Mapping the UAF1-binding site in USP1.
A. Schematic representation of USP1 deletion mutants used to map the UAF1-binding site. The critical USP1 nuclear localization signals NLS1 and NLS2 are depicted as green rectangles. The ability of each fragment to induce (+) or not (−) nuclear relocation of Xpress-UAF1 is indicated to the right. B. Immunoblot analysis demonstrating the expression and the correct size of the different USP1 mutant proteins. C. Confocal images show representative examples of 293T cells co-expressing Xpress-UAF1 (red) with full-length (FL) GFP-USP1 or with the different deletion mutants (green). Nuclear relocation of Xpress-UAF1 is induced by the fragments (1–672), (1–520), (420–785) and (420–520), but not by the fragments (1–500), (450–785), or the construct with the interstitial deletion Del(420–520). Cells were counterstained with Hoechst to show the nuclei (DNA panels). D. Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution in three of the samples shown in panel C. The number of cells counted in each sample (n) is indicated within the graph. E. Co-immunoprecipitation analysis, using GFP-trap, showing that full-length USP1 and the (420–520) fragment, but not the USP1 Del(420–520) mutant, interact with Xpress-UAF1 in co-transfected 293T cells. The upper panel shows that the three USP1 proteins were efficiently pulled down by the GFP-trap reagent (the dotted line indicates that the panel is a composite of two images from the same gel). The middle panel shows that Xpress-UAF1 was co-immunoprecipitated with FL USP1 and the (420–520) fragment, but not with the Del(420–520) mutant, an observation that is entirely consistent with the results obtained in the relocation assay. The lower panel shows the expression levels of Xpress-UAF1 in the whole-cell extract (WCE) as control. F. Alignment of human USP1 protein sequence encompassing the UAF1-binding domain (blue) with USP1 proteins from mouse, Xenopus (XENLA) and zebrafish (DANRE). The amino acid sequence VERIV, which resembles a UAF1-interacting motif in HPV E1 protein , is boxed.