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

SUMOylation is a post-translational modification regulating protein localization, stability, and activity, with effects varying depending on the identity of the conjugated SUMO protein (SUMO1 or SUMO2/3) and type of SUMOylation (mono-, multi-, or poly-). We previously developed a small 32 amino acids SUMOylation tag derived from the ZNF451 E3 ligase (hereafter “ZNF”) that biases substrates toward SUMO2/3 conjugation and modulates the transcriptional activity of p53. To determine how enhanced SUMOylation affects protein localization, we fused ZNF 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; using a chain-deficient SUMO2(K0) shifted the pattern toward multi-SUMOylation. VHH-ZNF did not modify HA-p53 alone but SUMOylated it in the presence of p53-GFP, consistent with proximity-based modification within p53 tetramers. In HEK293 cells co-expressing p53-GFP and VHH-ZNF, immunoblotting and proteomics confirmed increased SUMO2/3 conjugation at K386 that was abrogated by the E1 inhibitor ML792. 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) nuclear bodies. Overall, we developed a new method to increase the SUMOylation of GFP-tagged proteins and to visualize SUMOylation-dependent relocalization in cells.