Figure 4

Homologous amino acid motifs, mapping to the Fingers sub-domain, mediate binding of USP1 and USP46 to UAF1. A. Alignment of USP1, USP46 and USP12 aminoacid sequences using CLUSTALW. USP1 aminoacid segment 420–520 and the homologous regions of USP46 and USP12 are highlighted in blue. B. Schematic representation of different GFP-tagged USP46 deletion mutants used to map its UAF1-binding motif. A heterologous nuclear localization signal (SV40 NLS) was fused to the amino terminal end of each fragment to ensure its nuclear accumulation C. Results of the UAF1 nuclear relocation assay with USP46 fragments. Confocal microscopy images show representative examples of 293T cells co-expressing UAF1-mRFP (red) and the different USP46 protein fragments (green). Cells were counterstained with Hoechst to show the nuclei (DNA panels). Nuclear relocation of UAF1-mRFP is induced by full length (FL) and the 165–259 fragment, but not by the 1–164 or the 243–366 fragments. D. Co-IP analysis of co-trasfected 293T cells, showing that Xpress-UAF1 co-immunoprecipitates with full-length USP46 and with the fragment encompassing residues 165–259, but not with the other two fragments tested. The dotted line indicates that the panel is a composite of two images from a single exposure of the same gel. WCE, whole cell extract. A section of the membrane stained with Ponceau is shown to gauge protein loading. E. Modeled structure of USP1 (left) and USP46 (right) catalytic domains using SWISS-MODEL and the USP7 structure 1NB8 [2] as a template. The Thumb, Palm and Fingers sub-domains are indicated. The UAF1-binding sites are highlighted in red (USP1 residues 420–520) or blue (USP46 residues 165–259).