The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA

Nucleocytoplasmic partitioning of core clock components is essential for the proper operation of the circadian system. Previous work has shown that the F-box protein ZEITLUPE (ZTL) and clock element GIGANTEA (GI) heterodimerize in the cytosol, thereby stabilizing ZTL. Here, we report that ZTL post-translationally and reciprocally regulates protein levels and nucleocytoplasmic distribution of GI in Arabidopsis. We use ectopic expression of the N-terminus of ZTL, which contains the novel, light-absorbing region of ZTL (the LOV domain), transient expression assays and ztl mutants to establish that the levels of ZTL, a cytosolic protein, help govern the abundance and distribution of GI in the cytosol and nucleus. Ectopic expression of the ZTL N-terminus lengthens period, delays flowering time and alters hypocotyl length. We demonstrate that these phenotypes can be explained by the competitive interference of the LOV domain with endogenous GI-ZTL interactions. A complex of the ZTL N-terminus polypeptide with endogenous GI (LOV-GI) blocks normal GI function, causing degradation of endogenous ZTL and inhibition of other GI-related phenotypes. Increased cytosolic retention of GI by the LOV-GI complex additionally inhibits nuclear roles of GI, thereby lengthening flowering time. Hence, we conclude that under endogenous conditions, GI stabilization and cytoplasmic retention occurs naturally through a LOV domain-mediated GI-ZTL interaction, and that ZTL indirectly regulates GI nuclear pools by sequestering GI to the cytosol. As the absence of either GI or ZTL compromises clock function and diminishes the protein abundance of the other, our results highlight how their reciprocal co-stabilization is essential for robust circadian oscillations.