Ubiquitin-dependent Proteolytic Control of SUMO Conjugates

Abstract

Posttranslational protein modification with small ubiquitinrelated modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.Small ubiquitin-related modifier (SUMO), 5 which is structurally related to ubiquitin, is conjugated posttranslationally to a large number of substrates (1-5). The enzymes mediating SUMO conjugation are similar to those that catalyze the transfer of ubiquitin (3, 4). The functions of these two modifications, however, are distinct. Posttranslational modification of proteins with certain types of ubiquitin chains serves as a secondary degradation signal that targets such proteins for degradation by the 26 S proteasome (6). SUMO modification, in contrast, is not thought to result in proteolytic targeting (1-3, 7). Among the many functions of SUMO modification are regulation of transcription, nuclear transport, formation of subnuclear structures, cell cycle progression, and DNA repair (1-5, 7-9). Several substrates can be modified either by ubiquitin or SUMO (10). Modification of PCNA on a specific Lys residue by ubiquitylation mediates DNA repair (10, 11). Sumoylation of the same Lys, in contrast, mediates interaction with the Srs2 helicase, which results in inhibition of recombination during DNA replication (12, 13). In this example, sumoylation and ubiquitylation appear to direct proliferating cell nuclear antigen into distinct functions by promoting alternative interactions. Sumoylation of I B␣ on Lys 21 has been proposed to prevent its ubiquitylation and subsequent degradation (14). It has also been shown that SUMO-1 modification of a pathogenic fragment of Huntingtin enhances stability of this fragment, thereby increasing neurodegeneration, whereas ubiquitylation reduces fragment stability (15).Several recent studies suggested that, similar to ubiquitin, SUMO can form substrate-linked chains. In Saccharomyces cerevisiae, SUMO chain formation does not appear to serve an essential function (16). The reduced ability to remov...

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

“…Based on our observations, we can infer the existence of a mechanisms that controls nucleolar localization of a subset of given nuclear proteins by their targeting to the nucleolus via transient polysumoylation and their nucleolar retention via recognition by some SUMO-binding protein(s) residing in the nucleolus. As putative SUMObinding domains have recently been identified (Hecker et al 2006;Lin et al 2006;Kerscher 2007;Sun et al 2007;Uzunova 2007), it is conceivable that some nucleolar proteins may have an ability to serve as receptors for sumoylated proteins, which hence acquire a nucleolarspecific function through such interactions. The experimental approach to modeling polysumoylation in vivo.…”

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…Importantly, for certain substrates, SUMOylation with SUMO2/3 is known to trigger the formation of long poly-SUMO chains that are recognized by ubiquitin ligases and lead to the polyubiquitinylation of the SUMOylated substrate, which in turns leads to their proteasomal degradation (71)(72)(73)(74). Data obtained during the execution of these studies revealed the formation of polySUMOylated NS1 in the presence of wt-ASL and mut-ASL (see, for example, Fig.…”

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

“…Ulp2D yeasts are unable to grow at 37 1C, likely because of a toxic accumulation of polySUMOylated proteins (Uzunova et al, 2007). We reasoned that the interaction of E1A with the N-terminus of UBC9 would block the noncovalent interaction of SUMO necessary for polySUMOylation.…”

“…We tested this directly by constructing diploid yeast expressing the dominant-negative Smt3 K11/15/19R mutant. This mutant cannot form chains, as it lacks the target lysines necessary for SUMO/Smt3 branching (Uzunova et al, 2007). Incorporation of this mutant protein into existing SUMO/Smt3 chains blocks chain extension that should effectively reproduce the consequences of expressing E1A CR2.…”

“…We also constructed a diploid yeast strain heterozygous for the gene encoding the Ulp2 Smt3/SUMO protease. This strain would be expected to accumulate polySUMOylated proteins (Uzunova et al, 2007). In this background, the ability of E1A CR2 to stimulate pseudohyphal growth was compromised (Figure 8b).…”