Formation of an SCF^ZTL complex is required for proper regulation of circadian timing

Abstract

SummaryThe 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 SCF ZTL complex in detail. Yeast twohybrid 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-boxcontaining 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 SCF ZTL complex. Taken together, our data establish SCF ZTL as an essential SCF class E3 ligase controlling circadian period in plants.

Plant Circadian Rhythms

“…In the ztl mutant, protein levels of TOC1 are elevated and only weakly rhythmic, demonstrating that ZTL is critical for degradation of TOC1. Increasing expression of ZTL confers corresponding dosage-dependent period shortening (Han et al, 2004). Collectively, these data argue that the level of TOC1 activity, as regulated through transcriptional repression by CCA1 and LHY and via protein degradation by ZTL, is a key determinant of circadian period.…”

“…F-box proteins provide specificity to proteasomal degradation pathways by specific interaction with and polyubiquitination of targets for degradation. In this case, ZTL is a component of an SCF complex that recruits TOC1 for proteasomal degradation Má s et al, 2003a;Han et al, 2004). In the ztl mutant, protein levels of TOC1 are elevated and only weakly rhythmic, demonstrating that ZTL is critical for degradation of TOC1.…”

PRR3 Is a Vascular Regulator of TOC1 Stability in theArabidopsisCircadian Clock

“…ZEITLUPE (ZTL), the founding member of the protein family characterized by a LIGHT, OXYGEN, or VOLTAGE (LOV) domain, an F-box motif, and kelch repeats, mediates the degradation of TOC1 via the formation of a multiprotein Skp/Cullin1/F-box complex that catalyzes the ubiquitylation of proteins destined for proteasomal degradation (Kiyosue and Wada, 2000;Somers et al, 2000Somers et al, , 2004Schultz et al, 2001;Han et al, 2004;Fukamatsu et al, 2005;Kevei et al, 2006). In the absence of ZTL, the pace of the clock is delayed as a result of increased TOC1 stability (Mas et al, 2003a).…”

Arabidopsis Kelch Repeat F-Box Proteins Regulate Phenylpropanoid Biosynthesis via Controlling the Turnover of Phenylalanine Ammonia-Lyase

“…One subfamily, the Kelch motif (repeat) containing F-box (KFB) proteins, were initially identified in Drosophila melanogaster (Bork and Doolittle, 1994), and more than 270 members have been annotated from Arabidopsis, poplar (Populus trichocarpa), rice (Oryza sativa), maize (Zea mays), pine (Pinus taeda), and Physcomitrella patens (Sun et al, 2007). A few characterized KFBs mainly function in circadian control or in modulating flowering time (Nelson et al, 2000;Han et al, 2004;Somers et al, 2004;Yasuhara et al, 2004;Imaizumi et al, 2005), but their roles in regulating plant secondary metabolism remain unclear.…”