Formation of an SCFZTL complex is required for proper regulation of circadian timing

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Abstract
The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24-h periodicity. In Arabidopsis a novel F-box protein, ZEITLUPE (ZTL), plays an important role in the control of the free-running period of the circadian clock. As a class, F-box proteins are well-established components of the Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein. Here we identify and characterize the SCFZTL complex in detail. Yeast two-hybrid tests demonstrate the sufficiency and necessity of the F-box domain for Arabidopsis Skp-like protein (ASK) interactions and the dispensability of the unique N-terminal LOV domain in this association. Co-immunoprecipitation of full-length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo. F-box-containing truncated versions of ZTL (LOV-F and F-kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot. Stable expression of F-box-mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo. Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss-of-function mutations, demonstrating the functional significance of the SCFZTL complex. Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants.