FIG 6.

Mutations of CK1a phosphorylation sites on FRQ cause temperature-sensitive FRQ-CK1a interaction and partial loss of temperature compensation. (A, left) Representative photos of race tubes used to evaluate conidiation rhythms of wild-type (frq complementation strain) and M10 and M9 mutant strains at the indicated temperatures. (Right) Plot of period versus temperature for wild-type and mutant strains. Error bars indicate standard errors of means (n = 5). (B, left) Western blot analyses of FRQ immunoprecipitated with anti-CK1a antibody from extracts of the wild-type, M9, and M10 mutant strains grown in constant light at 21 and 29°C. (Right) Plot of relative amount of FRQ in wild-type, M9, and M10 mutant strains. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized to CK1a level. Error bars are standard deviations (n = 3). ns, not significant; *, P < 0.05; Student’s t test. (C, left) Western blot analyses of FRQ in the wild-type, M9, and M10 mutant strains grown in constant light at 21 or 29°C for the indicated number of hours after CHX addition. (Right) Plot of relative FRQ levels at the indicated hours after CHX treatment. Error bars are standard deviations (n = 3). *, P < 0.05; **, P < 0.01; Student’s t test. (D) FRQ-CK1a interaction is important for the maintenance of circadian period of the wild-type strain within the physiological temperature range (20 to 30°C). When temperature is above 34°C, the FRQ-CK1a interaction is enhanced, resulting in period shortening and impaired temperature compensation.