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In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO) protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood.
Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3)(C580S), that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3)(C580S) to purify Smt3-modified proteins from cell extracts.
Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3)(C580S) interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.
Cell division is a fundamental feature of all life and involves the controlled duplication and faithful segregation of an organism's genetic material from one cell to the next. In eukaryotes, each cell division cycle is therefore executed as a tightly regulated, stepwise program that relies on intact chromosomes. In humans, the consequences of faulty chromosome segregation and the inability to repair DNA damage have been implicated in cancer, aging and congenital birth defects.
Ubiquitin and small ubiquitin-like modifier (SUMO), two small proteins that can become attached to other cellular proteins in a reversible manner , control important aspects of the cell division program. Ubiquitin is best known for its role in the targeted, proteasome-mediated destruction of proteins, including key cell-cycle regulators, but also holds nonproteolytic functions . Sumoylation, on the other hand, does not directly target proteins for degradation. Rather, modification of proteins with SUMO has been shown to modulate various cellular processes, including cell-cycle regulation, transcriptional activation, nucleocytoplasmic transport, DNA replication and repair, chromosome dynamics, apoptosis, ribosome biogenesis, and the formation of nuclear bodies . Additionally, an unexpected role of SUMO in protein ubiquitination has been uncovered. Briefly, degradation of several nuclear proteins, including some that are involved in DNA repair and transcriptional regulation, are preceded by modification with SUMO. These sumoylated proteins are recognized by SUMO-targeted ubiquitin ligases (STUbLs), which mediate their ubiquitination .
SUMO proteins are highly conserved from yeast to humans. Yeast cells express one SUMO protein (Smt3), and vertebrates express three isoforms (SUMO1, SUMO2 and SUMO3) . SUMO2, SUMO3 and yeast Smt3 can form SUMO chains. SUMO1, on the other hand, lacks the internal lysine required for polymerization and may function as a chain terminator for SUMO2 and SUMO3 chains . All SUMO variants are conjugated to lysine residues of specific proteins, but only a fraction of these target proteins are modified with SUMO at any given time [7, 8]. In metazoans, the dysregulation of sumoylation adversely affects developmental processes and has been implicated in the progression of neurodegeneration, cancer and infectious diseases [9, 10]. More than 1, 000 sumoylated proteins have been identified in yeast and humans, but only in a few cases has the role of sumoylation been studied in detail .
In the budding yeast Saccharomyces cerevisiae, the ligation of SUMO to specific substrate proteins requires an E1 heterodimer (Aos1 and Uba2) that activates SUMO, as well as E2 (Ubc9) and E3 (Siz1, Siz2, and Mms21) enzymes that aid in the conjugation and ligation of SUMO to proper target proteins . Two yeast SUMO proteases, Ulp1 and Ulp2, contain a conserved cysteine protease domain that can remove the SUMO moiety from modified proteins. Recent evidence suggests that Ulp2, and its mammalian orthologs Susp1/SENP6 and SENP7, play a role in the removal of SUMO and SUMO chains from nuclear proteins [12–16]. Ulp1, on the other hand, has two contrasting cellular functions. Ulp1 facilitates sumoylation by processing precursor SUMO into its conjugation competent form. Conversely, Ulp1 also facilitates desumoylation by removing SUMO from nuclear and cytosolic proteins after conjugation . Therefore, impairment of Ulp1 results in the accumulation of SUMO conjugates and the inability to carry out de novo sumoylation. The resulting lack of mature SUMO has been shown to adversely affect cellular DNA repair processes, the processing and export of the 60S preribosomal particle, nucleus-cytoplasm trafficking and cell viability [18–21].
The substrate specificity of SUMO proteases is at least in part regulated through their localization . For example, certain yeast (Ulp2) and vertebrate (SENP6 and SENP7) SUMO proteases localize within the nucleus. In contrast, both yeast (Ulp1) and vertebrate (SENP1 and SENP2) SUMO proteases reside at the nuclear envelope (NE) through their interactions with the nuclear pore complex (NPC) [23–26]. Distinct domains have been identified that are required for Ulp1 NPC localization (amino acid residues 1 to 403) and SUMO processing (amino acid residues 404 to 620) [25–28]. The Ulp1 localization domain promotes interaction with karyopherins, which are soluble proteins that mediate transport across the NE and help localize Ulp1 to the nucleoplasmic side of the NPC. The Ulp1 localization domain can be subdivided into region 1 (Kap121-binding domain) and region 2 (Kap60- and Kap95-binding domain). Juxtaposed to the NPC localization domain of Ulp1 is a coiled-coil (cc) domain with a putative nuclear export signal and region 3, the catalytically active, conserved ubiquitin-like protease domain (UD) of Ulp1 [25–27]. Only regions 1 and 2 are involved in Ulp1 localization to the NPC, and the karyopherins seem to play a redundant role. NPC association of Ulp1 requires several proteins, including the nucleoporins Nup60 and Nup84, the silencing protein Esc1 and the myosin-like proteins Mlp1/2 [18, 19, 28]. Together these proteins may provide a scaffold for the functional regulation and substrate access of Ulp1 at the NPC.
The identification of NPC localization domains in Ulp1 has done little to aid our understanding of how SUMO proteases are targeted to their respective substrates . One possibility is that SUMO proteases may contain structural features which allow for noncovalent interactions with SUMO and SUMO-modified proteins as they enter the nucleus. Indeed, conserved SUMO-interacting motifs (SIMs) have been predicted to be localized in the yeast SUMO protease Ulp2, as well as in mammalian SENP1, SENP2, SENP6 and SENP7 [14, 22, 29, 30]. Even though SIMs have not been identified in Ulp1, the crystal structure of the catalytic domain (region 3) bound to Smt3 reveals that both proteins interact through multiple residues that are distributed across a SUMO-binding surface (SBS) on the SUMO protease . Only the carboxy terminus of bound Smt3 is inserted into a hydrophobic tunnel that leads toward Ulp1's active site. SUMO processing and deconjugation require an active site cysteine residue that resides at the end of this tunnel (see Additional files 1 and 2). It has been suggested that this configuration may allow for the accommodation of many different sumoylated proteins as well as SUMO precursors .
Ulp1 and several other SUMO proteases play important roles in mitosis [17, 32]. In budding yeast, loss of Ulp1-mediated desumoylation leads to cell-cycle progression defects and cell death . This observation suggests that Ulp1 plays a key role in the sumoylation dynamics of important cell-cycle regulatory proteins. Though these cell-cycle-specific targets have eluded identification, several nuclear and cytosolic proteins involved in DNA replication and mitosis have been identified as Ulp1 desumoylation substrates [33–35]. How the NPC-localized Ulp1 is targeted to these mitotic substrates, especially those that are localized in the cytosol, is not entirely clear. In budding yeast, the NE does not break down during mitosis, and access to cytosolic desumoylation targets is therefore not automatic. It has been reported that during mitosis, Kap121 blocks Ulp1's access to its NPC-binding site and thus promotes an interaction of Ulp1 with septins . A deletion mutant of Ulp1 lacking region 2 (Δ2), the Kap60- and Kap95-binding domain, has previously been shown to localize to septins in a Kap121-dependent manner . Curiously, it has recently been demonstrated that region 2 also plays a role in nucleolar accumulation of Ulp1 after ethanol-induced stress .
One set of cytosolic substrates of the Ulp1 SUMO protease are the septins [27, 35]. The septins comprise an evolutionarily conserved class of GTPases that are implicated in bud-site selection, bud emergence and growth, microtubule capture and spindle positioning . Members of the septin family in yeast include Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7. These proteins are unique because they can form filaments that assemble into a ring structure and mark the site of new bud formation during cell division. At the end of mitosis, this ring separates and resembles a double-collar residing at the junction between the mother and daughter cells.
The septins Cdc3, Cdc11 and Shs1 are subject to sumoylation. Sumoylation of the septins occurs very briefly from the onset of anaphase to cytokinesis, with SUMO being attached only to the mother side of the double-septin ring collar [27, 38, 39]. Cell-cycle (G2/M) arrest with nocodazole, a microtubule depolymerizing drug, greatly increases SUMO conjugation to septins . Septin sumoylation in budding yeast is mediated by the SUMO E3 ligase Siz1 [40, 41]. During most of the cell cycle, Siz1 resides in the nucleus. However, at the M phase, Siz1 exits the nucleus to sumoylate septin proteins and possibly other cytosolic substrates . Deletion of SIZ1 from cells abolishes septin sumoylation while causing only mild growth and cell-cycle progression defects. At the end of mitosis, the septins are desumoylated by Ulp1, even though Ulp1 remains visibly enriched at the NPC [27, 35, 38].
In the current study, we focused on the SUMO protease Ulp1. As detailed above, Ulp1 resides at the inner face of the NPC. This enrichment at the NPC depends on direct interactions with karyopherins and two domains in the amino terminus of Ulp1. In theory, this localization is well-suited to give Ulp1 access to some nuclear substrates and those in transit across the NE. However, both nuclear and cytosolic desumoylation targets of Ulp1 have been identified, posing an interesting question. How is Ulp1 directed to its cytosolic desumoylation targets? To answer these questions, we sought to identify features of Ulp1 required for substrate targeting in vivo and in vitro. Herein we describe our finding that the carboxy terminus of Ulp1 affects the targeting and retention of this SUMO protease to sumoylated target proteins, including septins, at the bud-neck of dividing cells. Specifically, we show that the interaction with SUMO comprises an important aspect of the subcellular targeting of Ulp1 to these substrates. Our findings are confirmed by biochemical analyses that focus on the SUMO-binding properties of Ulp1(3)C580S, a novel truncation mutant that interacts avidly with SUMO and sumoylated proteins in vivo and in vitro. Significantly, the results of this study add important new details to our understanding of how Ulp1 interacts dynamically with its substrates and also provides potentially useful new directions for the study of Ulp1-interacting proteins.
As part of a larger study to identify how Ulp1 is targeted to its mitotic desumoylation substrates, we analyzed the localization of GFP-tagged versions of both the full-length wild-type (WT) Ulp1 and a catalytically inactive mutant of Ulp1 (Ulp1C580S) in G2/M-arrested yeast cells (see Methods and Additional file 3). The C580S mutation replaces the catalytic cysteine with a serine residue, rendering the Ulp1 SUMO protease catalytically inactive . Both fusion proteins were expressed under the control of the Ulp1 promoter on low-copy plasmids, and images were collected using a fluorescence microscope. Consistent with its localization to NPCs, WT Ulp1 stained only the NE of arrested yeast cells (Figure 1A, left). Unexpectedly, however, full-length Ulp1C580S was enriched at both the bud-neck and the NE of G2/M-arrested cells (Figure 1A, right). This bud-neck localization of Ulp1C580S is reminiscent of the localization of the septin ring. Several sumoylated septins have been shown to be Ulp1 substrates, and we show in this study that the septin Cdc3 is highly sumoylated during G2/M arrest (Figure 1B). Furthermore, a catalytically inactive Ulp1 mutant colocalizes with the septin Cdc11 in G2/M-arrested (noc) cells (Figure 1C). Therefore, Ulp1C580S resides at the bud-neck localized septin ring.
Localization of Ulp1 and the catalytically inactive Ulp1 (C580S) in dividing yeast cells. (A) Yeast cells (MHY500) were transformed either with a low-copy plasmid expressing GFP fusions of Ulp1 or with the catalytically inactive Ulp1(C580S) mutant. Representative images indicating the localization of GFP-tagged Ulp1 and Ulp1(C580S) after nocodazole-induced G2/M arrest are shown (YOK 1611 and YOK 1474). Note that only the Ulp1(C580S) mutant can be seen at the bud-neck of arrested cells. The arrowhead indicates the position of the bud-neck. (B) Confirmation of sumoylation of Cdc3 was achieved. Whole-cell extracts (WCE) from yeast cells expressing the YFP-tagged septin Cdc3 (YOK 1398) were treated with nocodazole (noc) or grown logarithmically (log) prior to preparation of WCEs. Extracted proteins were then separated on SDS-PAGE gels and probed with the JL-8 antibody (see Methods) to detect Cdc3-YFP and more slowly migrating sumoylated Cdc3-YFP adducts. The identity of sumoylated Cdc3-YFP bands was confirmed by comparing gel shift assays with untagged and FLAG-tagged Smt3 (data not shown). (C) Colocalization of Cdc3 and Ulp1 is shown. A strain coexpressing full-length Ulp1(C580S)-GFP (green) and Cdc3-CFP (red) (strain YOK 2204) was arrested in G2/M and then observed under a fluorescence microscope with the appropriate filter sets (left panel). Arrowheads indicate septin-localized, pseudocolored Ulp1-GFP (green), Cdc3-CFP (red) and the merged image (overlay). Also shown for comparison (right panel) is the colocalization of the Ulp1(3)(C580S)-GFP truncation and Cdc3-CFP (strain YOK 2205). Ulp1(3)(C580S)-GFP is described in Figure 4.
Our data suggest that introducing the C580S mutation into the catalytic domain of Ulp1 somehow alters the subcellular distribution of this SUMO protease, causing it to localize with a bud-neck-associated substrate, possibly a sumoylated septin protein. Localization changes have also been reported for catalytically inactive, substrate-trapping mutants of phosphatases that form stable complexes with their substrates in vivo .
We tested whether the C580S mutation that visually increased the ability of Ulp1 to associate with the septin ring in vivo was, in fact, SUMO-dependent. For this purpose, the Ulp1C580S construct was expressed in two Smt3 mutants (smt3-331 and smt3-R11, 15, 19) or two SUMO pathway mutants (ubc9-1, siz1Δ siz2Δ) [13, 39, 41, 44–46]. Logarithmically growing cells of each mutant were arrested in G2/M, and images were collected to assess the septin ring localization of Ulp1C580S in comparison to an SMT3 WT strain. In our analyses, we found that in the absence of both SUMO chains (in the R11, 15, 19 mutants) and a mutant SUMO protein (in the smt3-331 mutant), the localization of Ulp1C580S to the septin ring was reduced (22% in smt3-331 and 36% in smt3-R11, 15, 19) in frequency and intensity but not abolished (Figure 2). We obtained different results in the ubc9-1 strain, a mutant of the SUMO E2-conjugating enzyme which impairs SUMO conjugation, and in the siz1Δ siz2Δ strain, a SUMO E3 ligase double-mutant that lacks sumoylation of septins and many other proteins [40, 41, 44]. Consistent with a role for Smt3 in the localization of Ulp1C580S, we were unable to detect septin ring localization of Ulp1C580S in ubc9-1 and siz1Δ siz2Δ strains. However, Ulp1C580S was retained at the NE (Figure 2A). As an additional control, the septin ring localization of GFP-tagged Smt3 was undetectable in both ubc9-1 and siz1Δ siz2Δ strains (Figure 2B).
Small ubiquitin-like modifier (SUMO) is required for the localization of Ulp1 (C580S) to the septin ring. (A) The indicated mutants smt3-331, ubc9-1, smt3-R11, 15, 19, siz1Δ siz2Δ (YOK 1995, YOK 2065, YOK 1910 and YOK 2067) and a WT control strain (WT) were transformed with a plasmid expressing GFP-tagged Ulp1(C580S). Representative images indicating the localization of GFP-tagged Ulp1(C580S) after G2/M arrest are shown. The septin ring localization of Ulp1(C580S) is indicated where present (arrowheads). Note that Ulp1(C580S) failed to localize to the septin ring in SUMO-conjugating and ligating enzyme mutants (ubc9-1 and siz1Δ siz2Δ, respectively). (B) Septin ring localization of Smt3-GFP is absent in ubc9-1 and siz1Δ siz2Δ strains. Localization of Smt3-GFP was visualized in G2/M-arrested WT, ubc9-1 and siz1Δ siz2Δ strains (YOK 1857, YOK 2144 and YOK 2143) using fluorescence microscopy. Arrowheads indicate the position of the septin ring.
Smt3 conjugation is required for Ulp1 localization to the septin ring. Therefore, Ulp1 is targeted to the septin ring of dividing cells in a SUMO-dependent fashion. Our data also suggest that the formation of SUMO chains on substrates may enhance this targeting of Ulp1.
Our finding that a single point mutation in Ulp1, C580S, dramatically enhanced the localization of full-length Ulp1 to the septin ring in a SUMO-dependent fashion warranted a more detailed analysis of the targeting domains in Ulp1. Therefore, we generated a collection of GFP-tagged Ulp1 truncations and domains that were expressed under control of the Ulp1 promoter. We reasoned that the truncations and domains of Ulp1 that retained substrate targeting information would also localize to the septin ring in G2/M-arrested cells. In all, we assessed the localization of ten GFP-tagged constructs in comparison to full-length WT Ulp1 and full-length Ulp1C580S (C580S). Our choice of individual constructs was guided by previous findings that Ulp1 consists of functionally separate domains. These domains include a Kap121-binding domain with a role in septin localization (region 1), a Kap95- and Kap60-binding domain with a role in NPC anchoring (region 2), a cc domain harboring a nuclear export signal (CC) and the catalytic UD (region 3) [25–27]. Representative images of these domains and their subcellular localization are shown in Figures 3A and 3B. As previously reported, we found that the Ulp1 protein lacking region 2 (Δ2) localized to the septin ring in the majority of large-budded, arrested cells . Therefore, region 2 of Ulp1 normally antagonizes localization and/or retention at the septin ring. This result is complemented by our novel finding that the full-length Ulp1C580S localized to the septin ring in 33% of all arrested, large-budded cells (Figures 1A and 3A).
Distinct and separate Ulp1 domains are required for localization to the septin ring. (A) and (B) Left: A schematic of Ulp1 deletion and truncation mutants used in this study is shown. The length of each construct (amino acid scale 1 to 621), the individual domains of Ulp1 and pertinent amino acid changes are shown. WT: full-length Ulp1; region 1: Ulp1(1 to 150); region 2: Ulp1(151 to 340); region 3: Ulp1(341 to 621); Δ2: Ulp1 lacking region 2; C580S: catalytically inactivating mutation; D451N: deleted salt bridge with small ubiquitin-like modifier (SUMO) (YOK 1611, YOK 1474, YOK 1490, YOK 1861, YOK 1479, YOK 2016, YOK 1839, YOK 1907, YOK 1903, YOK 2203, YOK 1828 and YOK 2157). Colored letters N, S and D summarize the observed nuclear, septin and diffuse localization of the indicated constructs, respectively. SBS corresponds to a shallow SUMO-binding surface on Ulp1 [31, 57, 58]. Right: Representative images of G2/M-arrested cells expressing the GFP-tagged Ulp1 constructs shown on the left. The arrowheads indicate the fraction of cells (%) with N, S or D localization and the presence and position of septin ring-localized Ulp1 constructs. (C) Quantification of distinct subcellular localization of wild-type and mutant Ulp1 region 3 constructs. Large-budded G2/M-arrested cells were imaged to assess diffuse, nuclear or septin ring localization (n > 100).
Next we investigated other residues of Ulp1 that could affect the septin ring localization of the Ulp1C580S mutant, possibly by interfering with its targeting to sumoylated substrates. Aspartate 451 (D451) in Ulp1 is required to form an essential salt bridge with arginine 64 of Smt3 [31, 47]. Therefore, we introduced a D451N mutation into Ulp1C580S and found that it abolished the accumulation of the full-length Ulp1 double-mutant (D451N and C580S) at the septin ring (Figure 3A). This finding underscores the importance of Smt3 in targeting full-length Ulp1 to the septin ring shown in Figure 2. Additionally, it may indicate that D451 is required for targeting of sumoylated proteins and the C580S mutation is required for retention of Ulp1 at the septin ring.
Most intriguingly, we found that a truncation consisting only of region 3 with the C580S mutation (Ulp1(3)(C580S)) displayed robust septin ring localization in 59% of cells (Figures 1C, right panel, and Figure 3B). In stark contrast, regions 1 and 2 and WT region 3, lacking the C580S mutation, failed to localize to the septin ring (Figures 3A and 3B). However, in strains with diffuse Ulp1 truncations, the septin ring stays intact. Therefore, necessary and sufficient SUMO-dependent targeting information is contained in region 3 of Ulp1, but not in regions 1 and 2. The latter conclusion is confirmed by two-hybrid assays with Smt3.
The previously published cocrystal structure of Ulp1 with Smt3 (MMDB database 13315) reveals that amino acids 418 to 447 of region 3 make extensive contact with Smt3 and constitute an exposed SBS  (see also Additional files 1 and 2). The SBS is situated next to, but does not include, the critical D451 residue that contacts Smt3 [31, 47]. Additionally, deletion of this SBS in region 3 of Ulp1 abolishes the complementation of a ulp1Δ deletion mutant . In an attempt to identify critical residues in the evolutionary conserved SBS domain, we used psi-blast to compare the protein sequence of the yeast Ulp1 catalytic domain with all nonredundant protein sequences in the National Center for Biotechnology Information database for seven iterations and limited the output to the top 250 matches. Our results contained 81 different species. Among these species, 61% of the sequences were identified as verified or predicted sentrin/SUMO protease/Ulp1 genes, 24% were identified as unnamed protein products or hypothetical genes and 15% were classified as "other" (crystal structures, unanalyzed sequences and so on). The alignment of these sequences allowed us to identify areas of strong conservation (see Additional file 1). Using this approach, we identified several highly conserved residues in the SBS. However, these amino acids did not contact Smt3 in the published cocrystal structure and likely play structural roles in Ulp1 folding .
We investigated the effect of deleting the entire SBS domain on the localization of Ulp1(3)(C580S). A Ulp1(3)(C580S)SBSΔ construct did not localize to the septin ring in the majority of cells (96%). These results match those obtained by Li and Hochstrasser  using a WT Ulp1(3)ΔSBS construct (C173). We confirmed that SBSΔ and other Ulp1(3) constructs are expressed as soluble proteins, suggesting that they are not grossly misfolded. We also cloned and expressed the SBS domain as a fusion with GFP (SBS-GFP). This construct was distributed diffusely throughout the cell and failed to localize to the septin ring (Figure 3, middle). These data suggest that the SBS domain of region 3 may be required for the initial interaction with sumoylated substrates, but additional features of Ulp1 are required for targeting (D451) and retention (C580S) of this SUMO protease at the septin ring.
Next we directed our attention to the temperature-sensitive ulp1ts-333 allele. This mutant allele causes cells to arrest in mitosis and accumulates unprocessed SUMO precursor and sumoylated proteins . Our ulp1ts construct of region 3, Ulp1(3)ts, contains three mutations (I435V, N450S and I504T), and introduction of C580S into Ulp1(3)ts showed a greatly reduced incidence and intensity of septin ring localization (compare panels in Figures 3B and 3C). We noted that the (N450S) mutation in the ts construct was located next to the salt bridge-forming residue D451 described above and that both residues were highly conserved in the consensus sequence of Ulp1-like molecules (Additional file 1). This suggests that residues altered in ulp1ts-333, specifically N450, may contribute to Smt3 interaction and possibly substrate targeting. It is possible that N450S perturbs the salt-bridge interaction formed between D451 of Ulp1 and R64 of Smt3, thus reducing the interaction with Smt3 and contributing to the temperature-sensitive phenotype. In support of this hypothesis, correction of the N450S mutation in Ulp1(3)ts (S450N) partially rescued the slow growth defect of a ulp1Δ strain at 30°C and 37°C (data not shown). The effect of the ulp1ts mutation on Ulp1's ability to interact with Smt3 is explored in more detail below.
We tested which domains of Ulp1 are required for targeting and retention at the septin ring in vivo. Using our region 1 and region 2 GFP-tagged constructs (see Figure 3A), we show that septin-targeting information is not contained in the domains that are known to interact with karyopherins. The Δ2 construct recapitulates the previous finding that Kap121-binding to region 1 regulates access of Ulp1 to the septin ring. The full-length Ulp1C580S mutant reveals that a single substrate-trapping mutation in Ulp1 suffices to enrich Ulp1 at the septin ring. To show that an Smt3 interaction is required for the septin localization of Ulp1C580S, we created the double-mutant (D451N and C580S). The D451N mutation is known to destroy an essential salt bridge formed between Smt3 and Ulp1. Next, using the Ulp1(3)C580S construct, we show that the septin-targeting information is limited to region 3 of Ulp1 (Figure 3B). Further truncating Ulp1(3)C580S revealed that a previously identified SBS domain in Ulp1(3)C580S is also involved in septin targeting and retention. To test whether mutations found in region 3 of the ulp1ts mutant play a role in septin localization, we introduced three additional mutations, I435V, N450S and I504T, into Ulp1(3)C580S. This Ulp1(3)ts C580S construct showed a reduced ability to enrich at the septin ring (Figure 3C), suggesting that its ability to interact with sumoylated septins may be reduced but not abolished.
In the preceding sections, we described our identification of necessary and sufficient substrate-targeting information in the catalytic domain (region 3) of Ulp1. However, region 3 of Ulp1 may not be the only domain involved in targeting to the septin ring. Region 1 of Ulp1, the Kap121-binding domain, has previously been implicated in septin targeting. Specifically, it has been reported that Kap121 is required for targeting Ulp1 to the septin ring during mitosis . Therefore, we decided to assess the role of Kap121 in the substrate-targeting of Ulp1(3)(C580S). Specifically, we used a kap121ts mutant  to assess the septin ring-targeting of WT Ulp1, full-length Ulp1C580S and Ulp1(3)(C580S). In our analysis, we found that full-length Ulp1C580S required Kap121 function for targeting to the septin ring. At the permissive temperature (30°C), Ulp1C580S demarcated the NE and septin ring of G2/M-arrested cells. After a shift to the nonpermissive temperature, however, Ulp1C580S could no longer be detected at the septin ring (Figure 4, middle). Surprisingly, the Ulp1(3)(C580S) truncation was localized to the septin ring at the permissive and nonpermissive temperatures in a kap121ts strain. As shown herein, Ulp1(3)(C580S) resided both inside the nucleus and at the septin ring at 30°C and 37°C (Figure 4, right).
Kap121-independent small ubiquitin-like modifier (SUMO)-targeting information resides in region 3 of Ulp1. kap121ts cells were transformed with plasmids expressing GFP-tagged wild-type (WT) Ulp1, Ulp1(C580S) and Ulp1(3)(C580S) under the control of the Ulp1 promoter (YOK 1487, YOK 1488 and YOK 1944). Shown are representative images indicating the localization of GFP-tagged Ulp1 constructs in large-budded cells at 30°C and 37°C, the nonpermissive temperature for kap121-ts. Arrowheads indicate the position of septin ring-localized Ulp1 constructs.
Our data suggest that Ulp1 contains both Kap121-dependent and Kap121-independent septin ring-targeting information. The only requirement to detect full-length Ulp1 at the septin ring is the C580S mutation and functional Kap121 (Figures 1, 2 and 4). In contrast Ulp1(3)(C580S), which lacks all domains required for NPC interaction through Kap121, Kap60 and Kap95, localizes to the septin ring and inside the nucleus. This finding provides strong evidence that Kap121-independent septin ring-targeting information resides in the catalytic domain (region 3) of Ulp1.
Our finding that a single amino acid change in the catalytic domain of Ulp1 results in greatly enhanced, SUMO-dependent localization to the septin ring also prompted us to investigate the two-hybrid interactions of Ulp1 with budding yeast SUMO (Smt3-BD; Smt3 fused to the Gal4 DNA-binding domain). Full-length WT Ulp1, the full-length catalytically inactive Ulp1C580S mutant, the Ulp1 Kap121-interacting domain (region 1), the Ulp1 Kap60/Kap95-interacting domain (region 2) and the catalytic domain (region 3) failed to interact with Smt3-BD (data not shown). However, the catalytically inactive Ulp1(3)(C580S) truncation interacted reproducibly and above background with Smt3 (see Figure 5, C580S).
Distinct and separate features of Ulp1 are required for interaction with SUMO. Two-hybrid analysis of various Ulp1(3) truncation mutants (C580S: catalytically inactive; D451N: deleted salt bridge with small ubiquitin-like modifier (SUMO); ts: mutations including S450N in ulp1ts-333) with SUMO/Smt3-BD. The presence of both Smt3 (pOBD2/TRP1) and Ulp1 constructs (pOAD/LEU2) was confirmed by growth on growth media lacking tryptophan and leucine (-T-L). The interaction between Ulp1 constructs and Smt3 is shown as triplicate patches of cells on media lacking adenine (-A). See Figure 3A for a graphic representation of individual constructs.
Ulp1(3)(C580S) appears to interact only weakly with our Smt3 two-hybrid bait construct, as indicated by fewer colonies on the reporter media (Figure 5, Ade). However, such an interpretation assumes an equally available pool of both bait and prey. One possible explanation for this result is that the Ulp1(3)(C580S) two-hybrid prey construct interacts with a number of available substrates in the cell (for example, free Smt3 and other sumoylated proteins), and, as a result, this sequestration is no longer available to the Smt3 two-hybrid bait construct, thus creating the appearance of a weak interaction. We reasoned that introduction of the ulp1ts mutations could weaken the potential substrate-trapping phenotype of Ulp1(3)(C580S), making more of the pool available to engage the Smt3 bait construct. Consistent with this model, when we introduced the ulp1ts mutations into the substrate-trapping Ulp1(3)(C580S) prey construct, we observed a more robust two-hybrid interaction with the Smt3 bait (Figure 5, compare C580S and ts(C580S). It must also be noted, however, that currently we cannot fully explain the variations in our in vivo and in vitro assays used to assess ability of Ulp1(3)(C580S) to interact with Smt3.
Next we focused on the D451N mutant of Ulp1 that prevents the interaction of Ulp1 with SUMO [31, 47]. As shown above, D451N, when introduced into Ulp1(C580S), prevents localization of this construct to the septin ring (Figure 3A, C580S/D451N). Correspondingly, we found that introduction of the D451N mutation into Ulp1(3)(C580S) completely abolished the two-hybrid interaction with Smt3 (Figure 5, compare C580S, D451N/C580S and D451N). These observations provide evidence that the targeting of Ulp1 to sumoylated substrates is a closely balanced act involving both Smt3 targeting and retention.
We hypothesized that if Ulp1(3)(C580S) were to interact avidly with Smt3, this mutated moiety of Ulp1 could efficiently interact with SUMO adducts in vitro. Therefore, to test the direct interaction of Ulp1(3)(C580S) with SUMO, we fused this domain to the carboxy terminus of maltose-binding protein (MBP) and expressed the recombinant fusion protein in bacteria. Subsequently, the MBP-Ulp1(3)(C580S) fusion protein was purified from bacterial extracts and bound to amylose resin (see Methods). As a control to assess the ability of MBP-Ulp1(3)(C580S) to interact with sumoylated proteins, we also purified a second MBP-fused Ulp1(3)(C580S) construct lacking the SBS domain (3(C580S)ΔSBS).
First we determined the ability of MBP-Ulp1(3)(C580S) to affinity-purify sumoylated proteins from crude yeast cell extracts. ulp1ts-333 cells expressing FLAG-tagged SMT3 were grown to log phase prior to preparation of yeast cell extracts (see Methods). These extracts were then incubated with resin-bound MBP-Ulp1(3)(C580S), MBP-Ulp1(3)(C580S)-ΔSBS or unbound amylose resin. After washing, bound yeast proteins were eluted, separated on SDS-PAGE gels and examined by Western blot analysis with an anti-FLAG antibody. Flag-SMT3-modified proteins present in the whole-cell extracts (WCEs) (Figure 6, lane 2) could clearly be detected bound to MBP-Ulp1(3)(C580S) (lane 5) but not to the MBP-Ulp1(3)(C580S)-ΔSBS control protein (lane 4). We identified both unconjugated Flag-Smt3 proteins as well as several higher-molecular-weight adducts. These data suggest that Ulp1(3)(C580S) can efficiently bind and enrich sumoylated proteins from crude yeast cell extracts. To demonstrate the versatility of Ulp1(3)(C580S)-aided Smt3 purification, we also purified monomeric and conjugated GFP-Smt3 from yeast cells (Figure 6B). Additionally, we probed the extracts and eluted proteins shown in Figure 6B with an anti-Cdc11 antibody, which revealed the specific copurification of Cdc11 with immobilized Ulp1(3)(C580S) (Figure 6C).
The Ulp1(3) (C580S) truncation binds SUMO and SUMO-modified proteins. (A) and (B) Immobilized Ulp1(3)(C580S) was analyzed for its ability to affinity-purify Smt3 from yeast whole-cell extracts (WCEs). WCEs containing FLAG-tagged Smt3 (YOK 428) (left) or GFP-Smt3 (YOK 1857) (right) (input) were prepared under nondenaturing conditions and incubated with immobilized maltose-binding protein (MBP)-Ulp1(3)(C580S) (3(C580S)), MBP-Ulp1(3)(C580S) lacking the small ubiquitin-like modifier (SUMO)-binding surface (3(C580S)ΔSBS) or unbound resin (amylose). After washing and elution, bound Smt3 and Smt3 conjugates were detected using either anti-Flag or anti-GFP antibody. (C) Immobilized Ulp1(3)(C580S) was analyzed for its ability to affinity-purify Cdc11 from yeast WCEs. WCE containing GFP-Smt3 (YOK 1857) was prepared under nondenaturing conditions and incubated with immobilized MBP-Ulp1(3)(C580S), MBP-Ulp1(3)(C580S) lacking the SUMO-binding surface (3(C580S)ΔSBS) or unbound resin (amylose). After washing and dilution, bound Cdc11 was detected using an anti-Cdc11 antibody (Santa Cruz Biotechnology). (D) WCEs from logarithmically growing yeast cells expressing GFP-tagged Ulp1(3), Ulp1(3)(C580S) and Ulp1(3)(C580S)ΔSBS (YOK 1839, YOK 1907, YOK 1903) (input) were prepared under nondenaturing conditions. Extracts were then incubated with SUMO2 immobilized on agarose beads (Boston Biochem). After washing and elution with sample buffer, bound proteins were detected using an anti-GFP antibody. (E) SUMO2 chains (Boston Biochem) were incubated with resin-bound MBP-Ulp1(3)(C580S) or unbound resin (amylose). After washing and elution with sample buffer, bound proteins were detected using an anti-SUMO2 antibody. SUMO2 chains loading control (input). Concentrations of immobilized MBP-Ulp1(3)(C580S) and MBP-Ulp1(3)(C580S) lacking the SUMO-binding surface (3(C580S)ΔSBS) were confirmed by Coomassie staining of eluted proteins and quantitation on an Agilent 2100 Bioanalyzer (Agilent Technologies).
In the reciprocal experiment, we tested whether a GFP-tagged Ulp1(3)(C580S) construct expressed in yeast cells could bind immobilized SUMO2, which is highly conserved to yeast Smt3. In this experiment, yeast cells expressing CEN-plasmid levels of GFP-tagged Ulp1(3), Ulp1(3)(C580S) or the Ulp1(3)(C580S)-ΔSBS (see Figure 3) were grown to log phase prior to preparation of yeast cell extracts. Individual extracts were then incubated with SUMO2 immobilized on agarose beads (see Methods). After washing, bound yeast proteins were eluted, separated on SDS-PAGE gels and examined by Western blot analysis with an anti-GFP antibody. This time the GFP-tagged Ulp1(3)(C580S) could be detected in the WCEs and bound to the SUMO2 agarose (Figure 6D). In contrast, neither the WT catalytic domain of Ulp1 (Ulp1(3)) nor Ulp1(3)(C580S)(SBSΔ) was bound to SUMO2 agarose. Similarly, the Ulp1(3)(C580S) could also be purified on SUMO1 agarose (data not shown).
We also tested whether immobilized Ulp1(3)(C580S) could be used to purify SUMO chains. In this experiment, we incubated purified SUMO2 chains with our immobilized Ulp1(3)(C580S) or the unbound amylose resin. After washing, bound SUMO2 chains were eluted, separated on SDS-PAGE gels and examined by Western blot analysis with an anti-SUMO2 antibody. SUMO2 chains could clearly be detected in the input (Figure 6E, lane 2) and bound the MBP-Ulp1(3)(C580S) (lane 4), but not the resin-only control (Figure 6E, lane 3). Both lower- and higher-molecular-weight adducts of SUMO2 were purified with a preference for higher-molecular-weight chains (5-mer, 6-mer or 7-mer). These data suggest that Ulp1(3)(C580S) can efficiently bind and enrich SUMO2 chains in vitro and that the MBP fusion of Ulp1(3)(C580S) may also be useful for the purification of sumoylated proteins from mammalian cells.
STUbLs such as the yeast Slx5/Slx8 heterodimer and the human RNF4 protein efficiently ubiquitinated proteins modified with SUMO chains [49, 50]. These proteins interact with their respective sumoylated ubiquitinated targets through SIMs. STUbL reactions have been reconstituted in vitro, but the purification of target proteins modified with SUMO chains has been technically difficult or prohibitively expensive. The ability of Ulp1(3)(C580S) to interact with SUMO may therefore provide a simple way to purify a SUMO chain-modified STUbL target of choice.
To test whether Ulp1(3)(C580S) can serve as a platform to modify a purified protein with SUMO2 chains, we incubated the immobilized MBP-Ulp1(3)(C580S) with SUMO2 chains. Unbound SUMO2 chains were removed by washing. The MBP-Ulp1(3)(C580S) SUMO2 chain complex was then eluted and added into a STUbL in vitro ubiquitinated reaction containing recombinant RNF4 (K A Fryrear and O Kerscher, unpublished reagents). Proteins in the STUbL-mediated ubiquitination assay were separated on SDS-PAGE gels and examined by Western blot analysis with an anti-SUMO2 antibody (Figure 7A). Consistent with previous observations, we were able to detect ubiquitinated SUMO2 chains after the STUbL reaction. This ubiquitination was dependent on RNF4 and SUMO2 chains. On the basis of these results, we propose that Ulp1(3)(C580S) may provide a useful, widely applicable tool for the study of sumoylated proteins and STUbL targets (Figure 7B).
MBP-Ulp1(3) (C580S) can serve as a SUMO2 binding platform for SUMO-targeted ubiquitin ligase-mediated substrate ubiquitination. (A) SUMO2 chains (Boston Biochem) were incubated with resin-bound maltose-binding protein (MBP)-Ulp1(3)(C580S). The complex of MBP-Ulp1(3)(C580S) with small ubiquitin-like modifier 2 (SUMO2) chains was then eluted and added to an in vitro ubiquitination reaction with the SUMO-targeted ubiquitin ligase (STUbL) RNF4, an E3 ubiquitin ligase. Proteins in the STUbL reactions were separated by SDS-PAGE and assessed by Western blot analysis with an anti-SUMO2 antibody. Arrows indicate modified SUMO2 chains. Lane 1: No SUMO chains; lane 2: no RNF4; lane 3: no Ulp1(3)(C580S); lane 4: all reagents. (B) Proposed model for using MBP-Ulp1(3)(C580S) as a SUMO2-binding platform for substrate ubiquitination. SUMO2, ubiquitin and RNF4 are indicated by spheres labeled S, spheres labeled Ub and the gray oval labeled RNF4, respectively.
In this study, we have demonstrated that region 3 of Ulp1, the catalytic domain, contains critical information for subcellular targeting to sumoylated substrates, including the septin Cdc11. To determine how Ulp1 is targeted to its substrates, we took advantage of a catalytically inactive Ulp1 mutant (C580S) that exhibited partial redistribution from the NE to the septin ring of dividing yeast cells. We found that the relocalization of Ulp1 depended on functional Smt3 and sumoylated proteins at the septin ring of dividing cells.
Importantly, using this novel Ulp1 in vivo septin-ring localization assay, we traced the critical targeting information to two features in region 3 of Ulp1, a previously identified SBS (amino acids 418 to 447) and a SUMO-contacting residue (D451) that resides near the carboxy terminus (see structure supplied). D451 of Ulp1 has previously been shown to contact Smt3 through a salt-bridge interaction [31, 47]. Indeed, in our analysis, we provide evidence that the salt bridge-disrupting D451N mutation abolishes the SUMO-dependent targeting of Ulp1 to septins and prevents the two-hybrid interaction of the catalytic domain of Ulp1 with Smt3 (Figures 3A, B and 5).
The sole requirements for the enrichment of full-length Ulp1 at the septin ring were the catalytically inactivating C580S mutation in the catalytic domain of Ulp1 and functional Kap121. This finding has important implications for the targeting role played by the amino-terminal karyopherin-binding domains of Ulp1 (discussed below). Additionally, Smt3 processing appears to be required for substrate release. The catalytically inactive Ulp1(3)(C580S) mutant is predominantly localized to the septin ring and nucleus of dividing yeast cells, whereas the catalytically active WT Ulp1(3) shows merely diffuse staining throughout the cell (compare Figure 3B, top). This is not due to differential expression or stability of either GFP fusion, as similar amounts of proteins were detected in cell extracts (see Figure 6D). We propose and show in our biochemical analysis that the C580S mutant may trap Smt3-modified proteins, allowing it to be observed in association with cellular desumoylation substrates. In support of this assessment, combining the D451N with the C580S mutation abolishes all visible bud-neck localization (Figure 3A). Therefore, we propose that Ulp1(C580S) first targets and docks Smt3 through the SBS domain and the salt bridge-forming D451 residue, then traps it in place because of its inability to cleave after the di-glycine motif of Smt3. We can assume that a trapped substrate prevents further catalysis or interactions with other Smt3 molecules, an assessment that is borne by our finding that, despite its septin-targeting and SUMO-binding properties, Ulp1(3)(C580S) interacts only weakly with Smt3 in a two-hybrid assay (Figure 5 and Additional file 4). A better understanding of how the sumoylated substrate is trapped by Ulp1(3)(C580S) may have important implications for the rational design of inhibitors for Ulp1-like SUMO proteases, but may await the elucidation of the cocrystal structure with a trapped substrate.
The interaction of budding yeast Ulp1 with Smt3 relies on multiple hydrophobic and salt-bridge interactions between the catalytic domain (region 3) of Ulp1 and the carboxy-terminal extension of Smt3. By making multiple contacts with Smt3, Ulp1 is particularly well-suited to interact with a wide variety of sumoylated substrates [22, 31]. Other SUMO proteases, Ulp2 and several SENP proteins (Senp1, Senp2, Senp6 and Senp7), are believed to interact noncovalently with their sumoylated substrates through dedicated SIMs [14, 22]. On the basis of our structure-function analysis of region 3, Ulp1 seems to employ a unique mode of interaction with Smt3 and sumoylated substrates. Ulp1 does not appear to contain bona fide canonical SIMs, and neither of the amino-terminal domains of Ulp1 (regions 1 and 2) interact with Smt3 or become enriched at the septin ring . This assessment is also underscored by the arrangement of Smt3 and Ulp1 in the cocrystal structure . The hydrophobic groove of Smt3 that would interact with a SIM-containing protein is turned away from the domains of Ulp1 that interact with Smt3. Interestingly, this may suggest that Ulp1 can be recruited to proteins that are covalently or noncovalently modified with SUMO and SUMO chains.
Our research demonstrates for the first time that noncovalent interactions between Ulp1 and SUMO are important not only for SUMO binding but also for the cytosolic targeting of this SUMO protease to the bud-neck and potentially to sumoylated septins. Septins are not the only cytosolic substrates of Ulp1 and are arguably the most prevalent [35, 38]; therefore, they may be readily scored in our septin ring targeting assay (Figures 3A and 3B). We predict that Ulp1 is also targeted to other cytosolic and septin-bound sumoylated substrate proteins, such as the karyogamy protein Kar9 . However, because of the low local concentrations in comparison to sumoylated septins, these proteins may be hard to detect. We propose, however, that sumoylated proteins that accumulate or aggregate in the cytosol of yeast cells may be readily detectable by Ulp1(3)(C580S).
As detailed below, Ulp1(3)(C580S) also provides a useful tool with which to purify these sumoylated proteins (Figure 6E), and such studies are underway (O Kerscher and Z C Elmore, unpublished data). Our findings provide strong evidence that SUMO, at least in the case of sumoylated proteins at the septin ring, is a required signal for the cytoplasmic targeting of Ulp1. Our alignments of the SBS domain and the juxtaposed salt bridge-forming D451 residue reveal that this mode of targeting may also be conserved in other metazoan Ulp1-like SUMO proteases (Additional file 1).
Though we clearly show herein that Ulp1 becomes enriched at the septin ring, we do not yet fully understand how Ulp1 arrives at this subcellular localization. Our findings support the previous observation  that Kap121 plays an important role in promoting Ulp1 targeting to the septin ring. Similar to a previously described Ulp1 mutant that lacks the Kap60/Kap95 binding domain (region 2) , the septin ring localization of the full-length Ulp1(C580S) protein described herein is dependent on functional Kap121. It is unlikely that the association with Kap121 shuttles Ulp1 to the septins. Rather, in mitosis, Kap121 becomes associated with a transport inhibitory nucleoporin, Nup53, and may thus exclude Ulp1 access to the inner phase of the NPC . This suggests that, in the absence of Kap121-binding, a fraction of Ulp1 is free to associate with sumoylated septins. Our studies confirm that Ulp1 lacking the Kap60/Kap95-binding domain (region 2) is enriched at the NPC and the septin ring (Figure 3A). We extend these observations by showing that the ability to target sumoylated septins resides in the catalytic domain (region 3) of Ulp1. We found that a Ulp1(3)(C580S) mutant, but not WT Ulp1(3), is enriched at the septin ring in the absence of Kap121. These data suggest that both the Ulp1(3)(C580S) mutant and WT Ulp1(3) can interact with sumoylated septins, but, unlike the subtrate-trapping Ulp1(3)(C580S) mutant, the catalytically active WT Ulp1(3) may be quickly released after desumoylation of the target protein, giving it a diffuse appearance in the cell. How Kap121 helps Ulp1-Δ2 to be retained at the septin ring in the absence of the C580S mutation is currently unclear. Kap121 may theoretically promote the interaction with a septin ring-localized protein. However, the localization of Kap121 to septins has not previously been reported.
One intriguing aspect of our study is the results of our analysis of the substrate-trapping Ulp1(3)(C580S) construct. Three lines of evidence reveal the avid interaction of Ulp1(3)(C580S) with SUMO proteins and sumoylated substrates. First, this Ulp1-derived construct shows a pronounced interaction with the bud-neck, which is composed of sumoylated septins in vivo. Second, Ulp1(3)C580S interacts with Smt3 in a two-hybrid assay, whereas WT Ulp1(3) does not. Third, the purified recombinant Ulp1(3)(C580S) protein is a potent affinity-tag for the purification of Smt3 conjugates and SUMO-modified proteins. Furthermore, in preliminary BiaCore analyses, we determined the Kd for the interaction between Ulp1(3)(C580S) and SUMO1 to be 12.8 nM, which is about 200 times stronger than the interaction between a SIM and SUMO, as previously reported by Hecker and co-workers . A related study involving the C603S mutant of the human SENP1 protease confirms our assessment of the substrate-trapping feature. Those authors observed relocalization of their SENP1(C603S) mutant in vivo to PML nuclear bodies and domains of the HDAC4 protein, suggesting that SUMO-dependent targeting may be a conserved feature of Ulp1-like SUMO proteases . The latter may also provide a useful strategy for the identification of mitotically important desumoylation substrates. Indeed, two-hybrid screens with Ulp1(3)(C580S) in the laboratory have already identified several novel cytosolic desumoylation targets (M Donaher and O Kerscher, unpublished data). We are also exploring the ability of Ulp1(3)(C580S) to act as a SUMO chain-binding tag that can be used to promote the interaction of putative STUbL target proteins with RNF4 and other STUbLs (Figure 7).
How Ulp1 and other SUMO proteases target specific mitotic substrates for desumoylation remains unknown. Our analysis of SUMO-dependent Ulp1 targeting to the septin ring provides important evidence that Ulp1-like SUMO proteases do not passively await their desumoylation substrates, but rather dynamically localize to them in a cell-cycle-specific manner. Future experiments that take advantage of the SUMO-binding properties of the substrate-trapping Ulp1(3)(C580S) construct may prove useful for the identification of clinically relevant targets of conserved Ulp1-like SUMO proteases in yeast and human cells.
Ulp1 remains one of the most enigmatic SUMO proteases with an essential role in cell-cycle progression. Our work focuses on the fundamental but unresolved question of how Ulp1, an NPC-associated SUMO protease, targets mitotic desumoylation targets in the cytosol. The current study of Ulp1 reveals for the first time that (1) specific determinants of Ulp1's catalytic domain are utilized to target the septins at the bud-neck of dividing cells, (2) Ulp1 requires SUMO and intact sumoylation for correct cytosolic targeting, (3) Ulp1, unlike Ulp2, relies on specific salt bridge-forming residues for substrate targeting, (4) septin-targeting information in the catalytic domain of Ulp1 is independent of Kap121 and (5) the substrate-targeting domain of Ulp1 can be modified to identify and purify sumoylated substrates of Ulp1 in genetic screens and in biochemical analyses.
The yeast strains and plasmids used in this study are listed in Table 1. smt3-331 expresses a temperature-sensitive Smt3 protein containing a L26S mutation ( and S Brill, personal communication). smt3R11, 15, 19 expresses a triple-mutant of Smt3 rendered unable to form SUMO chains through replacement of lysines 11, 15 and 19 with arginines . ulp1ts expresses a temperature-sensitive mutant of Ulp1 that contains three mutations (I435V, N450S and I504T) in region 3, the catalytic domain . Yeast media preparation and manipulation of yeast cells were performed as described previously . Yeast strains were grown at 30°C unless otherwise indicated. DNA fragments containing Ulp1 under the control of its endogenous promoter were amplified from yeast genomic DNA and placed in-frame with a carboxy-terminal GFP-tag in the CEN/LEU2 plasmid pAA3 . Primer pairs used for full-length Ulp1 amplification were OOK2 (ULP1 (-310 to -294)) and OOK3 (ULP1 (+1, 842 to +1, 863)).
To prepare the truncated and mutated Ulp1-GFP constructs listed in Table 1, the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies, La Jolla, CA, USA) and the Phusion Site-Directed Mutagenesis Kit (Finnzymes Oy/Thermo Fisher Scientific, Vantaa, Finland) were used according to the manufacturers' instructions. Primer sequence information for the construction of individual mutants and truncations are available upon request. All constructs were sequenced, verified and/or confirmed by performing complementation assays (for example, see Additional file 3). Additionally, we confirmed that septin rings are intact in strains expressing plasmid-borne Ulp1(3)C580, WT Ulp1 and Ulp1(3) (see Additional file 5). For two-hybrid constructs, ORFs of the indicated genes were PCR-amplified and recombined into gapped pOAD and pOBD2 vectors (Yeast Resource Center, Department of Biochemistry and Department of Genome Sciences, University of Washington, Seattle, WA, USA).
To overexpress and purify Ulp1 truncations from bacteria, the respective Ulp1 fragments were PCR-amplified and cloned into pMALc-HT (a gift from Sean T Prigge, Department of Molecular Microbiology and Immunology, The Johns Hopkins University School of Public Health, Baltimore, MD, USA), thereby adding an in-frame MBP module followed by a TEV protease cleavage site and a His6 epitope tag. Ulp1 derivatives were expressed as MBP fusions in BL21 Star (DE3) cells (Invitrogen) containing a pRIL plasmid. The YCp-111 Cdc3-CFP/LEU plasmid was a kind gift from Ryuichi Nishihama of John Pringle's laboratory (Stanford University School of Medicine, Stanford, CA, USA).
Gal4-activation domain (AD) fusions of ULP1 and the indicated ULP1 mutants in pOAD were transformed into the AH109 reporter strain expressing a Gal4 DNA-binding domain (BD) fusion of SMT3 in pOBD. Two-hybrid interactions of serially diluted cells were scored in duplicate on dropout media lacking adenine.
Frozen bacterial cell pellets from 200 ml of isopropyl β-D-1-thiogalactopyranoside-induced BL21 Star (DE3) cells were thawed on ice and resuspended in 2 ml of 1 × PBS containing 1 × Halt Protease Inhibitor Cocktail (catalog no. 78430; Pierce Biotechnology/Thermo Fisher Scientific, Rockford, IL, USA). Ice-cold cells were sonicated using a Branson Sonifier ultrasonic cell disruptor (Branson Ultrasonics, Danbury, CT, USA), and extracts were cleared by centrifugation at 15, 000 rpm for eight minutes at 4°C. Cleared bacterial extracts were added to 15-ml conical tubes and diluted using 4 ml of 1 × PBS containing the protease inhibitor cocktail. MBP-tagged proteins (MBP-Ulp1(3), MBP-Ulp1(3)(C580S) or MBP-Ulp1(3)(C580S)ΔSBS) were bound to 5-ml columns containing 300 μl of amylose resin (New England Biolabs, Ipswich, MA, USA) and washed extensively with 1 × PBS. Yeast cell protein extracts containing the indicated target proteins were passed over the amylose resin, and proteins bound to MBP-Ulp1(3), MBP-Ulp1(3)(C580S) or MBP-Ulp1(3)(C580S)ΔSBS were eluted with 100 mM maltose or SDS-PAGE sample buffer. Yeast cell protein extracts were generated by bead-beating about 50 nm of yeast cell pellets in 1 × cell lysis buffer (catalog no. 9803; Cell Signaling Technology, Danvers, MA, USA) containing 25 mM N-ethylmaleimide. "Bound" material (Figures 6A and 6B) corresponds to proteins affinity-purified from approximately 8 nm of extracted cells. Input (WCE) lysates correspond to 1.25 nm of extracted proteins (about one-sixth the material loaded onto the column). In SUMO pull-down experiments, recombinant MBP-Ulp1(3)(C580S) or MBP-Ulp1(3) was incubated with SUMO1 or SUMO2 agarose (Boston Biochem Inc, Cambridge, MA, USA) in 1 ml of 1 × PBS with protease inhibitors. Proteins bound to the agarose beads were washed in 1 × PBS and eluted with 1 × SDS-PAGE sample buffer. All protein extracts were separated on NuPage Novex 4-12% Bis-Tris gradient gels (NP0321; Invitrogen, Carlsbad, CA, USA) using 3-morpholinopropane-1-sulfonic acid-SDS running buffer (NP0001; Invitrogen). Equal loading and concentrations of recombinant proteins were confirmed by Coomassie staining and analysis using an Agilent 2100 Bioanalyzer using a Protein 230 Kit (Agilent Technologies, Santa Clara, CA, USA). BiaCore measurements were conducted by Affina Biotechnologies, Inc (Stamford, CT, USA) as a paid service.
Unless otherwise noted, cells were grown in rich media, arrested in G2/M using nocodazole (15 μg/ml/3 hours/30°C), washed in 2% dextrose and harvested by centrifugation. Images of live cells were collected using a Zeiss Axioskop microscope (Carl Zeiss USA, Thornwood, NY, USA) fitted with a QImaging Retiga™ SRV charge-coupled device digital camera (QImaging, Surrey, BC, Canada), i-Vision-Mac software (BioVision Technologies, Exton, PA, USA) and a Uniblitz electro-programmable shutter assembly system (Vincent Associates/UNIBLITZ, Rochester, NY, USA). Pertinent filter sets for the above applications include CZ909 (GFP), XF114-2 (CFP) and XF104-2 (YFP) (Chroma Technology Group, Bellows Falls, VT, USA). Images were normalized using i-Vision-Mac software and pseudocolored and adjusted using Adobe Photoshop software (Adobe Systems Inc, San Jose, CA, USA).
Enzymes and substrates used in our in vitro ubiquitination assays were quantified using a Protein 230 Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer's instructions. We used 10 × ubiquitination buffer, E1 enzyme (Uba1), ATP and 20 × ubiquitin provided in a commercial ubiquitin activating kit (BML-UW0400-0001; Enzo Life Sciences Inc, Farmingdale, NY, USA). Ubiquitination buffer, isopentenyl diphosphate (100 U/ml), dithiothreitol (DTT) (50 μM), E1 (Uba1), E2 (Ubc4) and E3 enzymes (RNF4) were combined with purified SUMO2 chains (ULC-210; Boston Biochem) and ubiquitin. Reactions totaled 27 μl and were incubated at 30°C for three hours. Reactions were stopped by adding an equal volume of SUMEB with Tris (SUTEB) sample buffer (0.01% bromophenol blue, 10 mM ethylenediaminetetraacetic acid, 1% SDS, 50 mM Tris, pH 6.8, 8 M urea) containing DTT (5 μl of 1 M DTT mixed with 1 ml SUTEB sample buffer). Protein products were boiled in a 65°C heat block for ten minutes and examined by Western blot analysis with anti-human SUMO2 antibody, anti-human SUMO2 (BML-PW0510-0025; Enzo Life Sciences Inc), anti-GFP (JL-8 632380; Clontech, Mountain View, CA), ANTI-FLAG M2 (F3165; Sigma-Aldrich, St Louis, MO, USA), anti-phosphoglycerate kinase (22C5D8; Invitrogen), anti-Cdc11 (y415) (sc-7170; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
We thank all members of the Kerscher lab for their support. We especially thank Cecilia Esteban for Figure 1B, Jeff Hollomon for an Ulp1 overexpression construct and Nora Foegeding and Chris Moad for help with experiments during revision of the manuscript. We are indebted to Diane Shakes, Rachael Felberbaum and Mark Hochstrasser for comments and/or edits of the manuscript. Additionally, we thank Vikram Panse, Maria Katariina Veisu, Sue Biggins, Mark Hochstrasser, Eric Rubenstein, Munira Basrai, Wei Chun Au, Rick Wozniak, Erica Johnson, John Pringle, Ryuichi Nishihama, Liz Allison, Mark Forsyth and Eric Engstrom for strains and reagents and Steve Brill for information on Smt3-331. This work was supported by National Institutes of Health grant R15-GM085792 (to OK), a William & Mary Howard Hughes Undergraduate Summer Research Fellowship (to MD and BCM), an ALSAM fellowship (to BCM) and a William and Mary Arts and Sciences Graduate Research grant (to ZE).
OK and ZE designed and interpreted all the results of the experiments and wrote the manuscript. ZE carried out all experiments with the following exceptions: MD and JW carried out the two-hybrid assays and helped to interpret the results, BM designed and conducted the STUbL assay and HM aligned Ulp SUMO protease sequences, confirmed conserved residues and helped with editing. All authors read and approved the final manuscript.


text:
Follow signs to I-40 West and Research Triangle Park.
Travel on I-40 West to Page Road (Exit 282).
At the bottom of the exit ramp, turn right onto Page Road.
Cross under I-40, then turn left at Emperor Blvd.
The first building on your right is Bristol Place.
SRC is located on the third floor of Bristol Place—Suite 300.
Follow signs to I-40 East.
Travel on I-40 East to Page Road (Exit 282).

industry_corpus2: Others


text:
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Anyway! There are lots of good stuff in cute colors. I like their sweaters and jeans--they've got stretchy, comfy jeans! Hope they would last me!
To end this post, I'm leaving you with something to be EXCITED about.
Isn't Aeropostale going to have a branch in Robinson's Magnolia already? I went there couple of months back and saw the brand name on one of the store under construction..
It looks like bright colored jeans are having a come-back on the fashion scene again. On the other hand, I'm excited for H&M!
Sounds great! I used to work at Amer. Eagle. Peddling AE jeans for minimum wage was the name of the game lol. Nowadays, I'm a big H&M fan!
This is good news! More choices mean more competition and could result to better price points for us consumers. I hope A and F, Old Navy and Sephora make it to our shores. I have hoodies from the former 2 and until now buhay pa ha, in fairness.
I still dream of wearing colorful jeans or pants pero I can't pull it off yet. Sayo Ms M. bagay talaga, how lucky!
Hmmm, the aforementioned brands aren't really my style but I'm sure hoping someone will pick this up and bring in Madewell, Joe's Jeans and Japanese brands in.
Great great news for shopaholics and brand enthusiasts as international brands make their way to our lovely country. I'm excited for H&M. Hope it will be sooner... And hopefully, A&F, Old Navy, and Aeropostale will have their flagship stores here soon.
I haven't been to AEO The Fort. But soon, very soon :) I've read a lot about the opening and it makes me wanting to visit ASAP.
crossing my fingers that H&M will partner with SM. sana sana!
Keeping my fingers crossed for H&M!
Jec: Hi there! :D A lot of my friends are loving it and too bad. wasn't able to check it when I was in HK. Hopefully they open H&M here this year!
Mapleapple: Same here! Can we be mates in Broke Apartel, Brokeland? Hahaha!
i love colored plain jeans as well as plain shirts hihi..
I've read from Kelly Misa's blog that the American Eagle has opened its Manila branch in BGC and I find their stuff (as shown on Kelyy's blog) not so attractive, its like a combination of Bench and SM clothes, or less attractive than F21. But the jackets are way too cool, nice quality indeed.


text: Add a stylish finish to your bedroom with a bed frame or headboard that speaks your style. From rustic to boho, we offer on-trend to classic beds that will spruce up the style in your bedroom. Creating the perfect accent, our bed frames and headboards are also durably constructed for a reliable design.

industry_corpus2: Manufacturing


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Gorgeous solo piano for meditation, relaxation and awakening.
I first heard about 11:11 in 1991 through Erik Berglund, who introduced me to Solara (https://www.tradebit.com) who organized and facilitated the 11:11 planetary activation in 1992. I have often found myself looking at a digital clock and seeing 11:11, and Iâve enjoyed resonating with the inner light and love within all whenever I see this symbol.
Then I was invited to play for a healing session at the United Research Light Center, in Black Mountain, NC, where I often perform, on 11/11/2007. The music just happened to be professionally recorded that day, and I was gifted with a CD of what I had played. After hearing that music and starting the process of producing the finished CD, I went to a theatre performance. The box office attendent gave me seat 11 in the 11th row! That was way - more confirmation than I expected!
People who are interested in Yanni George Winston Steven Halpern should consider this download.


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Scan this bar code with your mobile device and see what Cheetah Lounge is working on.
• Streams are free and easily accessible worldwide.
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industry_corpus2: Film & Entertainment


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Continuing his East Asian tour, Apple CEO Tim Cook arrived in Japan this week and on Wednesday visited a popular shrine in Kyoto, met with executives from Nintendo and scored an early preview of Super Mario Run for iOS.
At around 9 a.m. Japan time, Cook tweeted out "good morning" in Japanese, a routine for the CEO world-traveler who commonly uses the microblogging service to greet locals in their native tongue. A picture accompanying the message shows Cook and an unidentified woman walking through the famed torii, or traditional Japanese gates, at Fushimi Inari Taisha in Kyoto.
Cook later tweeted a picture of a meeting at Nintendo, likely at the gaming giant's headquarters in Kyoto, where he is seen playing the hotly anticipated iOS title "Super Mario Run" with legendary creator Shigeru Miyamoto. Scheduled to arrive before the end of 2016, Miyamoto presented the game onstage during a surprise appearance at Apple's iPhone 7 event last month.
Besides taking a first look at the most awaited iOS game of the year, what other business Cook plans to handle in Japan is unknown.
Cook makes his way to Japan via Shenzhen, China, where on Tuesday he revealed Apple's plans to open an R&D center in 2017. Reported by local media yesterday, the upcoming facility is expected to foster hardware production and software developing talent in China's manufacturing mecca.

industry_corpus2: Computing & Telecommunications


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The Wautoma High School presents the Rodger and Hammerstein’s musical classic, The Sound of Music, on Nov. 3 & 4 at 7 p.m. and Nov. 5 at 2 p.m. The beloved classic tells the story of Maria, who takes a job as a governess to a large family while she decides whether to become a nun. She falls in love with the children, and eventually their widowed father, Captain von Trapp.
Many songs from the musical have become standards and are still to this day, such as “Edelweiss”, “My Favorite Things”, “Climb Ev’ry Mountain”, “Do-Re-Mi”, and the title song “The Sound of Music.” Tickets are on sale now through the box office. Call 920-787-2189 between 10 a.m.-2 p.m., Monday through Friday.

industry_corpus2: Subject Education


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Optional: Narrow the search scope to a required company group by clicking on its name.
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Note: The prompt in the search field displays the current search scope according to the most recent selection. For example, if the prompt says "Search white label", this means your search will be conducted among all White Label companies of all available company groups and return only White Label companies.
Search for the required company by typing its name in the search field. The relevant search results are displayed.
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Optional: Select/Deselect the corresponding check box by the required Company to share your entity/prevent the required Company from gaining access to your entity.
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In an increasingly interdependent but uneven world, analyses of the role of ideas (theory), of power (politics), and of material interest (economics) must be brought together for an in-depth understanding of global political processes. The Major in Philosophy, Politics, and Economics has its origins in the long-established and highly successful PPE degree offered at the University of Oxford. Its speciﬁcity at The American University of Paris lies in its integrated approach, with courses across the three disciplines mutually reinforcing each other.
We are the thinkers, the travelers, the creators, the leaders – the individuals who have chosen to live and learn internationally, finding our placewithin a powerful community that begins in the heart of Paris.
We cultivate critical thinking through a strong academic foundation that finds synergybetween diverse disciplines and evolves to meet the needs of the contemporary job market.
We become translators of culture with the capacity to analyze, adapt, and thrive.
The American University of Paris (AUP) follows an American liberal arts model with over 20 bachelor degree programs and 15 master’s programs across four major divisions: Arts & Sciences; International Business Administration; International Politics, Economics, & Public Policy; and Communications & Film. Accredited by Middle States, AUP is urban, contemporary and multi-cultural. Students of nearly 100 nationalities share small classes with accomplished professors, fully leveraging AUP's location in the center of one of the world’s greatest cities. Merit and need-based scholarships are offered to those who qualify. Students with a minimum average grade of VG (15) on the Swedish Fullständigt Slutbetyg från Gymnasieskolanwill receive 32 advanced credits.
In theclassrooms: Students are encouraged to define and reachindividual academic goals within a deeply interdisciplinary curriculum that is cross-cultural and comparative at its core. In an average class of fifteen, over twelve different nationalities are represented, bringing together the diverse perspectives that create the AUP learning experience. Scholastic practice and industry skills are sharpened through theoretical and experiential learning. Faculty advises students throughout their academic experience in the choice of classes, majors, and self-directed studies.
Student clubs, government, and social responsibility initiatives are a chance to explore, to create, and to lead. Notably, our student media organization produces an industry-standard magazine and news site, supported by renowned international journalists. Sports and AUP discounts at gyms across the city help to justify French pastry consumption.
On Campus:extends from the 7th arrondissement into the city’s bookstores, cafés, libraries, and galleries. Building on everyday immersion in Parisian language and culture, our curriculum challenges students to engage in deeper academic and cultural learning sharpened by discussion, analysis, and research at over 200 institutions across the city, such as: The Louvre, The Sorbonne, UNESCO, InstitutFrançaise de la Mode, Palais de Tokyo, and L’Institut du Monde Arabe.
AUP housing partners organize apartments for incoming undergraduates before arrival. Homestays with Parisian families are also available. To ensure a smooth transition to life in Paris, we assist in the setup of utilities, phones, internet, and banking, as well as optional laundry and cleaning services.
The world as a classroom:Students engage directly with the histories, perspectives, and contemporary realities of cultures around the world on study trips led by faculty experts. Ranging from single-day to month-long expeditions, every program includes learning linked to international discovery – from the study of art history in Rome to fist-hand experience with human rights law in the courts of Sarajevo and Belgrade to immersion in the Moroccan business center of Fez.


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Football was given its most recent chance to reform as a result of the Culture, Media and Sport Select Committee review of Football Governance, last summer. The report provided some pretty radical thinking on the big issues affecting our game.
The report was the result of pledges secured by the Trust movement in the Conservative manifesto and the Coalition agreement. In their October 2011 response, the Government supported the the Committee’s recommendations from, threatening legislation if the authorities failed to act strongly enough.
SD Agenda for Expert Group – the purpose was to suggest the terms of reference for group to examine how recommendations for increased supporter engagement could be delivered.
These were submitted to the football authorities, Mr. Robertson and over 500 MPs. The FA, the Premier League, the Football League and National Game finally published their response to this review on March 9th. Although we saw the acceptance of basic principles such as licensing, and the introduction of Financial Fair Play in the Premier League and Championship – something SD called for – fundamental reform that would make football fit for years to come was avoided, and a whole series of issues, it would appear, are potentially being kicked into the long grass and in some cases avoided entirely.
An example is the debt issue, so important to all of us, and the whole of football. The word appears just once – but unfortunately it’s referring to the “debt of gratitude to volunteers” working across the country for football. Yet the word ‘debt’ appeared over 50 times in the Select Committee report alone. Guardian journalist David Conn, who knows more about these issues than most, accused the FA of surrendering their authority with ‘barely a whimper’.
The response is far too vague on far too many issues. Supporter rights and responsibilities – where supporters’ trusts would work towards a formal role at a club as part of the licence conditions – was a key issue for the Trust movement. We will be seeking discussions with the FA and Leagues in the football pyramid as well as the Minister for Sport.
The financial commitment alone of supporters’ trusts has averaged £2.5m a season since 2000 (money which has often been part of dealing with debt and insolvency problems that the authorities have failed to get to grips with – there have been 92 insolvencies in the top five divisions since 1992) that have cost the game £5m during this period, or a net investment of £25m.
We have outlined our views to our over 140 English and Welsh affiliated trusts. We also need to identify the opportunities we have as movement to force further change, working with trusts as partners, and their over 250,000 members – in other words you.

industry_corpus2: Sports


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San Antonio -- March 14, 2016 -- Rapid melting of ice and Arctic permafrost is altering tundra regions in Alaska, Canada and Russia, according to a new study released in the journal Nature Geoscience. Ice-wedge degradation has been observed before in individual locations, but this is the first study to determine that rapid melting has become widespread throughout the Arctic.
Southwest Research Institute® (SwRI®) provided time series and change detection analyses of high resolution remote sensing imagery acquired over several Arctic sites as part of this study. An international team of 19 researchers analyzed and compared these results with field data and climate observations to understand how degradation of ice deep underground is affecting surface topography and hydrology.
"The analysis clearly shows dramatic changes to this landscape, especially during relatively short periods in unusually warm summers in recent years," said Dr. Marius Necsoiu, a principal scientist in SwRI's Geosciences and Engineering Division.
If this trend continues, it will lead to widespread draining and differential subsidence of the landscape, having long-term effects on plants and wildlife across the Arctic.
"It's really the tipping point for the hydrology," said Anna Liljedahl, the lead researcher and an assistant professor at the University of Alaska Fairbanks (UAF) Water and Environmental Research Center.
The Arctic tundra has a unique landscape consisting of many interconnected polygon shapes roughly 15 to 30 meters wide. These polygons are shaped by large frozen ice wedges located meters into permafrost. Ice wedges gradually formed over hundreds or even thousands of years as water seeped into permafrost cracks. The micro-topographic features affect drainage, snow distribution and the general wetness or dryness of a landscape.
For his analysis, Necsoiu compared historical aerial photos and satellite images from 1948 to 1990 with recent images taken from 2005 and 2012. Combined with field observations, the team's analyses showed that deep troughs formed in the landscape as the tops of ice wedges melted. Field researchers also observed a decline in native plants such as lichen and moss. In early images, where ice wedges were not degraded, snow melt gathered in small shallow-ponds during summer months. In recent years, however, the ponds did not form in those areas as deep troughs from advanced ice degradation created channels for the water to run off.
Study sites across the Arctic showed ice-wedge degradation occurred in less than a decade. In some cases, a single unusually warm summer was enough to cause more than 10 centimeters of surface subsidence, which causes pooling and runoff in an otherwise relatively flat landscape.
Although these regions contain "cold permafrost," with an overall average temperature of about 7 degrees Fahrenheit, surface thawing still occurred at all of the 10 study sites.
Gradual warming of permafrost has been well-documented in the Arctic, but the polygon study indicates that even a brief period of unusual warmth can cause a rapid shift in a short time period. Researchers said this is a new trend for the Arctic, at least for the last 60 years covered in the study.
To learn more about Southwest Research Institute's Geosciences and Engineering Division, visit swri.org.

industry_corpus2: Water Resources & Marine


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Smiles team had a blast at Clover Avenue Elementary School last October. We participated in the “Halloween Hurrah” which the school organizes every year. The kids loved our “tooth vitamins” game. The object was to throw the vitamins in the mouth. This was no easy task but Clover goers proved to be great players! Winners received a toothbrush (to brush away that Halloween candy), toys and stickers. We also donated three tickets to visit Smiles as raffle prizes. We are already looking forward to next year’s Hurrah!
Every princess gets a balloon..


text: Rose Garden Pool is managed by American Pool Management. All employees of Rose Garden Pool are hired and managed by American Pool Management. For summer employment, call Jenn Bell at 866-630-7665 or apply on-line at American Pool Enterprises, Inc.


text:
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When there are big events, I like to pull together resources in one place — and, of course, this has been open government week — the Office of Management and Budget issued a series of policies, while agencies issued their open government plans.
You can find Federal News Radio 1500 AM’s ongoing coverage of the open government initiative here.
Before the plans were released, I posted DorobekINSIDER: Assessing transparency and open government.
* The White House open government site, which has a lot of good information but buries links to agency open government plans in the open government dashboard.
* OMB director Peter Orszag blog post: OMB and Open Government, which includes a link to the four OMB open government policies — also listed below — and to OMB’s open government plan.
Put simply, it’s increasingly clear government is not going to become more open and transparent without extraordinary public pressure. And WE are going to have to be the ones to put that pressure on them.
You can help right now by joining our campaign for open government and signing the pledge to demand all public government information be available ONLINE and in REAL-TIME.
Much of that discussion has revolved around the Paperwork Reduction Act — and a strong frustration that it really hinders agencies flexibilities.
More disappointing from my standpoint, it keeps in place the notion that citizen interaction with the government is essentially a “burden” and still codifies the position that significant interaction with the public should be minimized (this is clearly contrary to open government).
The discussion has spurred me to actually print out the Paperwork Reduction Act and read it for myself to get a sense of what it actually says. My sense is that some of what OMB is trying to do is work within the constraints of the law — a law enacted in the early 1980s before hardly anybody even had e-mail addresses.
The guidance makes it easier for agencies to use social media and requires steps to ensure better rule-making and spending transparency.

industry_corpus2: Politics & Administration


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Dr. Davletov’s team of researchers has developed a unique patented system of four distinct molecular helices which quickly assemble into a tight, rigid and stable nanostructure scaffold. The ends of the helices can be attached to a wide range of other materials, enabling these materials to be connected to each other in an ordered and hierarchical way. These connections are very stable, resistant to detergents and to temperatures up to 80C, but can also be controllably disconnected by applying suitable conditions. In effect the molecular scaffold can function as a strong but reversible staple between any two (or more) materials or molecules.
The helices are based on the naturally-occurring systems which fuse cell membranes together. They are very easy to attach to other protein molecules, and can also be connected to a flat surface and used to trap and immobilise particular proteins onto the surface. The scaffold can be used to bring new properties to molecular structures, for example adding luminescence, or creating molecular tags or barcodes. Long sequences of molecules can be joined together in a controlled order using multiple scaffolds.
There are many potential uses of the technology in any system where smaller components need to be combined to form larger structures. It is particularly useful for modular and combinatorial approaches – the researchers have calculated that a mixture of 100 individually-labelled helices would create 900,000 unique combinations. It also works well when used to bring together a number of distinct structures to combine proteins, create new drugs, add fluorofors, attach gold nanoparticles and many more.
In addition the scaffold can be used as a way to neutralise a toxic active molecule by dividing it into two non-toxic halves which can then be handled easily in a laboratory with no safety implications. The two halves can be recombined using the helical scaffold at the time that the toxic material is actually needed in its complete form, for example for medicinal use.
The challenge for the i-Team is to investigate the wide range of possible applications for this new and highly-innovation approach. By looking at applications ranging from the realms of physical nanostructures to the assembly of protein-based materials, and discussing the technology with relevant industry experts, the team will recommend the next steps and best practical uses for the researchers to focus their efforts.

industry_corpus2: Biopharmaceuticals


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This is easy! Under the Finances tab, click Add Bank Account. You can link more than one bank account to Rally.org and choose to withdraw and transfer your funds into these specified accounts.
You will need a valid U.S checking and routing account number to withdraw funds.
When adding a bank account, you will also be asked to enter an EIN for your organization. Don’t have an EIN? Just enter your Social Security Number and you’re good to go. Don’t worry: All your information is safe with us.

industry_corpus2: Finance & Economics


text: Implementation is first step towards making smooth running of college ERP software. Our technical experts will reach your campus to install software. We would also recommend you to make necessary changes in computer networks or establish network topography. Our team members has got expertise in software installation and implementing ERP systems as we have hundreds and hundreds of implementation experience. We cross check entire system after installation and ensure smooth working of systems. We confirm with your team prior to leaving campus that you have got system well in place. Our offline method of ERP implementation is free of cost. We strongly believe in software installation and support service without which all other stuff may go fruitless. All of your college data remains on your college server within your control. Once we deploy project our next step is of College ERP software training.


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For people who follow structure, trading is fun and profitable. Our Educational Products teach you sound market timing methods and rigorous discipline so you can acquire for yourself that “beforehand” perspective that will help you identify preemptively high-potential market moves. Integrating this perspective with a well-crafted trading plan founded upon reasonable money management principles will increase your chances to consistently beat the market over time.
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Each month we like to highlight a couple of amazing benefits of Royal Jelly, one of the most powerful ingredients in our Golden Royal Honey. This month we’re going to take a look at how Royal Jelly can help you have clear, soft, radiant skin. We’ll also discuss how research has shown that Royal Jelly is an effective natural remedy for lowering your blood pressure.
It is a substance secreted from the glands of worker bees. Rich in vitamins, minerals and amino acids, Royal Jelly is like bee super food and what the queen bee eats exclusively, and why she grows so much larger and lives so much longer than regular worker bees.
If you want to know what Royal Jelly can do for you, keep reading!
Summer’s in full swing, which means there are still week’s left of warm weather and wearing less clothes. And the less clothes you wear, the more skin you show and the better you want it to look!
But not all of us were born with flawless skin. Some of us have dry patches and/or little red bumps here and there. We’re human, not perfect supermodels.
Though we may be mere mortals, we can still have beautiful skin once we know a few tricks. We all know about hydration for beautiful skin – that’s a good trick.
But do you know about Royal Jelly?
How Can Royal Jelly Help Your Skin?
For centuries, different cultures around the world have highly prized Royal Jelly for its numerous health benefits. Royal Jelly is known to help heal skin while moisturizing and giving it needed nutrients.
This creamy white bee product is chock full of complex B vitamins, amino acids, fatty acids, minerals, enzymes, natural antibiotic properties, and antibacterial properties. Royal jelly is also known for its anti-aging benefits because of its ability to lower cholesterol and decrease inflammation.
It has antibiotic properties and has been used to treat wounds.
If you have sensitive, dry skin or skin that’s prone to eczema and rashes, give Royal Jelly a try.
Let’s face it, life isn’t always easy. Between working more hours than we should, dealing with awful commutes, scrambling to pay the bills and still trying to make time for real human connections, many of us feel overwhelmed and stressed to the max.
As if the feeling of being stressed weren’t bad enough, stress can wreak havoc on our physical bodies. When we feel stressed, our body releases stress hormones like cortisol and adrenaline as part of the “fight or flight” response.
These hormones get us ready to either fight or run like heck by making us more alert and getting our heart to beat faster thereby pump blood to our muscles quicker.
When this hormonal response happens day after day after day, the result is often high blood pressure, and that increases your risk of heart disease and stroke.
That’s the bad news. But the good news is, Royal Jelly can naturally lower your blood pressure, decreasing your chances of developing other deadly diseases.
Royal Jelly is the richest known natural source of vitamin B5, which lowers stress, and also provides other B vitamins plus vitamins A, C, D and E. Royal jelly also provides 20 amino acids, essential fatty acids, and important minerals such as potassium, calcium, zinc, iron and manganese.
But the main blood pressure lowering ingredients in Royal Jelly are short chains of amino acids, known as peptides, which block the effects of ACE (angiotensin converting enzymes). ACE is a powerful constrictor of blood vessels, and by blocking its action, constricted blood vessels can dilate to help reduce hypertension. This is the same mechanism used by prescribed ACE inhibitor drugs. But Royal Jelly is much safer because if has no nasty side effects.
If you’d like to naturally lower your blood pressure, try Royal Jelly for yourself.
*Always speak to your healthcare provider before starting any new treatment or protocol.

industry_corpus2: Health & Medicine


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Showing results for tags 'heres lucy '.
LittleRickyII posted a topic in There Just Wasn't Enough ROOM For All of You!
John Davidson's parents were Baptist ministers. He has apparently held these beliefs since he was a very young man, long before he was on Here's Lucy, but he had to keep quiet because of this career. I'm glad he now feels free to talk.