Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fu s) mutants of Arabidopsis exhibit features of light-grown seedlings when gr own in the dark. Cloning and biochemical analysis of COP9 have revealed tha t it is a component of a multiprotein complex, the COP9 signalosome (previo usly known as the COP9 complex). Here, we compare the immunoaffinity and th e biochemical purification of the COP9 signalosome from cauliflower and con firm its eight-subunit composition. Molecular cloning of subunit 4 of the c omplex revealed that it is a proteasome-COP9 complex-elf3 domain protein en coded by a gene that maps to chromosome 5, near the chromosomal location of the cops and fus4 mutations. Genetic complementation tests showed that the cops and fus4 mutations define the same locus, now designated as COP8. Mol ecular analysis of the subunit I-encoding gene in both cops and fus4 mutant s identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cops mutant background resulted in complete rescue of the mutan t phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 sign alosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUSE, and Arabidopsis JAB1 ho molog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated tha t COP8 is highly conserved among multicellular eukaryotes and is also simil ar to a subunit of the 19S regulatory particle of the 26S proteasome.