Small ubiquitin-related modifier (SUMO) family proteins function by becoming covalently attached to other proteins as post-translational modifications. SUMO modifies many proteins that participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction. Reversible attachment of SUMO is controlled by an enzyme pathway that is analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate, and in many cases are not understood at the molecular level. Frequently SUMO alters interactions of substrates with other proteins or with DNA, but SUMO can also act by blocking ubiquitin attachment sites. An unusual feature of SUMO modification is that, for most substrates, only a small fraction of the substrate is sumoylated at any given time. This review discusses our current understanding of how SUMO conjugation is controlled, as well as the roles of SUMO in a number of biological processes.

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.

Covalent attachment of the ubiquitin-related protein SUMO to other proteins participates in many processes including signal transduction, transcriptional regulation, and growth control. We report the characterization of Siz1 as an E3-like factor in the SUMO pathway. Siz1 is required for SUMO attachment to the S. cerevisiae septins in vivo and strongly stimulates septin sumoylation in vitro. Siz1 and the related protein Siz2 promote SUMO conjugation to different substrates at different stages of the cell cycle and, together, are required for most SUMO conjugation in yeast. Siz1, Siz2, and the PIAS (protein inhibitor of activated STAT) proteins form a conserved family defined by an unusual RING-related motif. Our results suggest that this family functions by promoting SUMO conjugation to specific substrates.

Smt3p and SUMO-1 contain Ub-like domains with much less sequence similarity to Ub than among genuine Ub SMT3 is an essential Saccharomyces cerevisiae gene homologs. Most of these, including the S.cerevisiae encoding a 11.5 kDa protein similar to the mammalian nucleotide excision repair protein Rad23p (Watkins et al., ubiquitin-like protein SUMO-1. We have found that 1993) and Dsk2p, involved in spindle pole body duplicSmt3p, like SUMO-1 and ubiquitin, can be attached ation (Biggins et al., 1996), do not become conjugated to to other proteins post-translationally and have characother proteins. The only Ub-like proteins other than terized the processes leading to the activation of the SUMO-1 shown to form conjugates are the interferonSmt3p C-terminus for conjugation. First, the SMT3 inducible protein UCRP (Loeb and Haas, 1992) and a translation product is cleaved endoproteolytically to baculovirus Ub variant (Haas et al., 1996). One distinexpose Gly98, the mature C-terminus. The presence of guishing feature of Ub-like proteins capable of conjugation Gly98 is critical for Smt3p's abilities to be conjugated is likely to be a diglycine sequence corresponding to the to protein substrates and to complement the lethality C-terminal Gly75 and Gly76 of Ub, which is critical for of a smt3Δ strain. Smt3p undergoes ATP-dependent interactions between Ub and Ub-pathway enzymes. Like activation by a novel heterodimeric enzyme consisting SUMO-1, UCRP and the baculovirus Ub variant, Smt3p of Uba2p, a previously identified 71 kDa protein similar contains these glycine residues. to the C-terminus of ubiquitin-activating enzymes Post-translational attachment of Ub to other proteins is (E1s), and Aos1p (activation of Smt3p), a 40 kDa carried out by a multi-step pathway culminating in formprotein similar to the N-terminus of E1s.

We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process. The S. cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome. Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes. We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1p's function in proteasome maturation.

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G2/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.

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...

Although the modification of cellular factors by SUMO is an essential process in Saccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes.Posttranslational modification of proteins is a central mechanism by which biological processes are regulated. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO 1 (Smt3p in budding yeast) to different cellular substrates (1, 2). SUMO conjugation is carried out by a multistep enzymatic pathway consisting of the heterodimeric SUMO-activating enzyme Aos1p/Uba2p, the SUMO-conjugating enzyme Ubc9p, and several different SUMO ligases including Siz1p and Siz2p in yeast (3-7). The end result of this enzymatic cascade is a covalent isopeptide bond linking the C-terminal glycine of SUMO to the ⑀-amino group of specific lysines in the target protein. Sumoylation of a target protein in this manner can regulate protein function by a number of different mechanisms including altering its subcellular localization, modulating its interaction with other proteins, or by antagonizing its attachment to ubiquitin or other lysine-targeting modifications. Desumoylation of substrates by SUMO isopeptidases, such as Ulp1p and Ulp2p in yeast, is also a central feature in regulation of protein function by . Genetic data in both budding and fission yeast, as well as the variety of known SUMO targets in mammalian cells, point to a broad role for sumoylation and desumoylation in the regulation of many biological processes including transcription, cell cycle progression, DNA damage response, and signal transduction (11,12).It is known that a large number of proteins are sumoylated in Saccharomyces cerevisiae, but very few of these substrates have been identified to date. This is thought to be due to the low abundance of the sumoylated targets and the fact that only a small fraction of a substrate is sumoylated under a given set of conditions. A critical step in the effort to gain a more complete picture of the role of SUMO in eukaryotic biology will be the identification and characterization of a wider range of SUMO targets. To this end, we have performed a global proteomics analysis of sumoylation in budding yeast. EXPERIMENTAL PROCEDURESYeast Strains-EJ337 contains at the chromosomal SMT3 locus a vers...