Abstract
Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue-light sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 ortholog in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N-terminal extension (termed CRY N-terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear- and nucleolar-localizing signals (NLS/NoLS), and a novel motif (termed DI-UIM) overlapping a double-sided ubiquitin-interacting motif (DUIM) and an inverted ubiquitin-interacting motif (IUIM). Potential activities of the NLS/NoLS and DI-UIM were examined to infer the molecular function of XtCRY6. GFP-NLS/NoLS fusion protein exogenously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP-XtCRY6 was observed in the cytoplasm. A glutathione S-transferase (GST) pull-down assay suggested that the DI-UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI-UIM binds two ubiquitin molecules in a relationship of a twofold rotational symmetry with the symmetry axis parallel or perpendicular to the DI-UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light-dependent protein degradation or gene expression through a CNE-mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus.