Misregulation of 2μm Circle Copy Number in a SUMO Pathway Mutant

Abstract

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...

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…The nibbled colony morphology of slx5Δ and slx8Δ mutants is reminiscent of that seen in SUMO mutants where it is known to be caused by overreplication of the 2μ circle plasmid (2μ) [24][25][26][27]. To determine whether 2μ was responsible for this phenotype in slx5Δ and slx8Δ mutants, we cured the cells of the plasmid by expressing a step-arrest mutant of the Flp recombinase [33].…”

“…Rather than being perfectly circular, these colonies have uneven edges due to an unstable population of enlarged slow-growing cells that exhibit clonal lethality [7,24]. The phenotypes of these cells are exacerbated at low temperature and include a delay in the G2/M phase of the cell cycle [22,23,25]. It has recently been shown that the nibbled phenotype of sumoylation mutants occurs due to over-replication of the 2μ circle which is normally an innocuous plasmid found in most laboratory strains of Saccharomyces [24][25][26].…”

Molecular Circuitry of the SUMO (Small Ubiquitin-like Modifier) Pathway in Controlling Sumoylation Homeostasis and Suppressing Genome Rearrangements

“…All tagged MCM strains and the indicated mutations were integrated into the chromosomal locus of the gene of interest (supplemental Table 1) using standard yeast genetic methods unless otherwise noted. The yeast strains used here also have their 2-m circles removed, according to a previous study (24). Fluctuation analysis of GCRs was performed as previously described (25).…”

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

“…The natural substrates of Slx5⅐Slx8-dependent ubiquitylation are unknown. The siz1⌬ siz2⌬ double mutant, slx5⌬, slx8⌬, ulp1, and certain NPC mutants accumulate as much as 10-fold higher than wt levels of 2 m DNA (Zhao et al, 2004;Chen et al, 2005;Dobson et al, 2005;Burgess et al, 2007;Ii et al, 2007b). A potential mechanism for SUMO's role in 2 m stability is suggested by the fact that Flp recombinase is modified by SUMO at a fairly high level (ϳ10%; Chen et al, 2005).…”