Macromolecular Assemblies of the Mammalian Circadian Clock

Mol Cell. 2017 Sep 7;67(5):770-782.e6. doi: 10.1016/j.molcel.2017.07.017.

Abstract

The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ∼750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ were distributed into several complexes of ∼0.9-1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ∼20-nm and ∼25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.

MeSH terms

  • ARNTL Transcription Factors / genetics
  • ARNTL Transcription Factors / metabolism
  • Animals
  • Casein Kinase Idelta / metabolism
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cell Nucleus / ultrastructure
  • Circadian Clocks*
  • Circadian Rhythm Signaling Peptides and Proteins / deficiency
  • Circadian Rhythm Signaling Peptides and Proteins / genetics
  • Circadian Rhythm Signaling Peptides and Proteins / metabolism*
  • Circadian Rhythm*
  • Cryptochromes / genetics
  • Cryptochromes / metabolism
  • Female
  • Genotype
  • Male
  • Mice, 129 Strain
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microscopy, Electron
  • Multiprotein Complexes
  • Particle Size
  • Period Circadian Proteins / genetics
  • Period Circadian Proteins / metabolism
  • Phenotype
  • RNA Interference
  • Signal Transduction
  • Single Molecule Imaging
  • Time Factors
  • Transfection

Substances

  • ARNTL Transcription Factors
  • Arntl protein, mouse
  • Circadian Rhythm Signaling Peptides and Proteins
  • Cry1 protein, mouse
  • Cry2 protein, mouse
  • Cryptochromes
  • Multiprotein Complexes
  • Per1 protein, mouse
  • Per2 protein, mouse
  • Per3 protein, mouse
  • Period Circadian Proteins
  • Casein Kinase Idelta