Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis

The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC (COP), DEETIOLATED (DET), and FUSCA (FUS) loci are essential regulatory genes involved in the light control of seedling developmental patterns in Arabidopsis. Although COP1, DET1, COP9, and FUS6 (also called COP11) have been cloned, their biochemical activities and interactions remain elusive. We have recently suggested that multiple pleiotropic COP, DET, and FUS genes may encode subunits of a large regulatory complex. In this study, we generated specific antibodies against Arabidopsis FUS6 and show that accumulation of both COP9 and FUS6 is coordinated in the pleiotropic cop, det, and fus mutant backgrounds and in wild-type plants throughout development. Both COP9 and FUS6 cofractionated into identical high molecular mass fractions in an analytical gel filtration assay, and neither was found in its monomeric form. Moreover, antibodies raised against either COP9 or FUS6 selectively coimmunoprecipitated both proteins. We have also developed an Arabidopsis protoplast immunolocalization assay and demonstrated that the COP9 complex is localized in the nucleus and that its nuclear localization is not affected by light conditions or tissue types. The integrated genetic and biochemical results strongly support the conclusion that both COP9 and FUS6 are components of the nuclear-localized COP9 complex. Therefore, we have provided the strongest evidence for the conclusion that at least some of the pleiotropic COP, DET, and FUS loci act in the same signaling pathway.