Identification of LOV KELCH PROTEIN2 (LKP2)-interacting factors that can recruit LKP2 to nuclear bodies

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.