Saccharomyces cerevisiae contains two Siz/PIAS SUMO E3 ligases, Siz1 and Siz2/Nfi1, and one other known ligase, Mms21. Although ubiquitin ligases are highly substrate-specific, the degree to which SUMO ligases target distinct sets of substrates is unknown. Here we show that although Siz1 and Siz2 each have unique substrates in vivo, sumoylation of many substrates can be stimulated by either protein. Furthermore, in the absence of both Siz proteins, many of the same substrates are still sumoylated at low levels. Some of this residual sumoylation depends on MMS21. Siz1 targets its unique substrates through at least two distinct domains. Sumoylation of PCNA (proliferating cell nuclear antigen) and the splicing factor Prp45 requires part of the N-terminal region of Siz1, the `PINIT' domain, whereas sumoylation of the bud neck-associated septin proteins Cdc3, Cdc11 and Shs1/Sep7 requires the C-terminal domain of Siz1, which is also sufficient for cell cycle-dependent localization of Siz1 to the bud neck. Remarkably, the non-sumoylated septins Cdc10 and Cdc12 also undergo Siz1-dependent sumoylation if they are fused to the short ΨKXE SUMO attachment-site sequence. Collectively, these results suggest that local concentration of the E3, rather than a single direct interaction with the substrate polypeptide, is the major factor in substrate selectivity by Siz proteins.
Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1⌬ siz2⌬ double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-typeappearing cells as well as sectors of enlarged cells that are arrested in G 2 /M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2m circle. siz1⌬ siz2⌬ mutants have up to 40-fold-higher levels of 2m than do wild-type strains. Furthermore, 2m is responsible for the siz1⌬ siz2⌬ mutant's obvious growth defects, as siz1⌬ siz2⌬ [cir 0 ] strains, which lack 2m, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2m are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2m, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2m copy number in the siz1⌬ siz2⌬ strain.The ubiquitin (Ub)-related protein SUMO (small ubiquitinrelated modifier) functions by being covalently attached to other proteins as a posttranslational modification (12,20,28). Many proteins with diverse cellular functions are modified by SUMO, including at least 300 proteins in Saccharomyces cerevisiae (6,13,30,41,46). SUMO conjugation is essential for viability in S. cerevisiae, and sumoylation-defective conditional mutants arrest in the cell cycle at G 2 /M. However, it is not known which substrates are involved in the essential function(s) of SUMO. SUMO conjugation is also essential in most other eukaryotic cells.SUMOs are a family of ϳ93-to 98-amino-acid proteins that are ϳ18% identical to Ub, a 76-residue modifier protein with several functions including targeting proteins for proteasomedependent proteolysis (15, 31). Like Ub, SUMO is attached to lysine residues in substrate proteins through an amide bond linking the C terminus of SUMO to the ε-amino group of the lysine residue. SUMO is often attached to the lysine in the sequence motif ⌿KXE, where ⌿ is a hydrophobic residue. However, on some substrates, SUMO is attached to lysine residues in sequences that do not match this motif (20). SUMO conjugation can have a variety of different effects, including modulating protein-protein interactions, altering enzymatic activity, or blocking ubiquitylation of the substrate by competing for its ubiquitylation site lysine (12,20,28). SUMO does not directly target proteins for proteasome-dependent proteolysis.SUMO is conjugated via a three-step enzyme pathway that first activates the SUMO C terminus and then modifies specific target proteins (12,20,28). This pathway consists of a heterodimeric SUMO-activating enzyme (E1) comprising Uba2 and Aos1, a SU...
Sumoylation, the process by which the ubiquitin-related SUMO protein is covalently attached to lysine side chains in other proteins, is involved in numerous processes in the eukaryotic cell, including transcriptional repression. In this study, we identify Gcn5, the histone-modifying subunit of the transcriptional regulatory complex SAGA, as a sumoylation substrate in yeast. In vitro, multiple sumoylation of recombinant Gcn5 alone or as a trimer with its interacting proteins Ada2 and Ada3 did not affect Gcn5's histone acetyltransferase (HAT) activity, suggesting that modification of Gcn5 with yeast SUMO (Smt3) may not directly regulate its HAT function. Through site-directed mutagenesis, the primary in vivo sumoylation site was identified as lysine-25, although an unsumoylatable K-to-R mutation of this residue led to no obvious in vivo effects. However, fusion of SUMO to the N-terminus of Gcn5 to mimic constitutive sumoylation resulted in defective growth on 3-aminotriazole media and reduced basal and activated transcription of the SAGA-dependent gene TRP3. Taken together with recent identification of multiple additional subunits of SAGA as sumoylated proteins in vivo, these data suggest that Gcn5 sumoylation may have an inhibitory role in transcriptional regulation.
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