1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient

Abstract

The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.