Identification of LOV KELCH PROTEIN2 (LKP2)-interacting Factors That Can Recruit LKP2 to Nuclear Bodies

Abstract

LOV KELCH PROTEIN2 (LKP2) is an F-box protein that has been postulated to function centrally, or near to the circadian clock oscillator. As a first step to determine which proteins act as substrates of LKP2, yeast two-hybrid screening was performed using LKP2 as bait, and two interaction factors, Di19 and COL1, were isolated. The transiently expressed Di19-GUS fusion protein was localized in the nucleus of Arabidopsis petiole cells. COL1 and other CO/COL family proteins could also interact with LKP1/ZTL, LKP2 or FKF1. The LKP2-binding site in CO or COL1 was near the center of each protein. The CCT motif in CO or COL1 was not sufficient for interaction with LKP2. LKP2 recognized CO with F-box and kelch repeat-containing regions, while it recognized COL1 with an LOV domain. When LKP2 was fused with cyan fluorescent proein (CFP) and transiently expressed in onion epidermal cells, CFP-LKP2 signals were localized in the nucleus and cytosol. Both yellow fluorescent protein (YFP)-CO and YFP-COL1 were located in the nucleus, forming nuclear bodies when they were transiently expressed. However, co-expression of CFP-LKP2 with YFP fused to either CO or COL1 resulted in the recruitment of CFP-LKP2 in nuclear bodies. Furthermore, the CFP-LKP2 and YFP-CO signals co-localized with signals for pU2B''-mRFP, which is a marker for Cajal bodies. These results suggest the possibility that LKP2 functions with CO/COL family proteins in the nuclear bodies.

Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of CONSTANS stability in Arabidopsis photoperiodic flowering

“…3 A and B and Fig. S7) (6,29). The lkp2 mutation enhances the early flowering phenotype of the ztl mutant (22).…”

“…Third, together with FKF1, ZTL also is involved in the regulation of CDF2 stability for flowering time regulation (17). Fourth, ZTL interacts with CO in yeast (29). Therefore, we hypothesized that ZTL also may regulate CO protein stability and that the gi phenotype is caused by the combinational effects of changes in FKF1 and ZTL functions.…”

Novel blue light receptors with an F-box: their direct control of the circadian clock and the flowering timing in Arabidopsis

“…LKP2 mRNA is detected in wide variety of tissues by RT-PCR and doesn't show diurnal oscillation (Shultz et al 2001). GFP associated fluorescence is detected in nuclei in the 35S:LKP2-GFP plant (Yasuhara et al 2004), while CFP-LKP2 signals are localized both in the nucleus and cytosol when transiently expressed in onion epidermal cells (Fukamatsu et al 2005). According to the yeast two hybrid assay, LKP2 interacts with TOC1 and PRR5 through its LOV domain and ASK proteins through its Fbox domain (Yasuhara et al 2004).…”

“…According to the yeast two hybrid assay, LKP2 interacts with TOC1 and PRR5 through its LOV domain and ASK proteins through its Fbox domain (Yasuhara et al 2004). Di19 and CONSTANS LIKE 1 (COL1) were also identified as interactors of LKP2 by yeast two hybrid screening to find candidate substrates of LKP2 (Fukamatsu et al 2005). COL1 and other CO/COL family members also interact with ZTL, FKF1 or LKP2.…”

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).…”