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...

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1D siz2D mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1D siz2D rad52D are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60D and nup133D, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1D siz2D mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1D cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.

The Hedgehog (Hh) receptor Patched-1 (PTCH1) opposes the activation of Gli transcription factors and induces cell death through a Gli-independent pathway. Here, we report that the C-terminal domain (CTD) of PTCH1 interacts with and is ubiquitylated on K1413 by the E3 ubiquitin-protein ligase Itchy homolog (Itch), a Nedd4 family member. Itch induces the ubiquitylation of K1413, the reduction of PTCH1 levels at the plasma membrane, and degradation, activating Gli transcriptional activity in the absence of Hh ligands. Silencing of Itch stabilizes PTCH1 and increases its level of retention at the plasma membrane. Itch is the preferential PTCH1 E3 ligase in the absence of Hh ligands, since of the other seven Nedd4 family members, only WW domain-containing protein 2 (WWP2) showed a minor redundant role. Like Itch depletion, mutation of the ubiquitylation site (K1314R) resulted in the accumulation of PTCH1 at the plasma membrane, prolongation of its half-life, and increased cell death by hyperactivation of caspase-9. Remarkably, Itch is the main determinant of PTCH1 stability under resting conditions but not in response to Sonic Hedgehog. In conclusion, our findings reveal that Itch is a key regulator of ligand-independent Gli activation and noncanonical Hh signaling by the governance of basal PTCH1 internalization and degradation.