Figure 1.
Three candidate nuclear localization signals in human USP1.
A. onfocal microscopy images showing nuclear localization of GFP-(left panels), and endogenous USP1 (right panels) B. Schematic representation of human USP1 protein showing the position of three candidate NLSs (cNLSs) identified by bioinformatics analysis (black rectangles). The amino acid sequence of each cNLS is indicated below. The position of the previously reported NES is also shown (white rectangle). C. Drawing shows a schematic representation of the NES-GFP construct used in the in vivo nuclear import assay. Each USP1 cNLS, and the positive (PKKKRKV) and negative (PAAARAV) control sequences were cloned upstream of the NES. Confocal images show representative examples of 293T cells transfected with each of these plasmids. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
Figure 2.
Identification of two NLSs that mediate USP1 nuclear import.
A. Schematic representation of USP1 deletion mutants. The positions of the cNLS and the NES are indicated. B. Immunoblot analysis demonstrating the expression and the correct size of the different mutant proteins. C. Confocal images of 293T cells showing the nucleocytoplasmic localization of each USP1 deletion mutant. All the fragments tested were nuclear, except for the (1–269) fragment, which was exclusively located to the cytoplasm. D. Images show that inhibition of the nuclear export receptor CRM1 using leptomycin B (LMB) induces a partial relocation of YFP-USP1(1–269) to the nucleus. E. Site directed mutagenesis of USP1 NLSs in the context of the full-length protein. On the left, drawings show a schematic representation of USP1 mutants bearing alanine substitutions (red) of several basic residues (green) in NLS1, NLS2 or both. Confocal microscopy images on the right show representative examples of the nucleocytoplasmic localization of each USP1 mutant in 293T cells. Mutation of both NLSs was necessary to abrogate USP1 nuclear import. Cells were counterstained with Hoechst to show the nuclei (DNA panels).
Figure 3.
USP1 NLSs mediate nuclear import of the USP1/UAF1 complex.
A. Confocal images showing that Xpress-UAF1 localizes to the cytoplasm in transfected 293T cells, and it does not relocate to the nucleus in the presence of LMB. B. Confocal images of 293T cells co-expressing Xpress-UAF1 with GFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Xpress-UAF1 (red) relocated to the nucleus when co-expressed with GFP-USP1 wild type, but remained in the cytoplasm when co-expressed with the import deficient mutant GFP-USP1NLS1/2m. Cells were counterstained with Hoechst to show the nuclei (DNA panels). C Semiquantitative analysis of Xpress-UAF1 nucleocytoplasmic distribution when co-expressed with YFP, GFP-USP1 wild type (WT) or GFP-USP1NLS1/2m. Graphs show the percentage of co-transfected cells showing nuclear (N), nuclear and cytoplasmic (NC) or cytoplasmic (C) localization of Xpress-UAF1. The number of cells counted in each sample (n) is indicated within the graph. D. A model illustrating the ability of USP1 to bind importins (imp) and UAF1, and thus, mediate the nuclear import of the USP1/UAF1 complex.
Figure 4.
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 [17], is boxed.
Table 1.
Amino acid identity across several functional domains between human USP1 protein and USP1 proteins from the mouse, the clawed frog Xenopus laevi (xenla), and the zebrafish Danio rerio (danre).
Figure 5.
A cellular relocation assay to screen for drugs that interfere with USP1/UAF1 complex formation.
In the upper part, the formation of the USP1/UAF1 complex can be assessed in cells co-transfected with GFP-USP1 and Xpress-UAF1, on the basis of the nuclear relocation of Xpress-UAF1. In the lower part, a drug that prevents USP1/UAF1 complex formation can be identified on the basis of the cytoplasmic localization of Xpress-UAF1.