The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RINGfinger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.The ubiquitin proteasome pathway participates in a broad variety of physiologically and developmentally controlled processes in plants (for an overview, see Smalle and Vierstra, 2004). A critical step involves E3 ubiquitin ligases that facilitate the transfer of ubiquitin moieties to a substrate protein, leading to degradation via the 26S proteasome.In Arabidopsis (Arabidopsis thaliana), the bestcharacterized E3s are the SCF (Skp1-cullin-F-box) complexes that consist of at least four subunits: a cullin protein (AtCUL1), an ASK (Arabidopsis Skp1 ortholog) protein, a RING finger protein (RBX1), and an F-box protein (Gray et al., 1999Lechner et al., 2002;Shen et al., 2002). The cullin is a scaffolding subunit for the SCF and binds the ASK-F-box and RBX1 subunits within NH 2 -and COOH-terminal domains, respectively (Zheng et al., 2002). F-box proteins are substrate adaptors that confer specificity to the assembled SCF complexes (Gagne et al., 2002). AtCUL1-dependent E3s are crucial regulators for phytohormone responses, flowering, embryo development, and other processes (Smalle and Vierstra, 2004).At least 11 cullins are encoded in the Arabidopsis genome . However, only six members contain the conserved RUB (related to ubiquitin)-modification site characteristic of cullins that assemble into an SCF or related E3 complex (del Querido et al., 2001;del Pozo et al., 2002). Based on homology to cullins in other organisms, these six Arabidopsis cullins can be classified into three groups. The first group is the CUL1 family, which includes AtCUL1 (At4g02570), AtCUL2 (At1g02980), and AtCUL5 (At1g43140). It is likely that all three members participate in an SCF complex Risseeuw et al., 2003). The second group comprises the CUL3 family with AtCUL3a (At1g26830) and AtCUL3b (At1g69670). These two cullins are approximately 88% identical to each other and represent potential Arabidopsis orthologs of Caenorhabditis...

SummaryCullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.