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. 2009 Apr;29(7):1694-706.
doi: 10.1128/MCB.01470-08. Epub 2009 Jan 12.

Quality control of a transcriptional regulator by SUMO-targeted degradation

Zheng Wang  1 , Gregory Prelich
Affiliations

Quality control of a transcriptional regulator by SUMO-targeted degradation

Zheng Wang et al. Mol Cell Biol. 2009 Apr.

Abstract

Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

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Figures

FIG. 1.
FIG. 1.
The mot1-301 phenotypes are due to reduced protein level. (A) Western blot showing the steady-state level of HA-tagged Mot1 and Mot1-301 during a time course after a temperature shift from 30°C to 39°C. Mot1 was detected by anti-HA antibody. G6PDH served as a loading control. (B) A mot1-301 strain was transformed with the indicated CEN and 2μm plasmids. Transformants were replica plated to SC-Leu plates at 30°C (-Leu) and 39°C (Ts) and to an SC-Leu plate containing galactose (Gal). Photos were taken after 2 days. The mot1-301 Ts− and Gal− phenotypes are reversed by CEN and 2μm mot1-301.
FIG. 2.
FIG. 2.
Mot1 is SUMOylated in vivo. (A) Mot1-HA was immunoprecipitated with anti-HA beads from an untagged strain (lane 1), an smt3Δ strain containing a CEN Myc3-SMT3 plasmid (lane 2), an SMT3+ strain containing empty vector (lane 3), or an SMT3+ strain containing a 2μm SMT3 plasmid (lane 4). The immunoprecipitated (IP) samples were subjected to SDS-PAGE and Western blot (WB) analysis with anti-HA antibody (left) or anti-SUMO antibody (right) to detect total Mot1 and SUMOylated Mot1, respectively. The numbers on the side of the gel are molecular mass markers. (B) Total lysates from the indicated strains overexpressing SMT3 were subjected to the same Mot1 in vivo SUMOylation assay as for panel A. WT, wild type.
FIG. 3.
FIG. 3.
SUMOylation of Mot1. (A) Summary of Mot1 deletions or point mutations and their in vivo SUMOylation statuses. A diagram of Mot1 is presented at the top, showing the locations of the conserved domains (A to D), the C-terminal ATPase domain, and the 11 consensus SUMO motifs (vertical lines). The SUMO consensus motifs within the N-terminal cluster are displayed. (B) Western blot (WB) of immunoprecipitated (IP) samples for assay of in vivo SUMOylation levels of Mot1 mutants within the N-terminal cluster, using anti-SUMO antibody (top) or anti-HA antibody (bottom). WT, wild type. (C) Similar in vivo SUMOylation assay for comparison of the SUMOylation levels of wild-type Mot1, Mot1-24, and Mot1-42.
FIG. 4.
FIG. 4.
Effects of disrupting SUMOylation of Mot1-301. (A) CEN LEU2 plasmids carrying the indicated mot1 alleles were transformed into a mot1Δ strain containing a CEN URA3 MOT1 plasmid. The CEN MOT1 plasmid was shuffled out using 5-FOA selection, and 10-fold serial dilutions of the resulting strains were plated onto yeast extract-peptone-dextrose plates and incubated at 30°C and 39°C for 3 days. (B) A complete test of all four phenotypes (Bur, Spt, Ts, and Gal) of the indicated MOT1 alleles. YPD, yeast extract-peptone-dextrose. (C) Total protein levels of genomic HA-tagged Mot1 and Mot1-301 in the indicated strains were examined by SDS-PAGE followed by anti-HA Western blot analysis. (D) CEN plasmids expressing HA-tagged Mot1, Mot1-301, or Mot1-301-K101R-K109R were transformed into SIZ1+ SIZ2+ or siz1Δ siz2Δ strains, as indicated, and their half-lives assayed during a cycloheximide chase time course. Quantification of Mot1 half-life is shown below the corresponding Western blots.
FIG. 5.
FIG. 5.
The ubiquitin-proteasome pathway is involved. (A) mot1-301 was crossed into strains containing the indicated ubiquitin E2 gene deletions, and the double-mutant phenotype was determined. Only ubc4Δ specifically suppressed the mot1-301 Ts− and Gal− phenotypes. YPD, yeast extract-peptone-dextrose. (B) A mot1-301 ubc4Δ strain was transformed with 2μm LEU2 plasmids carrying the indicated genes. Transformants were patched and replica plated to SC-Leu plates at 30°C (-Leu) and 39°C (Ts) and to an SC plate containing galactose (Gal). Photos were taken after 2 days. 2μm UBC5 reversed the suppression of mot1-301 by ubc4Δ for the Ts+ and Gal+ phenotypes. (C) A CEN plasmid containing mot1-301-HA or mot1-301-K101R-K109R-HA was transformed into strains with the indicated genotypes, and Mot1-301 stability was assayed by Western blotting during a cycloheximide chase. (D) A CEN mot1-301-HA plasmid was transformed into a pdr5Δ strain, and Mot1 stability was assayed upon addition of dimethyl sulfoxide (DMSO) or 50 μM MG132 in DMSO. (E) The levels of Mot1 SUMOylation in wild-type, slx5Δ, and slx8Δ strains were assayed using an anti-SUMO Western blot (WB). IP, immunoprecipitation. (F) Two-hybrid interactions between Slx5-Slx8 and Mot1. Wild-type and mutant alleles were cloned into the pGADT7 (Gal4AD, LEU2+) or pGBKT7 (Gal4BD, TRP1+) vector as indicated. Combinations of plasmids were then transformed into yeast strain PJ69-4A, which contains Gal UAS-driven ADE2 as a reporter gene, and selected on SC plates lacking leucine and tryptophan. Transformants were then patched and replica plated to SC-Leu-Trp (left) and SC-Leu-Trp-Ade (right) plates. Slx5-104 was found to interact with Mot1Δ14.
FIG. 6.
FIG. 6.
Differential regulation of Mot1 and Mot1-301 by SUMOylation. (A) Western blot for assaying the half-lives of wild-type Mot1 and Mot1-K101R-K109R during a cycloheximide chase in strains with the indicated genotypes. (B) Western blot showing the steady-state protein levels of wild-type Mot1 in strains with the indicated genotypes. (C) HA-tagged Mot1 and Mot1-301 strains were transformed with a 2μm SMT3 plasmid and assayed for SUMOylation. An anti-HA Western blot (WB) against crude extracts is shown in the left panel. Mot1-HA and Mot1-301-HA were immunoprecipitated (IP) with anti-HA beads and analyzed using anti-HA antibody (center) or anti-SUMO antibody (right). (D) Same as panel C, except in the absence of SUMO overexpression.
FIG. 7.
FIG. 7.
Canavanine-induced SUMO-targeted degradation of wild-type Mot1 protein. (A) Cells were grown overnight in medium lacking arginine before incubation in 30 μg/ml canavanine. Cultures were harvested every 30 min after canavanine addition for up to 2 h, followed by Western blotting (WB) for detection of in vivo SUMOylation of Mot1-HA. IP, immunoprecipitation. (B) Western blot for assaying the canavanine-induced SUMOylation of Mot1-HA expressed in wild-type or slx5Δ slx8Δ strains. The two major SUMO-Mot1 species are indicated by asterisks, and a ladder of additional SUMO-Mot1 species with reduced mobility are indicated by the bracket. (C) Cells were treated with canavanine, together with DMSO alone or 50 μM MG132 in DMSO, and total Mot1 protein levels were measured by anti-HA Western blot analysis during the time course. (D) Cells with the indicated genotypes were grown and treated with canavanine (CAN) as described for panel A and harvested every hour after canavanine treatment. Total lysates were prepared and probed with anti-HA antibody to detect Mot1 levels. Quantification of the Western blots is shown to the right. G6PDH served as a loading control for all experiments. WT, wild type.
FIG. 8.
FIG. 8.
Canavanine-induced SUMOylation and degradation of other proteins besides Mot1. (A) Crude lysates of cells grown overnight in medium lacking arginine, followed by a time course after addition of 30 μg/ml canavanine. Lysates were subjected to Western blotting (WB) with anti-SUMO antibody to determine the effect of canavanine on SUMOylation level in the crude extract. G6PDH served as a loading control. (B) Tenfold serial dilutions of the indicated strains were spotted onto SC-arginine (-CAN) and SC-arginine plus 2.5 μg/ml canavanine (+CAN) plates, showing the sensitivity to the drug. Photos were taken after incubation at 30°C for 3 days. WT, wild type. (C to F) Cells were grown overnight in medium lacking arginine. Half of the culture was collected (CAN-), and the other half was further incubated in the presence of 30 μg/ml canavanine for 2 h (CAN+). Crude lysates were prepared and analyzed by Western blotting using anti-HA antibody to detect HA-tagged Spt6, Rpb3, Spt20, Rtf1, Rco1, Set2, Bur2, and Paf1; anti-Rbp1 antibody to detect Rpb1; anti-Myc antibody to detect Myc-tagged Spt16; and anti-Flag antibody to detect Flag-tagged yDr1.
FIG. 9.
FIG. 9.
Siz1-Siz2 and Slx5 effects on canavanine-induced degradation. (A) Wild-type (WT), siz1Δ siz2Δ, and slx5Δ strains expressing HA-tagged Spt20 were grown and treated with 60 μg/ml canavanine (CAN) and harvested every 2 h after canavanine treatment. Crude lysates were prepared and probed with anti-HA antibody to detect Spt20 levels. HA-Spt20 was revealed as a doublet by anti-HA Western blotting, since both bands were absent in the lysates prepared from a SPT20 untagged strain. G6PDH served as a loading control. Quantification of the Western blots is shown to the right. (B) Wild-type, siz1Δ siz2Δ, and slx5Δ strains expressing HA-tagged Rco1 were grown and treated with 30 μg/ml canavanine and harvested every 30 min after canavanine treatment. Crude lysates were prepared and probed with anti-HA antibody to detect Rco1 levels. G6PDH served as a loading control.
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