Multiple domains in Siz SUMO ligases contribute to substrate selectivity.

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\u27 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 PsiKXE 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

The role of Schizosaccharomyces pombe SUMO ligases in genome stability

SUMOylation is a post-translational modification that affects a large number of proteins, many of which are nuclear. While the role of SUMOylation is beginning to be elucidated, it is clear that understanding the mechanisms that regulate the process is likely to be important. Control of the levels of SUMOylation is brought about through a balance of conjugating and deconjugating activities, i.e. of SUMO (small ubiquitin-related modifier) conjugators and ligases versus SUMO proteases. Although conjugation of SUMO to proteins can occur in the absence of a SUMO ligase, it is apparent that SUMO ligases facilitate the SUMOylation of specific subsets of proteins. Two SUMO ligases in Schizosaccharomyces pombe, Pli1 and Nse2, have been identified, both of which have roles in genome stability. We report here on a comparison between the properties of the two proteins and discuss potential roles for the proteins

Cdk1 and SUMO Regulate Swe1 Stability

The Swe1/Wee1 kinase phosphorylates and inhibits Cdk1-Clb2 and is a major mitotic switch. Swe1 levels are controlled by ubiquitin mediated degradation, which is regulated by interactions with various mitotic kinases. We have recently reported that Swe1 levels are capable of sensing the progress of the cell cycle by measuring the levels of Cdk1-Clb2, Cdc5 and Hsl1. We report here a novel mechanism that regulates the levels of Swe1. We show that S.cerevisiae Swe1 is modified by Smt3/SUMO on residue K594 in a Cdk1 dependant manner. A degradation of the swe1K594R mutant that cannot be modified by Smt3 is considerably delayed in comparison to wild type Swe1. Swe1K594R cells express elevated levels of Swe1 protein and demonstrate higher levels of Swe1 activity as manifested by Cdk1-Y19 phosphorylation. Interestingly this mutant is not targeted, like wild type Swe1, to the bud neck where Swe1 degradation takes place. We show that Swe1 is SUMOylated by the Siz1 SUMO ligase, and consequently siz1Δ cells express elevated levels of Swe1 protein and activity. Finally we show that swe1K594R cells are sensitive to osmotic stress, which is in line with their compromised regulation of Swe1 degradation

Functional Reconstitution of a Tunable E3-Dependent Sumoylation Pathway in Escherichia coli

SUMO (small ubiquitin-related modifier) is a reversible post-translational protein modifier that alters the localization, activity, or stability of proteins to which it is attached. Many enzymes participate in regulated SUMO-conjugation and SUMO-deconjugation pathways. Hundreds of SUMO targets are currently known, with the majority being nuclear proteins. However, the dynamic and reversible nature of this modification and the large number of natively sumoylated proteins in eukaryotic proteomes makes molecular dissection of sumoylation in eukaryotic cells challenging. Here, we have reconstituted a complete mammalian SUMO-conjugation cascade in Escherichia coli cells that involves a functional SUMO E3 ligase, which effectively biases the sumoylation of both native and engineered substrate proteins. Our sumo-engineered E. coli cells have several advantages including efficient protein conjugation and physiologically relevant sumoylation patterns. Overall, this system provides a rapid and controllable platform for studying the enzymology of the entire sumoylation cascade directly in living cells

SUMO modification of PCNA is controlled by DNA

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and—compared with the diversity of sumoylation substrates—their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated

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

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 siz1Δ siz2Δ double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing 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 2μm circle. siz1Δ siz2Δ mutants have up to 40-fold-higher levels of 2μm than do wild-type strains. Furthermore, 2μm is responsible for the siz1Δ siz2Δ mutant's obvious growth defects, as siz1Δ siz2Δ [cir(0)] strains, which lack 2μm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2μm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2μm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2μm copy number in the siz1Δ siz2Δ strain

The Early Independent Problem Solving Survey (EIPSS): Its Psychometric Properties in Children Aged 12-47 Months

Independent problem solving (IPS) involves solving problems alone; with motivation and persistence; without watching others; or requesting or receiving help. The Early Independent Problem Solving Survey (EIPSS) was developed for 12- to 47-month-olds. Study 1 (N = 272) found good internal reliability and a 2-factor structure: Repetitive (repeatedly solvable problems, e.g., jigsaws) and Novel IPS (one-off problems, e.g., reaching out-of-reach toys). Study 2 (N = 567) confirmed good internal reliability and the 2-factor structure. Study 3 (N = 85) found a positive correlation between a divergent thinking lab measure and Novel IPS. Study 4 found good 6-month-longitudinal stability (N = 110) for the EIPSS and its subscales; and good agreement between parents (N = 32) for the Repetitive subscale. Study 5 (all data combined) demonstrated no item functioning differences across demographic variables. Differences for child age, child gender, parent age, and parent education were found for the EIPSS and subscales

Evaluation of the effects of ultrafiltration on the quality and stability of pre-filtered and pasteurized Philrice tapuy

Tapuy is a Philippine rice wine originating from Batad, Ifugao made from interaction of microorganisms found in a culture called bubod and rice. Compounds responsible for flavor are produced by the decomposition of proteins by proteases, which are produced by some microorganisms from bubod, and the formation of glycerol and fatty acids by the yeasts. However, the proteins cause turbidity and discoloration due to protein instability, and the development of a patis-like off-flavor and aroma during storage. The Philippine Rice Research Institute (PhilRice) conducted extensive research to significantly improve the quality of this product and was successful in extending its shelf life from less than a week to about six months. However, persistent darkening and development of patislike off-flavor and aroma still existed after six months of storage. With the use of membrane technology, specifically ultrafiltration, these problems can be addressed by removing the compounds that cause the darkening and off-flavor development of the stored tapuy. Pre-filtered and pasteurized batches of tapuy were passed through ultrafiltration membranes with different molecular weight cut-offs. The performance of each membrane, and its effect on the physicochemical properties and stability of the wine were evaluated. It was found that the ultrafiltration process did not affect most of the physicochemical properties, except for the protein and polyphenol contents. Flux decline was also shown to increase with increasing molecular weight cut-off for the pasteurized wine. It was concluded that ultrafiltration is a viable option for the production of tapuy