Characterization of SUMO-conjugating enzyme mutants in Schizosaccharomyces pombe identifies a dominant-negative allele that severely reduces SUMO conjugation.

The phenotypes of mutants defective in the Schizosaccharomyces pombe SUMO conjugator Hus5 (the homologue of Ubc9) show that it is required for recovery from S phase arrest. Unlike the case with ubiquitination, where ligases are required, SUMO conjugators are sufficient for substrate recognition and conjugation of SUMO onto target proteins, at least in vitro. Thus SUMO conjugators are likely to be important regulators of sumoylation. We report here on the characterisation of two hus5 alleles. Although hus5.17 and hus5.62 respond in a similar manner to UV and ionising radiation, they have different responses to the DNA synthesis inhibitor, hydroxyurea (HU). In addition, SUMO (Pmt3) is mis-localised in hus5.17 but not in hus5.62 mutant cells. The mutations in hus5.62 and hus5.17 map to A129 and to the 5' splice site of intron 2 respectively. We have characterised the Hus5.62 protein and shown in vitro, that it still interacts with SUMO and at least one protein, Rad22, which is a SUMO-modified target. The Hus5.62 protein is also capable of forming a thioester link with SUMO, but it does not function in sumoylation assays, either in the modification of Rad22 or in SUMO chain formation. When over-expressed in wild type S. pombe cells, the Hus5.62 protein has a dominant negative effect on sumoylation.