Fig. 1. Sequence and constructs for Xenopus Cullin-1. (A) Protein alignment of the mouse, human and X. tropicalis Cullin-1. The full-length protein is 99.6% identical at the amino acid level. The Rbx-1- and Skp1-binding domains are boxed in red and blue, respectively. (B) Diagrammatic representation of Cullin-1 constructs. Two C-terminally truncated versions of Cullin-1 were used. Cul1-C75 and Cul1-C477 are missing 75 and 477 amino acids from the C terminus respectively (C-terminal truncations indicated by purple bars in A). As a consequence, Cul1-C477 contains none and Cul1-C75 only contains a subset of the Rbx-1-binding sites. Moreover, lysine 720, the neddylation site (green star in A) is missing in both constructs. These constructs should lose or have reduced binding ability to Rbx-1, one of the components of the SCF complex, and are likely to interfere with the correct formation of the endogenous SCF. A fourth construct (N148-Cul1-C477), missing the 148 N-terminal and the 477 C-terminal amino acids, was also created. This deletes both the Skp-1- (yellow) and the Rbx-1- (red) binding domains and should not interfere with the endogenous SCF complex. (C) A wild-type SCF complex (with its substrate ß-catenin) versus a complex based on the truncated Cullin proteins (Cul1-C75). In the wild-type scenario, Cullin proteins provide the bridge between the F-box protein that binds ß-catenin and the E2 conjugating enzyme that supplies ubiquitin molecules. The truncation is likely to inhibit the interaction between the Cul1-C75 and Rbx-1/E2, hence preventing SCF function. Polyubiquitination of ß-catenin is shown by a series of grey circles, which are missing in the Cul1-C75-based SCF complex.