The circadian clock is a highly conserved timer which allows organisms to anticipate future conditions driven by the 24-hour day on planet Earth. Circadian clocks from fungi to mammals are based on a transcription-translation feedback loop (TTFL) molecular architecture. Progressive phosphorylation of core clock proteins can alter both their activity and stability and is required for all known circadian TTFLs. The mechanism for kinase control of circadian period has been extensively studied; however, the mechanism(s) whereby phosphatases alter period remain less studied. Based on the observation that strains of Neurospora crassa lacking phosphatase pph-4 display a short circadian period, we investigated regulation of pph-4 and its role in the clockworks. In addition to period shortening, loss of pph-4 results in a significant loss of both temperature and nutritional compensation, consistent with substrates within the core clock. We identify the clock-relevant PP4 phosphatase holoenzyme as a heterotrimer and confirm that catalytic activity of the phosphatase is required for normal period. Two activators of PP4 identified by homology were shown also to regulate circadian period. Biochemical and cell biological analyses suggest that PP4 acts in the nucleus, and a mass spectrometry-based screen identified NCU07414, a member of the HSP40 chaperone system, as a novel PP4 binding partner whose loss results in a dramatically shortened period of the clock when ablated. A model consistent with the data suggests that PP4 may act in opposition to kinases to influence the rate of accumulation of the clock-relevant phosphorylations that determine circadian period.
Mehalow, A. K., Wang, Z., Gerber, S. A., Loros, J. J., Dunlap, J. C.
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