The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein

Abstract

Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. Like SMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1(ts) strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Delta mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of a ulp1(ts) mutation or overproduction of catalytically inactive Ulp1 can substantially overcome the ulp2Delta defects. Inactivation of Ulp2 also suppresses several ulp1(ts) defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

FIG. 1

Yeast Ulp enzymes. (A) UD in S. cerevisiae Ulp1 and Ulp2. Positions of the catalytic His (H) and Cys (C) residues and of the Ulp2-specific insertion in the UD (solid bar) are indicated. (B) Sequence alignment of the UDs of yeast Ulp1 and Ulp2. The arrowheads mark presumptive catalytic residues; black and grey boxes mark identical and similar residues, respectively.

FIG. 2

S. cerevisiae Ulp2 is an Smt3-cleaving enzyme. (A) Cleavage of radiolabeled His₆-ubiquitin-Smt3-HA by GST-Ulp2 and yeast Ubp1 analyzed after 2 h at 30°C by SDS-polyacrylamide gel electrophoresis (12.5% gel). Left lane, no added enzyme. Inhibitor preincubations were done for 15 min at room temperature. Ald, ubiquitin aldehyde; −, absent. (B) In vitro cleavage by purified GST-Ulp2 or GST-Ulp1 of yeast Smt3-protein conjugates. The blotting conditions did not allow visualization of free Smt3. NEM-treated extracts (25 μg) from ulp2Δ cells grown at 23°C (two left lanes) or 37°C (three right lanes) are shown. A species of ∼85 kDa was reproducibly enhanced following Ulp2 digestion in vitro; this might represent a multiply modified protein from which not all Smt3 molecules were cleaved. A rabbit anti-Smt3 antibody was used for immunoblot analysis. In the lower gel, filters were reprobed with anti-PGK (3-phosphoglycerate kinase) to compare protein loadings. The positions of molecular mass markers are indicated in each panel (in kilodaltons).

FIG. 3

Growth of ulp2Δ cells. (A) Tetrad analysis of an ulp2Δ/ULP2 heterozygote. One His⁺ segregant from the 10 tetrads shown eventually grew into a small colony. (B) Growth on rich medium at 37°C for 5 days or 30°C for 4 days of ulp2Δ cells carrying high-copy-number (HC) plasmids with the indicated genes. The Smt3 proteins were tagged with HA.

FIG. 4

Cell morphology of ulp2Δ cells. Wild-type and congenic ulp2Δ cells grown at 30°C were viewed with Nomarski optics (DIC). Mutant cells were enlarged relative to the wild type, frequently had elongated buds, and occasionally formed daisy chains. Microtubules were visualized by antitubulin immunofluorescence, and nuclei were visualized by DAPI fluorescence.

FIG. 5

Comparison of ulp2Δ, ulp1^ts, and ulp2Δ ulp1^ts mutants. Tenfold serial dilutions of logarithmically growing cultures were spotted onto YPD plates that were placed at 30°C (A) or 37°C (B). Alternatively, cells were incubated at 30°C in the presence of 15 μg of benomyl sulfate/ml (C), 0.1 M hydroxyurea (D), or 0.005% MMS (E) or they were exposed to 1.3 J/m² of UV radiation (F) or 200 kilorads of γ rays (G).

FIG. 6

Evaluation of checkpoint function in the ulp2Δ mutant. (A) Growth of cells after incubation for the indicated times in benomyl. The treated cells were spread on YPD plates and incubated at 30°C. Survival was calculated as the percentage of colonies formed from each time point sample relative to the number counted at the outset of the treatment for each strain. The strains used were MHY501, MHY1380, and MHY1628. (B) Growth of cells after incubation in MMS. The ulp2Δ and ubc9-1 mutants are congenic with MHY501, and the mec1 mutant MHY1621 is congenic with W303a. wt, wild type. (C) Growth of cells after incubation in hydroxyurea. (D) Normal block to spindle elongation in ulp2Δ cells exposed to 0.1 M hydroxyurea. Cell budding and spindles were evaluated from micrographs of anti-tubulin-stained cells. The mean numbers of cells counted for each time point were 51 and 68 for MHY501 (wild type) and MHY1380 (ulp2Δ), respectively. (E) Abnormal spindle elongation in the mec1 checkpoint mutant evaluated as for panel D. The mean numbers of cells counted for each time point were 56 and 44 for W303a (wild type) and MHY1621 (mec1), respectively. (F) Average number of cell bodies (cells plus buds) observed in microcolonies that initiated from single cells of the indicated genotype, which were isolated from an α-factor synchronized cell population. Strains were isogenic except as indicated (11). Plates contained 2% galactose. (G) Inhibition of colony formation of ulp2Δ cells overexpressing MPS1.

FIG. 6

Evaluation of checkpoint function in the ulp2Δ mutant. (A) Growth of cells after incubation for the indicated times in benomyl. The treated cells were spread on YPD plates and incubated at 30°C. Survival was calculated as the percentage of colonies formed from each time point sample relative to the number counted at the outset of the treatment for each strain. The strains used were MHY501, MHY1380, and MHY1628. (B) Growth of cells after incubation in MMS. The ulp2Δ and ubc9-1 mutants are congenic with MHY501, and the mec1 mutant MHY1621 is congenic with W303a. wt, wild type. (C) Growth of cells after incubation in hydroxyurea. (D) Normal block to spindle elongation in ulp2Δ cells exposed to 0.1 M hydroxyurea. Cell budding and spindles were evaluated from micrographs of anti-tubulin-stained cells. The mean numbers of cells counted for each time point were 51 and 68 for MHY501 (wild type) and MHY1380 (ulp2Δ), respectively. (E) Abnormal spindle elongation in the mec1 checkpoint mutant evaluated as for panel D. The mean numbers of cells counted for each time point were 56 and 44 for W303a (wild type) and MHY1621 (mec1), respectively. (F) Average number of cell bodies (cells plus buds) observed in microcolonies that initiated from single cells of the indicated genotype, which were isolated from an α-factor synchronized cell population. Strains were isogenic except as indicated (11). Plates contained 2% galactose. (G) Inhibition of colony formation of ulp2Δ cells overexpressing MPS1.

FIG. 7

Smt3-protein conjugates in ulp2Δ cells. (A) Effect on Smt3-protein conjugates of overproduction of Ulp2 or Ulp1. vector, pRS424; all alleles are in pRS424. The arrowheads indicate prominent Smt3 conjugates that accumulate in ulp2Δ cells. Samples were run on an SDS–7.5 to 18% polyacrylamide gradient gel followed by anti-Smt3 immunoblotting. Free Smt3 was not detected under the blotting conditions used. (B) Cleavage of Smt3-protein conjugates in yeast extracts. Spheroplasts were incubated at 37°C for 30 min and then lysed with (+) or without (−) NEM. After 30 min at 30°C, 90 μg of protein from each sample was loaded on a 10 to 15% gradient gel, followed by immunoblot analysis. The asterisk and bracket denote Smt3 conjugates from ulp1^ts cells that, unlike most of the conjugates, disappeared during in vitro incubation without NEM. (C) Smt3-precursor processing analyzed by anti-Smt3 immunoblotting with enhanced chemiluminescent detection. The strains used were MHY1614 to MHY1617 that expressed SMT3 from a plasmid-borne CUP1 promoter.

FIG. 8

Localization of Ulp1 and Ulp2. (A) Anti-myc immunoblot analysis of myc9-tagged Ulps. Lane 1, untagged control MHY500 cells. (B) Indirect immunofluorescence localization of tagged Ulps. The nuclei were visualized with DAPI.

FIG. 9

Smt3 processing and Smt3-protein conjugates in ulp mutants. (A) Suppression of ulp2Δ temperature sensitivity by catalytically defective Ulp1 mutants. (B) Smt3-protein conjugates in ulp mutants at 30°C. The strains used were MHY1614 to MHY1617. (C) Reciprocal suppression of ulp2Δ and ubc9-1 growth defects. (D) Provision of additional mature Smt3 does not impair the suppression of benomyl sensitivity in the ulp2Δ ulp1^ts mutant. The YPD plates included 100 μM CuSO₄ to induce His₆-Smt3 expression from the YRTAG310-His6-SMT3-gg plasmid.