Slx5 promotes transcriptional silencing and is required for robust growth in the absence of Sir2.

In the budding yeast Saccharomyces cerevisiae, transcriptional silencing is defined as constitutive, chromatin-mediated repression of transcription that occurs at HM loci, ribosomal DNA (rDNA), and telomeres. The Sir2 protein, essential for S. cerevisiae transcriptional silencing, is of particular interest as the founding member of the sirtuin family of NAD-dependent protein deacetylases that is conserved throughout eukaryotes. Increased expression of SIR2 or its closest homologs increases the life span in yeast and animals, respectively (reviewed in references 31 and 44). Although these studies suggest the conservation of an aging pathway, molecular mechanisms underlying the conserved function of sirtuins in aging remain unclear. In yeast, the only reported targets of Sir2 activity are histones. By contrast, the mammalian sirtuin SIRT1 deacetylates many different targets, including p53 (reviewed in reference 55). In yeast, Sir2 binds the telomeres and HM loci in complex with Sir3 and Sir4. Deacetylation of histone H4 lysine 16 (H4K16) promotes binding of Sir3 to chromatin and spreading of transcriptional silencing (reviewed in reference 54). However, Sir3 and Sir4 have no known metazoan homologs. Thus, it seems likely that some Sir2 molecular interactions remain undiscovered. Indeed, molecular mechanisms that contribute to sir2Δ phenotypes, such as suppression of replication onset (47) and unequal inheritance of oxidative damage between mother and daughter cells (1), are undefined. To identify novel Sir2-interacting proteins that might participate in these and other uncharacterized molecular pathways, we performed a screen for Sir2 physical interactions. We identified Slx5 as a novel Sir2 interactor by two-hybrid analysis with Sir2 fused to the Gal4 DNA-binding domain (GBD). Slx5 is a RING domain protein that was first identified as essential for viability in the absence of Sgs1 (45). Sgs1 is the sole S. cerevisiae representative of the RecQ helicase family that includes human WRN and BLM. Slx5 forms a complex with another RING domain protein, Slx8 (45, 76). Mutants lacking Slx5 or Slx8 show an increase in global sumoylation (6, 28, 70). SUMO is a ubiquitin-like protein moiety that is covalently attached to lysine residues. The Slx5/8 complex may function as a SUMO E3 ligase (28). Recently, it was discovered that Slx5 contains SUMO interaction motifs and that Slx8 is a ubiquitin E3 ligase (49, 67, 73). The Slx5/8 complex is thus a member of a newly defined conserved family of factors, known as STUbLs (SUMO-targeted ubiquitin ligases), present in humans as a single protein, RNF4 (49, 62). A function shared by some members of this protein family may be ubiquitination of polysumoylated proteins. The polysumoylated and ubiquitinated proteins are then degraded by the proteasome (67), providing a previously unsuspected mechanism for down-regulation. Although Sir3 and Sir4, but not Sir2, are known to be sumoylated (11, 72), the Sir2-Slx5 two-hybrid interaction required neither protein and the GAD-Slx5 interactor lacked the N-terminal SUMO interaction motifs. Furthermore, we recapitulated the physical interaction in vitro by affinity precipitation under conditions in which protein sumoylation is not preserved. Thus, the physical interaction was not likely to be bridged by sumoylation. The proteins have a functional overlap defined by several independent assays. Yeast lacking the SLX5 gene grew poorly, and additionally deleting SIR2, SIR3, or SIR4 exacerbated this defect, indicating that Slx5 and the Sir2/3/4 complex may have parallel roles in a vital process. Deletion of the SLX5 gene caused a previously unsuspected loss of telomeric and rDNA silencing, as does sir2Δ. Furthermore, ulp2Δ mutant cells, which share the slx5Δ phenotype of increased global sumoylation, also shared the silencing defect and loss of viability in combination with the sir2Δ or sir4Δ mutation. Therefore, the role of Slx5 in silencing appears to be linked to its role in regulating sumoylation, such that excess accumulation of sumoylated proteins is toxic in the absence of silenced chromatin.