Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops 1, 2. CRYPTOCHROME (CRY) proteins are critical components of these clocks 3, 4 and repress the activity of the transcription factor heterodimer CLOCK/BMAL1 5, 6, 7. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, CRYs have extended carboxyl-terminal tails and cannot repair DNA damage (reviewed in [8]). Unlike mammals, Xenopus laevis contains both CRYs (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study CRY repressive function. We can extend findings to CRYs in general because xCRYs share high sequence homology with mammalian CRYs [9]. We show here that deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. Our results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins.
