Analysis of mammalian circadian clock protein complexes over a circadian cycle

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK18. In mice, peak expression of the repressors in the early morning reduces CLOCK-and BMAL1-mediated transcription/translation of the repressors them-selves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK18. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography- tandem mass spectrometry to identify components of circa-dian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK18 complexes at the different circadian time points, provide masses of 255 and 707 kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.