Eukaryotic circadian clocks employ autoregulatory negative feedback loops to control daily rhythms. In the filamentous fungus Neurospora, FRQ, FRH, WC-1, and WC-2 are the core components of the circadian negative feedback loop. To close the transcription-based negative feedback loop, the FRQ-FRH complex inhibits the activity of the WC complex in the nucleus by promoting the casein kinases-mediated WC phosphorylation. Despite its essential role in the nucleus, most FRQ is found in the cytoplasm. In this study, we mapped the FRQ regions that are important for its cellular localization. We show that the C-terminal part of FRQ, particularly the FRQ-FRH interaction domain, plays a major role in controlling FRQ localization. Both the mutation of the FRQ-FRH interaction domain and the down-regulation of FRH result in the nuclear enrichment of FRQ, suggesting that FRH regulates FRQ localization via a physical interaction. To study the role of FRQ phosphorylation, we examined the FRQ localization in wild-type as well as an array of FRQ kinase, FRQ phosphatase, and FRQ phosphorylation site mutants. Collectively, our results suggest that FRQ phosphorylation does not play a significant role in regulating its cellular localization. Instead, we find that phosphorylation of FRQ inhibits its transcriptional repressor activity in the circadian negative feedback loop. Such an effect is achieved by inhibiting the ability of FRQ to interact with WCC and casein kinase 1a. Our results indicate that the rhythmic FRQ phosphorylation profile observed is an important part of the negative feedback mechanism that drives robust circadian gene expression.
