SUMO-targeted ubiquitin ligases in genome stability

Abstract

We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.

Figure 1

Identification and characterization of the Slx8-Rfp complex. (A) The indicated yeast two-hybrid strains were spotted onto selective plates, which were untreated (No drug), or drug treated (20 mM 3-AT), to identify interacting proteins. Key indicates genes placed into the Gal4 DNA-binding (DBD) or the Gal4-activating (AD) domains. Empty vector (−) and positive controls (Nse5:Nse6 interaction) are shown. (B) Ectopically expressed GST-Rfp1/2 (or GST alone) were induced in an Slx8-myc strain, and subjected to GST pulldown, Inputs and Pulldowns were immunoblotted with anti-myc antisera, amido black staining is shown as a GST loading control. (C) Left panels: localization of ectopically expressed EGFP-Rfp1 or EGFP-Slx8 was detected in live cells. Right panels: DNA staining with DAPI (4′,6′-diamidino-2-phenylindole). Arrowheads indicate Rfp1 subnuclear foci. (D) Serial dilutions of the indicated strains grown at the semipermissive temperature (32°C), which were nontreated (YES), or treated with the indicated concentrations of hydroxyurea (HU), methylmethane sulfonate (MMS), camptothecin (CPT), or ultraviolet (UV) irradiated. (E) The indicated strains were serially diluted on selective media at 32°C, and either untreated, or treated with HU, under induced (−B1) or repressed (+B1) conditions.

Figure 2

Analysis of the slx8-1 mutant phenotypes. (A) The indicated strains were serially diluted onto YES plates at permissive (25°C) or semipermissive (34°C) temperatures. Two independent isolates of slx8-1 rad60-4 are shown. (B) Fluorescence microscopy was used to analyze spontaneous Rad22-YFP foci formation in wild-type and slx8-1 cells, grown at the semipermissive temperature (32°C). DNA staining with DAPI is shown. Arrowheads indicate Rad22-YFP foci. At least 300 cells were scored for each strain. (C) The indicated strains were serially diluted onto YES plates at permissive (25°C) or semipermissive (34°C) temperatures.

Figure 3

SIM-dependent Rfp interactiom with SUMO. (A) S. pombe Rfp1/2 SUMO interacting motifs (SIMs; red) and C-terminal hydrophobic regions (blue) are shown. Identical (^*) and conserved residues (:) are indicated. (B, C) The indicated yeast two-hybrid strains were spotted onto selective plates, which were untreated (No drug) or drug treated (20 mM 3-AT), to identify interacting proteins. Key indicates genes in the Gal4 DNA-binding (DBD) or Gal4-activating (AD) domains. Positive (Nse5:Nse6) and empty vector (−) controls are shown. (D) The indicated strains were serially diluted onto selective media at 32°C, and either untreated or HU treated. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 4

Slx8, but not Rfp1/2, is a RING-dependent E3 ubiquitin ligase in vitro. (A, B) In vitro ubiquitination assays containing (+) or not (−) E1, E2, ubiquitin, and GST-Slx8, GST-Slx8 deleted for its RING motif (ΔRING) or GST-Rfp1, as indicated, were resolved by SDS–PAGE and immunoblotted with antisera for ubiquitin or GST. Polyubiquitin (Ubⁿ) and di-ubiquitin (Ub²) chains are indicated. (C) Total lysates were prepared following 7 h incubation at 36°C, resolved by SDS–PAGE and immunoblotted with antisera for SUMO (Pmt3) or Cdc2 (loading control). (D) Upper panels: the indicated strains were serially diluted at the permissive temperature (25°C), with no drug, or semipermissive temperature (34°C) in the presence of 5 mM HU. Western blotting to determine total sumoylation levels in the indicated strains was as described in (C).

Figure 5

Rfp stimulates Slx8-dependent ubiquitination of Rad60. (A) Upper panel: S. pombe Rad60 and H. Sapiens NIP45 tandem C-terminal SUMO-Like Domains (SLDs; boxed) are shown. Lower panel: the indicated yeast two-hybrid strains were spotted onto selective plates, which were either untreated (No drug), or drug treated (20 mM 3-AT), to identify interacting proteins. Key indicates the genes placed either into the Gal4 DNA-binding (DBD) or the Gal4-activating (AD) domains. (−) Denotes an empty vector. (B) Ectopically expressed GST-Full Length-Rad60 (GST-F.L.), GST-Rad60 SUMO-Like domains (GST-SLDs), or GST alone were induced in an Slx8-myc strain. Upper panel: (Pulldowns): GST pulldowns were immunoblotted with anti-myc antisera. Amido black staining is shown as a GST loading control. Lower panel (inputs): loading controls were immunoblotted with anti-myc antisera. Two independent strains expressing Rad60 SUMO-Like domains (GST-SLDs) are shown. (C, D) In vitro ubiquitination assays containing (+) or not (−) E1, E2, ubiquitin, Rad60-TAP, GST-Slx8, GST-Slx8 deleted for its RING motif (ΔRING), GST-Rfp1 or GST-Rfp1 deleted for its SIM motif (ΔSIM), as indicated, were resolved by SDS–PAGE and immunoblotted with antisera for Rad60-TAP, ubiquitin, or GST. Polyubiquitin chains (Ubⁿ) are indicated.

Figure 6

Identification of a Human homolog of Slx8-Rfp. (A) Top left: the SUMO interacting motifs (SIMs; red), C3HC4 RING domain (green), RING-like domain (Blue), and C-terminal hydrophobic regions (black) are shown. Top right: alignments of the indicated proteins, depicting their SIM domains (red). An acidic stretch of amino acids in PIAS, which makes additional contacts with SUMO is underlined. Residues of each protein shown: PIAS, 454–485; Rfp1, 9–44; Rfp2, 14–48; RNF4, 41–68; MIP1, 160–231, and Slx5, 18–34. Lower Panel: C-terminal hydrophobic residues (shaded region) and RING domains (boxed residues) are shown. A region of basic residues common to the Slx8 proteins is underlined. Residues of each protein shown: Rfp2, 193–205; RNF4, 130–190; S. pombe Slx8, 204–269, and S. cerevisiae Slx8, 203–274. (B) The indicated strains were streaked on selective plates, which all contained 5 mM HU. Left panel: slx8-1 transformed with the indicated vectors, incubated at permissive (25°C; upper section) or semipermissive (34°C; lower section) temperatures. Middle and right panels: pRNF4 was either induced (−B1; upper sections) or shut off (+B1; lower sections) in two independent slx8Δ or rfp1Δ rfp2Δ strains. (C) Total lysates were immunoblotted with antisera for SUMO (Pmt3) or Cdc2 (loading control). (D, E) HEK293T cells were transfected with (+) or without (−) myc-RNF4, FLAG-NIP45, HA-SUMO1ΔC4, NIP45 mutants (FLAG-Nterm or FLAG-Nterm-SUMO1ΔC4), or treatment with 2 mM HU, as indicated. Upper panels (Pulldowns): FLAG pulldown, immunoblotted with myc or FLAG antisera. Lower panel (Input): loading controls immunoblotted with myc antisera. (^*) Denotes background bands. A full-colour version of this figure is available at the EMBO Journal Online.

Figure 7

Model for SUMO-directed ubiquitination by the conserved STUbL family. Following the appropriate stimulus and priming modification, e.g. phosphorylation, a target protein X is subject to sumoylation. Upon completion of the function associated with sumoylated target X (// on arrow indicates variable time elapsed), it is subject to simultaneous desumoylation by the Ulp isopeptidases and STUbL-dependent ubiquitination. STUbL-dependent ubiquitination of X may also require a priming modification, e.g. phosphorylation/dephosphorylation of X. STUbL-dependent ubiquitination may directly target X to the proteasome for degradation, thus clearing the pool of ‘sumoylation competent' target X (e.g. phosphorylated X). In the absence of STUbLs (STUbLΔ), target X may be locked in a cycle of sumoylation and desumoylation, resulting in a net increase in sumoylated proteins in the cell. In this model, ubiquitin and SUMO do not coexist on the target. Indeed, ubiquitination by STUbLs could be on the same (or proximal) lysine that was sumoylated, thus blocking resumoylation.