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...

Posttranslational modification of proteins with the small ubiquitin-related modifier (SUMO) has been implicated in many important physiological functions, including the regulation of transcription and DNA repair. In most cases, only a small fraction of the total cellular amounts of a given protein is sumoylated at a certain point in time. Sensitive detection of sumoylated forms of proteins by western blotting is, therefore, an important step in the identification and/or characterization of a protein control by sumoylation. Polysumoylated proteins are recognized and targeted to the proteasome by specific ubiquitin ligases bearing SUMO interaction motifs. Sumoylation itself is reversible by the action of desumoylating enzymes. Their activities cause a rapid loss of SUMO conjugates in most standard cell extracts. To preserve SUMO-protein conjugates, therefore, a preparation of extracts under denaturing conditions is recommended. Here, we describe the application of an alkaline lysis procedure and a western blot protocol for the analysis of SUMO conjugates in yeast and human cells. In addition, we describe the application of another extraction procedure combined with immobilized metal affinity chromatography for the analysis of ubiquitin-SUMO hybrid conjugates from yeast and human cells.