A ZNF-nanobody fusion reveals SUMOylation-dependent changes in p53 protein localization

SUMOylation is a post-translational modification regulating protein localization, stability, and activity, with effects varying depending on the conjugated SUMO protein and type of SUMOylation. To determine how enhanced SUMOylation affects protein localization, we fused ZNF, a SUMOylation tag derived from SUMO E3 ligase ZNF451 that biases substrates toward SUMO2/3, to the GFP-binding nanobody vhhGFP4 (VHH), creating VHH-ZNF to drive SUMOylation of GFP-tagged substrates in trans. In vitro, VHH-ZNF increased SUMO2/3 modification of p53-GFP, preferentially generating polySUMO2 chains at the canonical K386 site. In HEK293 cells co-expressing p53-GFP and VHH-ZNF, immunoblotting and proteomics confirmed increased SUMO2/3 conjugation of p53 at K386. Fluorescence microscopy analyses revealed that SUMOylated p53 transitions from a diffuse nuclear distribution to SUMO-positive nuclear foci that partially overlap with promyelocytic leukemia (PML) and, less so, to 53BP1 nuclear bodies. Overall, we developed a method to increase the SUMOylation of GFP-tagged proteins and to visualize SUMOylation-dependent relocalization in cells.