Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins

The function of small ubiquitin- like modifier ( SUMO)binding proteins is key to understanding how SUMOylation regulates cellular processes. We identified two related Schizosaccharomyces pombe proteins, Rfp1 and Rfp2, each having an N- terminal SUMO- interacting motif ( SIM) and a C- terminal RING- finger domain. Genetic analysis shows that Rfp1 and Rfp2 have redundant functions; together, they are essential for cell growth and genome stability. Mammalian RNF4, an active ubiquitin E3 ligase, is an orthologue of Rfp1/ Rfp2. Rfp1 and Rfp2 lack E3 activity but recruit Slx8, an active RING- finger ubiquitin ligase, through a RING - RING interaction, to form a functional E3. RNF4 complements the growth and genomic stability defects of rfp1rfp2, slx8, and rfp1rfp2slx8 mutant cells. Both the Rfp- Slx8 complex and RNF4 specifically ubiquitylate artificial SUMO- containing substrates in vitro in a SUMO binding- dependent manner. SUMOylated proteins accumulate in rfp1rfp2 double- null cells, suggesting that Rfp/ Slx8 proteins may promote ubiquitin- dependent degradation of SUMOylated targets. Hence, we describe a family of SIM- containing RING- finger proteins that potentially regulates eukaryotic genome stability through linking SUMO- interaction with ubiquitin conjugation.