. The result shown is a representative example of multiple independent experiments.

Fig. 6. A model for FWD1-mediated FRQ degradation by the ubiquitin–proteasome pathway. FRQ, WC-1 and WC-2 form the circadian negative feedback loop. FRQ is progressively phosphorylated by CKI, CKII and possibly other kinases. After FRQ is phosphorylated extensively, it interacts with FWD1 and is rapidly ubiquitylated and degraded.