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. 2010 May;24(9):893-903.
doi: 10.1101/gad.1906510. Epub 2010 Apr 13.

SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor

Yang Xie  1 , Eric M Rubenstein, Tanja Matt, Mark Hochstrasser
Affiliations

SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor

Yang Xie et al. Genes Dev. 2010 May.

Abstract

Many proteins are regulated by ubiquitin-dependent proteolysis. Substrate ubiquitylation can be stimulated by additional post-translational modifications, including small ubiquitin-like modifier (SUMO) conjugation. The recently discovered SUMO-targeted ubiquitin ligases (STUbLs) mediate the latter effect; however, no endogenous substrates of STUbLs that are degraded under normal conditions are known. From a targeted genomic screen, we now identify the yeast STUbL Slx5-Slx8, a heterodimeric RING protein complex, as a key ligase mediating degradation of the MATalpha2 (alpha2) repressor. The ubiquitin-conjugating enzyme Ubc4 was found in the same screen. Surprisingly, mutants with severe defects in SUMO-protein conjugation were not impaired for alpha2 turnover. Unmodified alpha2 also bound to and was ubiquitylated efficiently by Slx5-Slx8. Nevertheless, when we inactivated four SUMO-interacting motifs (SIMs) in Slx5 that together account for its noncovalent SUMO binding, both in vitro Slx5-Slx8-dependent ubiquitylation and in vivo degradation of alpha2 were inhibited. These data identify alpha2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that alpha2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation.

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Figures

Figure 1.
Figure 1.
SLX5 and SLX8 are required for degradation of the α2*-Ura3-3HA reporter protein. (A) Schematic of the two major ubiquitin conjugation pathways that regulate α2 degradation. (B) Growth assay for α2*-Ura3-3HA metabolic stabilization. The slx5Δ and slx8Δ mutants were transformed with a centromeric plasmid carrying the α2*-URA3-3HA allele, grown in liquid medium lacking leucine (to maintain the LEU2 plasmid), and then spotted on both SD-leucine and SD-uracil plates in 10-fold serial dilutions. The α2* protein carries the I4T and L10S substitutions, which inhibit degradation by the Ubc6/7–Doa10 pathway. Pictures were taken after 1 d (SD-leucine) or 3 d (SD-uracil) at 30°C. (C) Cycloheximide-chase/immunoblot analysis of α2*-Ura3-3HA degradation. Cell extracts from the indicated strains were harvested at the indicated times after addition of cycloheximide, and were resolved by 8% SDS-PAGE followed by anti-HA immunoblotting. Anti-Pgk1immunoblotting allowed comparison of protein loading between samples. (α2*-UH) α2*-Ura3-3HA.
Figure 2.
Figure 2.
Slx5 and Slx8 contribute to degradation of endogenous α2 as part of the Ubc4-dependent pathway. (A) Representative pulse-chase analysis of α2 degradation in wild-type (WT) and mutant yeast strains. (B) Quantitation of α2 degradation rates in the indicated strains (error bars depict standard deviations; n = 3).
Figure 3.
Figure 3.
Slx5–Slx8-dependent ubiquitylation of α2 in vivo and in vitro. (A) Ubiquitylation of α2*-Ura3-3HA in yeast cells. A plasmid expressing α2*-Ura3-3HA was transformed along with YEp105, a plasmid encoding Myc epitope-tagged ubiquitin, into wild-type (WT) (MHY501), ubc4Δ (MHY498), slx5Δ (MHY3712), and slx8Δ cells (MHY3716). The α2*-Ura3-3HA protein was precipitated with anti-α2 antibodies bound to Protein A-agarose, and the precipitated species were analyzed by immunoblotting. (α2*-UH) α2*-Ura3-3HA. (B) Ubiquitin conjugation to α2 with recombinant proteins. (Lanes 3–7) Purified α2 substrate (1 μM; from Escherichia coli) was incubated for 2 h at 30°C with Uba1 (E1) (0.1 μM), Ubc4 (E2) (0.37 μM), and 1 μM each the indicated recombinant proteins. Control reactions lacked α2 (lane 1) or ubiquitin (lane 2). Reaction samples were resolved by 10% SDS-PAGE and visualized by anti-α2 immunoblotting. (RΔ) Recombinant Slx5 or Slx8 protein, with the respective RING domain deleted (Xie et al. 2007); (**) nonspecific contaminating band from the purified Slx5 and Slx8 preparations; (*) nonspecific cross-reacting species that copurified with α2.
Figure 4.
Figure 4.
Mutations in the SUMO pathway do not impair degradation of cellular α2. (A) A plasmid expressing α2-3HA was transformed into the indicated congenic strains. Cells were grown at 30°C and shifted for 30 min to 37°C before the addition of cycloheximide. Whole-cell extract was prepared at various time points after cycloheximide addition, and the disappearance of α2-3HA was followed by anti-HA immunoblot analysis. (B) Plot of α2-3HA degradation rates for the experiment in A. (C) SUMO conjugation profiles of yeast strains shown in A. Extracts from logarithmically growing cells at 30°C were analyzed by anti-SUMO immunoblotting. A portion of the Gelcode Blue-stained membrane is shown to verify comparable sample loading. (D) A SUMO mutant defective for SIM binding does not impair α2 degradation. Representative pulse-chase analyses of endogenous α2 degradation at 30°C in doa10Δ cells or doa10Δ smt3Δ cells harboring the smt3-F37A allele on a centromeric plasmid. (E) Plot of α2 degradation for the experiment in D. (F) Yeast two-hybrid analysis of interaction of Slx5 (pGAD-Slx51–443) (Hannich et al. 2005) with Smt3ΔGG (pGBD-UC1-Smt3ΔGG) or Smt3ΔGG-F37A (pGBD-Smt3ΔGG-F37A). PJ69-4a cells were spotted in sixfold serial dilutions. Two-hybrid interaction was determined by analyzing growth on medium lacking histidine. The F37A mutation did not affect SM3ΔGG protein levels (not shown). (AD) Gal4 transcriptional activation domain; (BD) Gal4 DNA-binding domain.
Figure 5.
Figure 5.
Slx5, but not Slx8, interacts physically with α2. Yeast ubc4Δ ubc6Δ matα2Δ cells (MHY3765) cells were cotransformed with pRS425-GAL1-α2 and a pYES2.1 (2 μm, GAL1) plasmid expressing V5-His6-tagged Slx5, Slx8, or Cdc48 (negative control). After induction with galactose, cells were lysed under native conditions, and V5-tagged proteins were immunoprecipitated with anti-V5-agarose. Slx5, Slx8, and Cdc48 proteins were detected by anti-V5 (Invitrogen), and coprecipitated α2 by anti-α2 immunoblotting. For lane 3, half of each of the number of cells used for lanes 4 and 5 were mixed and lysed together, so the amount of input Slx8 and Slx5 was half of that used for each of the proteins in lanes 4 and 5. (Lane 1) A control immunoprecipitation with cells that overexpressed α2 but lacked any V5-tagged protein.
Figure 6.
Figure 6.
The SIMs of Slx5 are required for SUMO binding and α2 degradation. (A) Schematic of Slx5 (wild type and mutants) with its SIMs and RING domain highlighted. Four residues that constitute the hydrophobic core of each SIM are indicated, with the altered sequences indicated for each mutant. The SIM core sequences that were tested were SIM-1, 24VILI27; SIM-2, 93ITII96; SIM-3, 116VDLD119; SIM-4, 155LTIV158; and SIM-5, 476TIIV479. (B) Protein binding to SUMO1-agarose. (Lanes 1–3) Purified recombinant MBP-Slx5 (wild type and sim-1234) or MBP2 proteins were incubated with SUMO1-agarose, and proteins that bound to the resin were eluted, resolved by SDS-PAGE, and detected by anti-MBP immunoblotting. Input proteins (20%) are shown in lanes 4–6. (C) Slx5–Slx8-mediated ubiquitylation of α2 in vitro (conditions as in Fig. 3). Reactions were stopped at the indicated times by addition of 2× SDS gel loading buffer. Proteins were resolved by SDS-PAGE, and were detected by anti-α2 immunoblotting. Asterisks denote the same two cross-reacting proteins noted in Figure 3. (D) Quantitation of α2 degradation in doa10Δ slx5Δ strains transformed with plasmid-borne SLX5 alleles measured by pulse-chase analysis at 30°C. (E) Coimmunoprecipitation of T7-tagged Slx8 with variants of Slx5. Analysis was done as in Figure 5. Slx5 variants were detected by anti-V5 immunoblotting, and coprecipitated T7-Slx8 was detected by anti-T7 immunoblotting. Protein staining of a portion of the membrane shows similar loading.
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