The SUMO Isopeptidase Ulp2 Prevents Accumulation of SUMO Chains in Yeast

Abstract

The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wildtype strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2⌬, consistent with the possibility that some ulp2⌬ phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitinlike domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae.SUMOs are ubiquitin-related proteins that function by being covalently attached to other proteins as post-translational modifications. SUMO conjugation is essential for viability of most eukaryotic cells and participates in many cellular processes including transcription, DNA repair, chromatin organization, nuclear transport, signal transduction, and the cell cycle (1-3). SUMO conjugation acts by different mechanisms on different substrates but does not directly target proteins for proteasome-dependent proteolysis.SUMO, like ubiquitin (Ub), 1 is linked to its substrates via an amide bond between its C-terminal carboxyl group and the ⑀-amino group of a lysine residue in the substrate (1-3). SUMOs share only ϳ18% sequence identity with Ub but contain a C-terminal domain with a Ub-fold that is virtually superimposable on the structure of Ub. SUMOs also contain a ϳ20-residue non-Ub-related N-terminal extension. Saccharomyces cerevisiae contains a single SUMO protein encoded by the SMT3 gene, while mammals contain three different SUMOs: SUMO-2 and SUMO-3, which are 95% identical to each other, and SUMO-1. SUMO attachment is catalyzed by a three-step enzyme pathway, analogous to the Ub pathway, consisting of the heterodimeric SUMO-activating enzyme (E1) Uba2⅐Aos1, the SUMO-conjugating enzyme (E2) Ubc9, and several different SUM...

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

“…In yeast, the Ulp2 SUMO/Smt3 protease counteracts polySUMOylation (Schwienhorst et al, 2000;Bylebyl et al, 2003). Ulp2D yeasts are unable to grow at 37 1C, likely because of a toxic accumulation of polySUMOylated proteins (Uzunova et al, 2007).…”

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

“…Sumoylation normally takes place at lysine residues that fall within the consensus sequence ΨKXE/D, where Ψ is a hydrophobic residue. 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].…”

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications

“…One of these peptide spectra indicated unambiguous acetylation of Lys6. The sequence surrounding K6/7 bears some similarity to the N-terminal extension of yeast SUMO, which is itself sumoylated (Bylebyl et al 2003). In H2B this sequence with surrounding alanines (AEKKPA) is repeated, generating another putative sumoylation/acetylation site at Lys16 and Lys17 (K16/17).…”