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 (small, ubiquitin-like modifier)-conjugating enzyme 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-conjugating enzymes are sufficient for substrate recognition and conjugation of SUMO on to target proteins, at least in vitro. Thus SUMO-conjugating enzymes are likely to be important regulators of sumoylation. Here, we report on the characterization of two hus5 alleles. Although hus5.17 and hus5.62 respond in a similar manner to UV and ionizing radiation, they have different responses to the DNA-synthesis inhibitor, hydroxyurea. In addition, SUMO (Pmt3) is mislocalized in hus5.17 cells, but not in hus5.62 mutant cells. The mutations in hus5.62 and hus5.17 map to Ala(129) and the 5' splice site of intron 2 respectively. We have characterized 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 overexpressed in wild-type S. pombe cells, the Hus5.62 protein has a dominant-negative effect on sumoylation.
Ho
Jenny C Y
JC
Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
Watts
Felicity Z
FZ
D004268:DNA-Binding Proteins / Q000378:metabolism; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D012097:Repressor Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes
D004268:DNA-Binding Proteins; C402486:Pmt3 protein, S pombe; C105272:RAD31 protein, S pombe; D012097:Repressor Proteins; D029702:Schizosaccharomyces pombe Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C085412:rad52 protein, S pombe; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; C093855:HUS5 protein, S pombe