A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1 epsilon. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frq Delta PEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frq Delta PEST-1 RNA and FRQ Delta PEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.