Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

Abstract

The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2μ circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Δ and slx8Δ mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.