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 ulp2⌬ 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 DNAdamaging 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 ulp2⌬ 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.The ubiquitin system is central to many biological regulatory mechanisms, including aspects of signal transduction, cell cycle progression, differentiation, and the stress response (reviewed in references 12, 13, 19, and 38). Covalent attachment of the ubiquitin polypeptide to cellular proteins is achieved through a highly conserved enzymatic pathway. In an ATP-consuming reaction, the C terminus of ubiquitin is first activated by an enzyme called E1, to which it becomes attached by a thiolester bond. The ubiquitin is then transferred to an E2 ubiquitinconjugating enzyme. Together with an additional factor called E3, or ubiquitin-protein ligase, E2 enzymes catalyze formation of an amide (isopeptide) bond between the C-terminal carboxyl group of ubiquitin and a lysine side chain(s) of the acceptor protein. Most frequently, the modified protein is targeted to the 26S proteasome, a protease that degrades the substrate into small peptides but allows recycling of ubiquitin.Eukaryotes express a set of ubiquitin-like proteins (Ubls) that diverge significantly from ubiquitin yet in some cases are also ligated to other proteins (reviewed in references 10 and 14). A recently discovered Ubl that is ligated to cellular proteins is the vertebrate SUMO-1 protein (also called UBL1, PIC1, sentrin, SMT3C, or GMP1) (17, 30). Human SUMO-1 is only 18% identical to ubiquitin but is 48% identical to a yeast protein called Smt3 (25), and the human and yeast proteins are functional homologs. Despite its limited sequence similarity to ubiquitin, SUMO-1 shares the ubiquitin superfold, alt...

In vivo modeling of polysumoylation uncovers targeting of Topoisomerase II to the nucleolus via optimal level of SUMO modification

“…In another control experiment, we tested whether a deletion of SMT4/ULP2, the major nuclear SUMO isopepti-dase-encoding gene (Li and Hochstrasser 2000;Strunnikov et al 2001), would mimic the accumulation of sumoylated substrates in the nucleolus, similarly to Top2p-5SUMO. We constructed the Smt4 + (wild-type) and Smt4 − (smt4Δ) strains, which expressed the GFP-Smt3p fusion from the native SMT3 promoter (see "Materials and methods"), as a sole source of SUMO.…”

“…Substantial deregulation of such targeting, e.g., by over-or undersumoylation of several substrates, may result in some nucleolar structure and segregation defects (Li and Hochstrasser 2000;Strunnikov et al 2001). Our analysis of rDNA content in the top2ΔC mutant indicates that the absence of sumoylated Top2p alone leads to the loss of tandem rDNA repeats (Fig.…”

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…Unlike Ub, typical SUMO modifications are believed to be monomeric although SUMO chains can form in vitro and in vivo [1,6]. Sumoylation is also reversible due to the activity of the SUMO-specific isopeptidases Ulp1 and Ulp2/Smt4 [6][7][8][9].…”

“…Rather than being perfectly circular, these colonies have uneven edges due to an unstable population of enlarged slow-growing cells that exhibit clonal lethality [7,24]. The phenotypes of these cells are exacerbated at low temperature and include a delay in the G2/M phase of the cell cycle [22,23,25].…”

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

“…In addition to the classical UBP/USP (ubiquitin-specific protease) and UCH (ubiquitin C-terminal hydrolase) enzymes, there are at least three distinct groups of enzymes capable of removing ubiquitin or a related UBL (e.g., SUMO) from a protein substrate. Proteases specific for SUMO conjugates include yeast Ulp1 and Ulp2 [9,10] and human SENP1 and SENP2 [11,12]. Other known ULPs include UBP43, which cleaves ISG15 conjugates [13], and DEN1 (SENP8), which deconjugates Nedd8 from protein substrates [14,15].…”