Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies.

Four nanobodies (VHH1-4(SMT3)) that target the yeast SUMO protein Smt3p were isolated and characterized. VHH1-4(SMT3) bind to Smt3p and Smt3p-tagged proteins with high affinity (Kd: low nM). NMR analysis shows that the four nanobodies all bind near the C-terminus of Smt3p, partially overlapping with the binding site for the SUMO protease Ulp1p. Binding of Smt3p-specific nanobodies impairs Ulp1-mediated cleavage of Smt3p-tagged proteins, with VHH1(SMT3) showing complete inhibition. The use of immobilized VHH2(SMT3) enabled efficient purification of Smt3p-tagged proteins, while VHH1(SMT3) can be used for immunoblotting and detects both Smt3p-tagged and free Smt3p. When expressed in yeast, VHH1(SMT3) causes significant growth defects, particularly when targeted to the nucleus or fused with GFP, indicative of interference with essential SUMOylation-dependent processes.