Nuclear localization is required for function of the essential clock protein FRQ.
The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. Here we report that FRQ is a nuclear protein and nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter into the nucleus and abolishes overt circadian rhythmicity, while reinsertion of the NLS at a novel site near the N-terminus of FRQ restores its function. Each form of FRQ enters the nucleus soon after its synthesis in the early subjective day; there is no evidence for regulated sequestration in the cytoplasm prior to nuclear entry. The kinetics of the nuclear entry are consistent with previous data showing rapid depression of frq transcript levels following the synthesis of FRQ, and suggest that early in each circadian cycle, when FRQ is synthesized, it enters the nucleus and depresses the level of its own transcript.
Luo
C
C
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Loros
J J
JJ
Dunlap
J C
JC
D001683:Biological Clocks / Q000502:physiology*; D001692:Biological Transport; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D002940:Circadian Rhythm / Q000502:physiology; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009491:Neurospora / Q000737:chemistry / Q000502:physiology*; D019913:Nuclear Localization Signals / Q000502:physiology*; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis
C087405:FRQ protein, Neurospora crassa; D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger