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...

We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMObinding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO-dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.

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Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca−/− mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect ofFanca−/−progenitors, either when total BM or when purified Lin−Sca-1+ cells were subjected to in vitro stimulation. Liquid cultures ofFanca−/−BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca−/−progenitors, purified Lin−Sca-1+ cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCAgenes. Lin−Sca-1+ cells fromFanca−/−mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors ofFanca−/−mice.