BACKGROUND
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 , two small proteins that can become attached to other cellular proteins in a reversible manner  <cit> , 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  <cit> .
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  <cit> .
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 , which mediate their ubiquitination  <cit> .
sumo proteins are highly conserved from yeast to humans.
yeast cells express one sumo protein , and vertebrates express three isoforms   <cit> .
sumo <dig>  sumo <dig> and yeast smt <dig> can form sumo chains.
sumo <dig>  on the other hand, lacks the internal lysine required for polymerization and may function as a chain terminator for sumo <dig> and sumo <dig> chains  <cit> .
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  <cit> .
in metazoans, the dysregulation of sumoylation adversely affects developmental processes and has been implicated in the progression of neurodegeneration, cancer and infectious diseases  <cit> .
more than  <dig>   <dig> sumoylated proteins have been identified in yeast and humans, but only in a few cases has the role of sumoylation been studied in detail  <cit> .
in the budding yeast saccharomyces cerevisiae, the ligation of sumo to specific substrate proteins requires an e <dig> heterodimer  that activates sumo, as well as e <dig>  and e <dig>  enzymes that aid in the conjugation and ligation of sumo to proper target proteins  <cit> .
two yeast sumo proteases, ulp <dig> and ulp <dig>  contain a conserved cysteine protease domain that can remove the sumo moiety from modified proteins.
recent evidence suggests that ulp <dig>  and its mammalian orthologs susp1/senp <dig> and senp <dig>  play a role in the removal of sumo and sumo chains from nuclear proteins  <cit> .
ulp <dig>  on the other hand, has two contrasting cellular functions.
ulp <dig> facilitates sumoylation by processing precursor sumo into its conjugation competent form.
conversely, ulp <dig> also facilitates desumoylation by removing sumo from nuclear and cytosolic proteins after conjugation  <cit> .
therefore, impairment of ulp <dig> 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  <cit> .
the substrate specificity of sumo proteases is at least in part regulated through their localization  <cit> .
for example, certain yeast  and vertebrate  sumo proteases localize within the nucleus.
in contrast, both yeast  and vertebrate  sumo proteases reside at the nuclear envelope  through their interactions with the nuclear pore complex   <cit> .
distinct domains have been identified that are required for ulp <dig> npc localization  and sumo processing   <cit> .
the ulp <dig> localization domain promotes interaction with karyopherins, which are soluble proteins that mediate transport across the ne and help localize ulp <dig> to the nucleoplasmic side of the npc.
the ulp <dig> localization domain can be subdivided into region  <dig>  and region  <dig> .
juxtaposed to the npc localization domain of ulp <dig> is a coiled-coil  domain with a putative nuclear export signal and region  <dig>  the catalytically active, conserved ubiquitin-like protease domain  of ulp <dig>  <cit> .
only regions  <dig> and  <dig> are involved in ulp <dig> localization to the npc, and the karyopherins seem to play a redundant role.
npc association of ulp <dig> requires several proteins, including the nucleoporins nup <dig> and nup <dig>  the silencing protein esc <dig> and the myosin-like proteins mlp1/ <dig>  <cit> .
together these proteins may provide a scaffold for the functional regulation and substrate access of ulp <dig> at the npc.
the identification of npc localization domains in ulp <dig> has done little to aid our understanding of how sumo proteases are targeted to their respective substrates  <cit> .
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  have been predicted to be localized in the yeast sumo protease ulp <dig>  as well as in mammalian senp <dig>  senp <dig>  senp <dig> and senp <dig>  <cit> .
even though sims have not been identified in ulp <dig>  the crystal structure of the catalytic domain  bound to smt <dig> reveals that both proteins interact through multiple residues that are distributed across a sumo-binding surface  on the sumo protease  <cit> .
only the carboxy terminus of bound smt <dig> 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 .
it has been suggested that this configuration may allow for the accommodation of many different sumoylated proteins as well as sumo precursors  <cit> .
ulp <dig> and several other sumo proteases play important roles in mitosis  <cit> .
in budding yeast, loss of ulp1-mediated desumoylation leads to cell-cycle progression defects and cell death  <cit> .
this observation suggests that ulp <dig> 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 ulp <dig> desumoylation substrates  <cit> .
how the npc-localized ulp <dig> 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, kap <dig> blocks ulp1's access to its npc-binding site and thus promotes an interaction of ulp <dig> with septins  <cit> .
a deletion mutant of ulp <dig> lacking region  <dig> , the kap60- and kap95-binding domain, has previously been shown to localize to septins in a kap121-dependent manner  <cit> .
curiously, it has recently been demonstrated that region  <dig> also plays a role in nucleolar accumulation of ulp <dig> after ethanol-induced stress  <cit> .
one set of cytosolic substrates of the ulp <dig> sumo protease are the septins  <cit> .
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  <cit> .
members of the septin family in yeast include cdc <dig>  cdc <dig>  cdc <dig>  cdc <dig> and shs1/sep <dig>  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 cdc <dig>  cdc <dig> and shs <dig> 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  <cit> .
cell-cycle  arrest with nocodazole, a microtubule depolymerizing drug, greatly increases sumo conjugation to septins  <cit> .
septin sumoylation in budding yeast is mediated by the sumo e <dig> ligase siz <dig>  <cit> .
during most of the cell cycle, siz <dig> resides in the nucleus.
however, at the m phase, siz <dig> exits the nucleus to sumoylate septin proteins and possibly other cytosolic substrates  <cit> .
deletion of siz <dig> 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 ulp <dig>  even though ulp <dig> remains visibly enriched at the npc  <cit> .
in the current study, we focused on the sumo protease ulp <dig>  as detailed above, ulp <dig> 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 ulp <dig>  in theory, this localization is well-suited to give ulp <dig> access to some nuclear substrates and those in transit across the ne.
however, both nuclear and cytosolic desumoylation targets of ulp <dig> have been identified, posing an interesting question.
how is ulp <dig> directed to its cytosolic desumoylation targets?
to answer these questions, we sought to identify features of ulp <dig> required for substrate targeting in vivo and in vitro.
herein we describe our finding that the carboxy terminus of ulp <dig> 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 ulp <dig> to these substrates.
our findings are confirmed by biochemical analyses that focus on the sumo-binding properties of ulp1c580s, 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 ulp <dig> interacts dynamically with its substrates and also provides potentially useful new directions for the study of ulp1-interacting proteins.
RESULTS
ulp <dig> localization to the nuclear envelope and the septin ring
as part of a larger study to identify how ulp <dig> is targeted to its mitotic desumoylation substrates, we analyzed the localization of gfp-tagged versions of both the full-length wild-type  ulp <dig> and a catalytically inactive mutant of ulp <dig>  in g2/m-arrested yeast cells .
the c580s mutation replaces the catalytic cysteine with a serine residue, rendering the ulp <dig> sumo protease catalytically inactive  <cit> .
both fusion proteins were expressed under the control of the ulp <dig> promoter on low-copy plasmids, and images were collected using a fluorescence microscope.
consistent with its localization to npcs, wt ulp <dig> stained only the ne of arrested yeast cells .
unexpectedly, however, full-length ulp1c580s was enriched at both the bud-neck and the ne of g2/m-arrested cells .
this bud-neck localization of ulp1c580s is reminiscent of the localization of the septin ring.
several sumoylated septins have been shown to be ulp <dig> substrates, and we show in this study that the septin cdc <dig> is highly sumoylated during g2/m arrest .
furthermore, a catalytically inactive ulp <dig> mutant colocalizes with the septin cdc <dig> in g2/m-arrested  cells .
therefore, ulp1c580s resides at the bud-neck localized septin ring.
our data suggest that introducing the c580s mutation into the catalytic domain of ulp <dig> 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  <cit> .
sumo conjugation is required for ulp <dig> localization to the septin ring
we tested whether the c580s mutation that visually increased the ability of ulp <dig> to associate with the septin ring in vivo was, in fact, sumo-dependent.
for this purpose, the ulp1c580s construct was expressed in two smt <dig> mutants  or two sumo pathway mutants   <cit> .
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 smt <dig> wt strain.
in our analyses, we found that in the absence of both sumo chains  and a mutant sumo protein , the localization of ulp1c580s to the septin ring was reduced  in frequency and intensity but not abolished .
we obtained different results in the ubc9- <dig> strain, a mutant of the sumo e2-conjugating enzyme which impairs sumo conjugation, and in the siz1Δ siz2Δ strain, a sumo e <dig> ligase double-mutant that lacks sumoylation of septins and many other proteins  <cit> .
consistent with a role for smt <dig> in the localization of ulp1c580s, we were unable to detect septin ring localization of ulp1c580s in ubc9- <dig> and siz1Δ siz2Δ strains.
however, ulp1c580s was retained at the ne .
as an additional control, the septin ring localization of gfp-tagged smt <dig> was undetectable in both ubc9- <dig> and siz1Δ siz2Δ strains .
smt <dig> conjugation is required for ulp <dig> localization to the septin ring.
therefore, ulp <dig> 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 ulp <dig>
distinct and separate ulp <dig> domains are required for localization to the septin ring
our finding that a single point mutation in ulp <dig>  c580s, dramatically enhanced the localization of full-length ulp <dig> to the septin ring in a sumo-dependent fashion warranted a more detailed analysis of the targeting domains in ulp <dig>  therefore, we generated a collection of gfp-tagged ulp <dig> truncations and domains that were expressed under control of the ulp <dig> promoter.
we reasoned that the truncations and domains of ulp <dig> 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 ulp <dig> and full-length ulp1c580s .
our choice of individual constructs was guided by previous findings that ulp <dig> consists of functionally separate domains.
these domains include a kap121-binding domain with a role in septin localization , a kap95- and kap60-binding domain with a role in npc anchoring , a cc domain harboring a nuclear export signal  and the catalytic ud   <cit> .
representative images of these domains and their subcellular localization are shown in figures 3a and 3b.
as previously reported, we found that the ulp <dig> protein lacking region  <dig>  localized to the septin ring in the majority of large-budded, arrested cells  <cit> .
therefore, region  <dig> of ulp <dig> 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 .
next we investigated other residues of ulp <dig> that could affect the septin ring localization of the ulp1c580s mutant, possibly by interfering with its targeting to sumoylated substrates.
aspartate  <dig>  in ulp <dig> is required to form an essential salt bridge with arginine  <dig> of smt <dig>  <cit> .
therefore, we introduced a d451n mutation into ulp1c580s and found that it abolished the accumulation of the full-length ulp <dig> double-mutant  at the septin ring .
this finding underscores the importance of smt <dig> in targeting full-length ulp <dig> to the septin ring shown in figure  <dig>  additionally, it may indicate that d <dig> is required for targeting of sumoylated proteins and the c580s mutation is required for retention of ulp <dig> at the septin ring.
most intriguingly, we found that a truncation consisting only of region  <dig> with the c580s mutation ) displayed robust septin ring localization in 59% of cells .
in stark contrast, regions  <dig> and  <dig> and wt region  <dig>  lacking the c580s mutation, failed to localize to the septin ring .
however, in strains with diffuse ulp <dig> truncations, the septin ring stays intact.
therefore, necessary and sufficient sumo-dependent targeting information is contained in region  <dig> of ulp <dig>  but not in regions  <dig> and  <dig>  the latter conclusion is confirmed by two-hybrid assays with smt <dig>
the previously published cocrystal structure of ulp <dig> with smt <dig>  reveals that amino acids  <dig> to  <dig> of region  <dig> make extensive contact with smt <dig> and constitute an exposed sbs  <cit>  .
the sbs is situated next to, but does not include, the critical d <dig> residue that contacts smt <dig>  <cit> .
additionally, deletion of this sbs in region  <dig> of ulp <dig> abolishes the complementation of a ulp1Δ deletion mutant  <cit> .
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 ulp <dig> 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  <dig> matches.
our results contained  <dig> different species.
among these species, 61% of the sequences were identified as verified or predicted sentrin/sumo protease/ulp <dig> genes, 24% were identified as unnamed protein products or hypothetical genes and 15% were classified as "other" .
the alignment of these sequences allowed us to identify areas of strong conservation .
using this approach, we identified several highly conserved residues in the sbs.
however, these amino acids did not contact smt <dig> in the published cocrystal structure and likely play structural roles in ulp <dig> folding  <cit> .
we investigated the effect of deleting the entire sbs domain on the localization of ulp <dig>  a ulp1sbsΔ construct did not localize to the septin ring in the majority of cells .
these results match those obtained by li and hochstrasser  <cit>  using a wt ulp1Δsbs construct .
we confirmed that sbsΔ and other ulp <dig> 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 .
this construct was distributed diffusely throughout the cell and failed to localize to the septin ring .
these data suggest that the sbs domain of region  <dig> may be required for the initial interaction with sumoylated substrates, but additional features of ulp <dig> are required for targeting  and retention  of this sumo protease at the septin ring.
next we directed our attention to the temperature-sensitive ulp1ts- <dig> allele.
this mutant allele causes cells to arrest in mitosis and accumulates unprocessed sumo precursor and sumoylated proteins  <cit> .
our ulp1ts construct of region  <dig>  ulp1ts, contains three mutations , and introduction of c580s into ulp1ts showed a greatly reduced incidence and intensity of septin ring localization .
we noted that the  mutation in the ts construct was located next to the salt bridge-forming residue d <dig> described above and that both residues were highly conserved in the consensus sequence of ulp1-like molecules .
this suggests that residues altered in ulp1ts- <dig>  specifically n <dig>  may contribute to smt <dig> interaction and possibly substrate targeting.
it is possible that n450s perturbs the salt-bridge interaction formed between d <dig> of ulp <dig> and r <dig> of smt <dig>  thus reducing the interaction with smt <dig> and contributing to the temperature-sensitive phenotype.
in support of this hypothesis, correction of the n450s mutation in ulp1ts  partially rescued the slow growth defect of a ulp1Δ strain at 30°c and 37°c .
the effect of the ulp1ts mutation on ulp1's ability to interact with smt <dig> is explored in more detail below.
we tested which domains of ulp <dig> are required for targeting and retention at the septin ring in vivo.
using our region  <dig> and region  <dig> gfp-tagged constructs , we show that septin-targeting information is not contained in the domains that are known to interact with karyopherins.
the Δ <dig> construct recapitulates the previous finding that kap121-binding to region  <dig> regulates access of ulp <dig> to the septin ring.
the full-length ulp1c580s mutant reveals that a single substrate-trapping mutation in ulp <dig> suffices to enrich ulp <dig> at the septin ring.
to show that an smt <dig> interaction is required for the septin localization of ulp1c580s, we created the double-mutant .
the d451n mutation is known to destroy an essential salt bridge formed between smt <dig> and ulp <dig>  next, using the ulp1c580s construct, we show that the septin-targeting information is limited to region  <dig> of ulp <dig> .
further truncating ulp1c580s revealed that a previously identified sbs domain in ulp1c580s is also involved in septin targeting and retention.
to test whether mutations found in region  <dig> of the ulp1ts mutant play a role in septin localization, we introduced three additional mutations, i435v, n450s and i504t, into ulp1c580s.
this ulp1ts c580s construct showed a reduced ability to enrich at the septin ring , suggesting that its ability to interact with sumoylated septins may be reduced but not abolished.
kap121-independent sumo-targeting information resides in the catalytic domain of ulp1
in the preceding sections, we described our identification of necessary and sufficient substrate-targeting information in the catalytic domain  of ulp <dig>  however, region  <dig> of ulp <dig> may not be the only domain involved in targeting to the septin ring.
region  <dig> of ulp <dig>  the kap121-binding domain, has previously been implicated in septin targeting.
specifically, it has been reported that kap <dig> is required for targeting ulp <dig> to the septin ring during mitosis  <cit> .
therefore, we decided to assess the role of kap <dig> in the substrate-targeting of ulp <dig>  specifically, we used a kap121ts mutant  <cit>  to assess the septin ring-targeting of wt ulp <dig>  full-length ulp1c580s and ulp <dig>  in our analysis, we found that full-length ulp1c580s required kap <dig> function for targeting to the septin ring.
at the permissive temperature , 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 .
surprisingly, the ulp <dig> truncation was localized to the septin ring at the permissive and nonpermissive temperatures in a kap121ts strain.
as shown herein, ulp <dig> resided both inside the nucleus and at the septin ring at 30°c and 37°c .
our data suggest that ulp <dig> contains both kap121-dependent and kap121-independent septin ring-targeting information.
the only requirement to detect full-length ulp <dig> at the septin ring is the c580s mutation and functional kap <dig> .
in contrast ulp <dig>  which lacks all domains required for npc interaction through kap <dig>  kap <dig> and kap <dig>  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  of ulp <dig>
multiple features in the catalytic domain of ulp <dig> affect sumo interactions
our finding that a single amino acid change in the catalytic domain of ulp <dig> results in greatly enhanced, sumo-dependent localization to the septin ring also prompted us to investigate the two-hybrid interactions of ulp <dig> with budding yeast sumo .
full-length wt ulp <dig>  the full-length catalytically inactive ulp1c580s mutant, the ulp <dig> kap121-interacting domain , the ulp <dig> kap60/kap95-interacting domain  and the catalytic domain  failed to interact with smt3-bd .
however, the catalytically inactive ulp <dig> truncation interacted reproducibly and above background with smt <dig> .
ulp <dig> appears to interact only weakly with our smt <dig> two-hybrid bait construct, as indicated by fewer colonies on the reporter media .
however, such an interpretation assumes an equally available pool of both bait and prey.
one possible explanation for this result is that the ulp <dig> two-hybrid prey construct interacts with a number of available substrates in the cell , and, as a result, this sequestration is no longer available to the smt <dig> 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 ulp <dig>  making more of the pool available to engage the smt <dig> bait construct.
consistent with this model, when we introduced the ulp1ts mutations into the substrate-trapping ulp <dig> prey construct, we observed a more robust two-hybrid interaction with the smt <dig> bait .
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 ulp <dig> to interact with smt <dig>
next we focused on the d451n mutant of ulp <dig> that prevents the interaction of ulp <dig> with sumo  <cit> .
as shown above, d451n, when introduced into ulp <dig>  prevents localization of this construct to the septin ring .
correspondingly, we found that introduction of the d451n mutation into ulp <dig> completely abolished the two-hybrid interaction with smt <dig> .
these observations provide evidence that the targeting of ulp <dig> to sumoylated substrates is a closely balanced act involving both smt <dig> targeting and retention.
ulp <dig> truncation binds sumo and sumo-modified proteins
we hypothesized that if ulp <dig> were to interact avidly with smt <dig>  this mutated moiety of ulp <dig> could efficiently interact with sumo adducts in vitro.
therefore, to test the direct interaction of ulp <dig> with sumo, we fused this domain to the carboxy terminus of maltose-binding protein  and expressed the recombinant fusion protein in bacteria.
subsequently, the mbp-ulp <dig> fusion protein was purified from bacterial extracts and bound to amylose resin .
as a control to assess the ability of mbp-ulp <dig> to interact with sumoylated proteins, we also purified a second mbp-fused ulp <dig> construct lacking the sbs domain Δsbs).
first we determined the ability of mbp-ulp <dig> to affinity-purify sumoylated proteins from crude yeast cell extracts.
ulp1ts- <dig> cells expressing flag-tagged smt <dig> were grown to log phase prior to preparation of yeast cell extracts .
these extracts were then incubated with resin-bound mbp-ulp <dig>  mbp-ulp1-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   could clearly be detected bound to mbp-ulp <dig>  but not to the mbp-ulp1-Δsbs control protein .
we identified both unconjugated flag-smt <dig> proteins as well as several higher-molecular-weight adducts.
these data suggest that ulp <dig> can efficiently bind and enrich sumoylated proteins from crude yeast cell extracts.
to demonstrate the versatility of ulp1-aided smt <dig> purification, we also purified monomeric and conjugated gfp-smt <dig> from yeast cells .
additionally, we probed the extracts and eluted proteins shown in figure 6b with an anti-cdc <dig> antibody, which revealed the specific copurification of cdc <dig> with immobilized ulp <dig> .
in the reciprocal experiment, we tested whether a gfp-tagged ulp <dig> construct expressed in yeast cells could bind immobilized sumo <dig>  which is highly conserved to yeast smt <dig>  in this experiment, yeast cells expressing cen-plasmid levels of gfp-tagged ulp <dig>  ulp <dig> or the ulp1-Δsbs  were grown to log phase prior to preparation of yeast cell extracts.
individual extracts were then incubated with sumo <dig> immobilized on agarose beads .
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 ulp <dig> could be detected in the wces and bound to the sumo <dig> agarose .
in contrast, neither the wt catalytic domain of ulp <dig> ) nor ulp <dig> was bound to sumo <dig> agarose.
similarly, the ulp <dig> could also be purified on sumo <dig> agarose .
we also tested whether immobilized ulp <dig> could be used to purify sumo chains.
in this experiment, we incubated purified sumo <dig> chains with our immobilized ulp <dig> or the unbound amylose resin.
after washing, bound sumo <dig> chains were eluted, separated on sds-page gels and examined by western blot analysis with an anti-sumo <dig> antibody.
sumo <dig> chains could clearly be detected in the input  and bound the mbp-ulp <dig> , but not the resin-only control .
both lower- and higher-molecular-weight adducts of sumo <dig> were purified with a preference for higher-molecular-weight chains .
these data suggest that ulp <dig> can efficiently bind and enrich sumo <dig> chains in vitro and that the mbp fusion of ulp <dig> may also be useful for the purification of sumoylated proteins from mammalian cells.
a sumo2-binding platform for substrate ubiquitination
stubls such as the yeast slx5/slx <dig> heterodimer and the human rnf <dig> protein efficiently ubiquitinated proteins modified with sumo chains  <cit> .
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 ulp <dig> to interact with sumo may therefore provide a simple way to purify a sumo chain-modified stubl target of choice.
to test whether ulp <dig> can serve as a platform to modify a purified protein with sumo <dig> chains, we incubated the immobilized mbp-ulp <dig> with sumo <dig> chains.
unbound sumo <dig> chains were removed by washing.
the mbp-ulp <dig> sumo <dig> chain complex was then eluted and added into a stubl in vitro ubiquitinated reaction containing recombinant rnf <dig> .
proteins in the stubl-mediated ubiquitination assay were separated on sds-page gels and examined by western blot analysis with an anti-sumo <dig> antibody .
consistent with previous observations, we were able to detect ubiquitinated sumo <dig> chains after the stubl reaction.
this ubiquitination was dependent on rnf <dig> and sumo <dig> chains.
on the basis of these results, we propose that ulp <dig> may provide a useful, widely applicable tool for the study of sumoylated proteins and stubl targets .
discussion
in this study, we have demonstrated that region  <dig> of ulp <dig>  the catalytic domain, contains critical information for subcellular targeting to sumoylated substrates, including the septin cdc <dig>  to determine how ulp <dig> is targeted to its substrates, we took advantage of a catalytically inactive ulp <dig> mutant  that exhibited partial redistribution from the ne to the septin ring of dividing yeast cells.
we found that the relocalization of ulp <dig> depended on functional smt <dig> and sumoylated proteins at the septin ring of dividing cells.
importantly, using this novel ulp <dig> in vivo septin-ring localization assay, we traced the critical targeting information to two features in region  <dig> of ulp <dig>  a previously identified sbs  and a sumo-contacting residue  that resides near the carboxy terminus .
d <dig> of ulp <dig> has previously been shown to contact smt <dig> through a salt-bridge interaction  <cit> .
indeed, in our analysis, we provide evidence that the salt bridge-disrupting d451n mutation abolishes the sumo-dependent targeting of ulp <dig> to septins and prevents the two-hybrid interaction of the catalytic domain of ulp <dig> with smt <dig> .
the sole requirements for the enrichment of full-length ulp <dig> at the septin ring were the catalytically inactivating c580s mutation in the catalytic domain of ulp <dig> and functional kap <dig>  this finding has important implications for the targeting role played by the amino-terminal karyopherin-binding domains of ulp <dig> .
additionally, smt <dig> processing appears to be required for substrate release.
the catalytically inactive ulp <dig> mutant is predominantly localized to the septin ring and nucleus of dividing yeast cells, whereas the catalytically active wt ulp <dig> shows merely diffuse staining throughout the cell .
this is not due to differential expression or stability of either gfp fusion, as similar amounts of proteins were detected in cell extracts .
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 .
therefore, we propose that ulp <dig> first targets and docks smt <dig> through the sbs domain and the salt bridge-forming d <dig> residue, then traps it in place because of its inability to cleave after the di-glycine motif of smt <dig>  we can assume that a trapped substrate prevents further catalysis or interactions with other smt <dig> molecules, an assessment that is borne by our finding that, despite its septin-targeting and sumo-binding properties, ulp <dig> interacts only weakly with smt <dig> in a two-hybrid assay .
a better understanding of how the sumoylated substrate is trapped by ulp <dig> 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 ulp <dig> with smt <dig> relies on multiple hydrophobic and salt-bridge interactions between the catalytic domain  of ulp <dig> and the carboxy-terminal extension of smt <dig>  by making multiple contacts with smt <dig>  ulp <dig> is particularly well-suited to interact with a wide variety of sumoylated substrates  <cit> .
other sumo proteases, ulp <dig> and several senp proteins , are believed to interact noncovalently with their sumoylated substrates through dedicated sims  <cit> .
on the basis of our structure-function analysis of region  <dig>  ulp <dig> seems to employ a unique mode of interaction with smt <dig> and sumoylated substrates.
ulp <dig> does not appear to contain bona fide canonical sims, and neither of the amino-terminal domains of ulp <dig>  interact with smt <dig> or become enriched at the septin ring  <cit> .
this assessment is also underscored by the arrangement of smt <dig> and ulp <dig> in the cocrystal structure  <cit> .
the hydrophobic groove of smt <dig> that would interact with a sim-containing protein is turned away from the domains of ulp <dig> that interact with smt <dig>  interestingly, this may suggest that ulp <dig> 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 ulp <dig> 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 ulp <dig> and are arguably the most prevalent  <cit> ; therefore, they may be readily scored in our septin ring targeting assay .
we predict that ulp <dig> is also targeted to other cytosolic and septin-bound sumoylated substrate proteins, such as the karyogamy protein kar <dig>  <cit> .
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 ulp <dig>
as detailed below, ulp <dig> also provides a useful tool with which to purify these sumoylated proteins , and such studies are underway .
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 ulp <dig>  our alignments of the sbs domain and the juxtaposed salt bridge-forming d <dig> residue reveal that this mode of targeting may also be conserved in other metazoan ulp1-like sumo proteases .
though we clearly show herein that ulp <dig> becomes enriched at the septin ring, we do not yet fully understand how ulp <dig> arrives at this subcellular localization.
our findings support the previous observation  <cit>  that kap <dig> plays an important role in promoting ulp <dig> targeting to the septin ring.
similar to a previously described ulp <dig> mutant that lacks the kap60/kap <dig> binding domain   <cit> , the septin ring localization of the full-length ulp <dig> protein described herein is dependent on functional kap <dig>  it is unlikely that the association with kap <dig> shuttles ulp <dig> to the septins.
rather, in mitosis, kap <dig> becomes associated with a transport inhibitory nucleoporin, nup <dig>  and may thus exclude ulp <dig> access to the inner phase of the npc  <cit> .
this suggests that, in the absence of kap121-binding, a fraction of ulp <dig> is free to associate with sumoylated septins.
our studies confirm that ulp <dig> lacking the kap60/kap95-binding domain  is enriched at the npc and the septin ring .
we extend these observations by showing that the ability to target sumoylated septins resides in the catalytic domain  of ulp <dig>  we found that a ulp <dig> mutant, but not wt ulp <dig>  is enriched at the septin ring in the absence of kap <dig>  these data suggest that both the ulp <dig> mutant and wt ulp <dig> can interact with sumoylated septins, but, unlike the subtrate-trapping ulp <dig> mutant, the catalytically active wt ulp <dig> may be quickly released after desumoylation of the target protein, giving it a diffuse appearance in the cell.
how kap <dig> helps ulp1-Δ <dig> to be retained at the septin ring in the absence of the c580s mutation is currently unclear.
kap <dig> may theoretically promote the interaction with a septin ring-localized protein.
however, the localization of kap <dig> to septins has not previously been reported.
one intriguing aspect of our study is the results of our analysis of the substrate-trapping ulp <dig> construct.
three lines of evidence reveal the avid interaction of ulp <dig> 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, ulp1c580s interacts with smt <dig> in a two-hybrid assay, whereas wt ulp <dig> does not.
third, the purified recombinant ulp <dig> protein is a potent affinity-tag for the purification of smt <dig> conjugates and sumo-modified proteins.
furthermore, in preliminary biacore analyses, we determined the kd for the interaction between ulp <dig> and sumo <dig> to be  <dig>  nm, which is about  <dig> times stronger than the interaction between a sim and sumo, as previously reported by hecker and co-workers  <cit> .
a related study involving the c603s mutant of the human senp <dig> protease confirms our assessment of the substrate-trapping feature.
those authors observed relocalization of their senp <dig> mutant in vivo to pml nuclear bodies and domains of the hdac <dig> protein, suggesting that sumo-dependent targeting may be a conserved feature of ulp1-like sumo proteases  <cit> .
the latter may also provide a useful strategy for the identification of mitotically important desumoylation substrates.
indeed, two-hybrid screens with ulp <dig> in the laboratory have already identified several novel cytosolic desumoylation targets .
we are also exploring the ability of ulp <dig> to act as a sumo chain-binding tag that can be used to promote the interaction of putative stubl target proteins with rnf <dig> and other stubls .
how ulp <dig> and other sumo proteases target specific mitotic substrates for desumoylation remains unknown.
our analysis of sumo-dependent ulp <dig> 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 ulp <dig> construct may prove useful for the identification of clinically relevant targets of conserved ulp1-like sumo proteases in yeast and human cells.
CONCLUSIONS
ulp <dig> 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 ulp <dig>  an npc-associated sumo protease, targets mitotic desumoylation targets in the cytosol.
the current study of ulp <dig> reveals for the first time that  specific determinants of ulp1's catalytic domain are utilized to target the septins at the bud-neck of dividing cells,  ulp <dig> requires sumo and intact sumoylation for correct cytosolic targeting,  ulp <dig>  unlike ulp <dig>  relies on specific salt bridge-forming residues for substrate targeting,  septin-targeting information in the catalytic domain of ulp <dig> is independent of kap <dig> and  the substrate-targeting domain of ulp <dig> can be modified to identify and purify sumoylated substrates of ulp <dig> in genetic screens and in biochemical analyses.

 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, 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, which mediate their ubiquitination. sumo proteins are highly conserved from yeast to humans. yeast cells express one sumo protein, and vertebrates express three isoforms. sumo sumo and yeast smt can form sumo chains. sumo on the other hand, lacks the internal lysine required for polymerization and may function as a chain terminator for sumo and sumo 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. in metazoans, the dysregulation of sumoylation adversely affects developmental processes and has been implicated in the progression of neurodegeneration, cancer and infectious diseases. more than 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 e heterodimer that activates sumo, as well as e and e enzymes that aid in the conjugation and ligation of sumo to proper target proteins. two yeast sumo proteases, ulp and ulp contain a conserved cysteine protease domain that can remove the sumo moiety from modified proteins. recent evidence suggests that ulp and its mammalian orthologs susp1/senp and senp play a role in the removal of sumo and sumo chains from nuclear proteins. ulp on the other hand, has two contrasting cellular functions. ulp facilitates sumoylation by processing precursor sumo into its conjugation competent form. conversely, ulp also facilitates desumoylation by removing sumo from nuclear and cytosolic proteins after conjugation. therefore, impairment of ulp 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. the substrate specificity of sumo proteases is at least in part regulated through their localization. for example, certain yeast and vertebrate sumo proteases localize within the nucleus. in contrast, both yeast and vertebrate sumo proteases reside at the nuclear envelope through their interactions with the nuclear pore complex. distinct domains have been identified that are required for ulp npc localization and sumo processing. the ulp localization domain promotes interaction with karyopherins, which are soluble proteins that mediate transport across the ne and help localize ulp to the nucleoplasmic side of the npc. the ulp localization domain can be subdivided into region and region. juxtaposed to the npc localization domain of ulp is a coiled-coil domain with a putative nuclear export signal and region the catalytically active, conserved ubiquitin-like protease domain of ulp. only regions and are involved in ulp localization to the npc, and the karyopherins seem to play a redundant role. npc association of ulp requires several proteins, including the nucleoporins nup and nup the silencing protein esc and the myosin-like proteins mlp1/. together these proteins may provide a scaffold for the functional regulation and substrate access of ulp at the npc. the identification of npc localization domains in ulp 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 have been predicted to be localized in the yeast sumo protease ulp as well as in mammalian senp senp senp and senp. even though sims have not been identified in ulp the crystal structure of the catalytic domain bound to smt reveals that both proteins interact through multiple residues that are distributed across a sumo-binding surface on the sumo protease. only the carboxy terminus of bound smt 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. it has been suggested that this configuration may allow for the accommodation of many different sumoylated proteins as well as sumo precursors. ulp and several other sumo proteases play important roles in mitosis. in budding yeast, loss of ulp1-mediated desumoylation leads to cell-cycle progression defects and cell death. this observation suggests that ulp 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 ulp desumoylation substrates. how the npc-localized ulp 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, kap blocks ulp1's access to its npc-binding site and thus promotes an interaction of ulp with septins. a deletion mutant of ulp lacking region, 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 also plays a role in nucleolar accumulation of ulp after ethanol-induced stress. one set of cytosolic substrates of the ulp sumo protease are the septins. 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 cdc cdc cdc cdc and shs1/sep 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 cdc cdc and shs 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. cell-cycle arrest with nocodazole, a microtubule depolymerizing drug, greatly increases sumo conjugation to septins. septin sumoylation in budding yeast is mediated by the sumo e ligase siz. during most of the cell cycle, siz resides in the nucleus. however, at the m phase, siz exits the nucleus to sumoylate septin proteins and possibly other cytosolic substrates. deletion of siz 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 ulp even though ulp remains visibly enriched at the npc. in the current study, we focused on the sumo protease ulp as detailed above, ulp 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 ulp in theory, this localization is well-suited to give ulp access to some nuclear substrates and those in transit across the ne. however, both nuclear and cytosolic desumoylation targets of ulp have been identified, posing an interesting question. how is ulp directed to its cytosolic desumoylation targets? to answer these questions, we sought to identify features of ulp required for substrate targeting in vivo and in vitro. herein we describe our finding that the carboxy terminus of ulp 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 ulp to these substrates. our findings are confirmed by biochemical analyses that focus on the sumo-binding properties of ulp1c580s, 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 ulp interacts dynamically with its substrates and also provides potentially useful new directions for the study of ulp1-interacting proteins.  ulp localization to the nuclear envelope and the septin ring as part of a larger study to identify how ulp is targeted to its mitotic desumoylation substrates, we analyzed the localization of gfp-tagged versions of both the full-length wild-type ulp and a catalytically inactive mutant of ulp in g2/m-arrested yeast cells. the c580s mutation replaces the catalytic cysteine with a serine residue, rendering the ulp sumo protease catalytically inactive. both fusion proteins were expressed under the control of the ulp promoter on low-copy plasmids, and images were collected using a fluorescence microscope. consistent with its localization to npcs, wt ulp stained only the ne of arrested yeast cells. unexpectedly, however, full-length ulp1c580s was enriched at both the bud-neck and the ne of g2/m-arrested cells. this bud-neck localization of ulp1c580s is reminiscent of the localization of the septin ring. several sumoylated septins have been shown to be ulp substrates, and we show in this study that the septin cdc is highly sumoylated during g2/m arrest. furthermore, a catalytically inactive ulp mutant colocalizes with the septin cdc in g2/m-arrested cells. therefore, ulp1c580s resides at the bud-neck localized septin ring. our data suggest that introducing the c580s mutation into the catalytic domain of ulp 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. sumo conjugation is required for ulp localization to the septin ring we tested whether the c580s mutation that visually increased the ability of ulp to associate with the septin ring in vivo was, in fact, sumo-dependent. for this purpose, the ulp1c580s construct was expressed in two smt mutants or two sumo pathway mutants. 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 smt wt strain. in our analyses, we found that in the absence of both sumo chains and a mutant sumo protein, the localization of ulp1c580s to the septin ring was reduced in frequency and intensity but not abolished. we obtained different results in the ubc9- strain, a mutant of the sumo e2-conjugating enzyme which impairs sumo conjugation, and in the siz1Δ siz2Δ strain, a sumo e ligase double-mutant that lacks sumoylation of septins and many other proteins. consistent with a role for smt in the localization of ulp1c580s, we were unable to detect septin ring localization of ulp1c580s in ubc9- and siz1Δ siz2Δ strains. however, ulp1c580s was retained at the ne. as an additional control, the septin ring localization of gfp-tagged smt was undetectable in both ubc9- and siz1Δ siz2Δ strains. smt conjugation is required for ulp localization to the septin ring. therefore, ulp 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 ulp distinct and separate ulp domains are required for localization to the septin ring our finding that a single point mutation in ulp c580s, dramatically enhanced the localization of full-length ulp to the septin ring in a sumo-dependent fashion warranted a more detailed analysis of the targeting domains in ulp therefore, we generated a collection of gfp-tagged ulp truncations and domains that were expressed under control of the ulp promoter. we reasoned that the truncations and domains of ulp 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 ulp and full-length ulp1c580s. our choice of individual constructs was guided by previous findings that ulp consists of functionally separate domains. these domains include a kap121-binding domain with a role in septin localization, a kap95- and kap60-binding domain with a role in npc anchoring, a cc domain harboring a nuclear export signal and the catalytic ud. representative images of these domains and their subcellular localization are shown in figures 3a and 3b. as previously reported, we found that the ulp protein lacking region localized to the septin ring in the majority of large-budded, arrested cells. therefore, region of ulp 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. next we investigated other residues of ulp that could affect the septin ring localization of the ulp1c580s mutant, possibly by interfering with its targeting to sumoylated substrates. aspartate in ulp is required to form an essential salt bridge with arginine of smt. therefore, we introduced a d451n mutation into ulp1c580s and found that it abolished the accumulation of the full-length ulp double-mutant at the septin ring. this finding underscores the importance of smt in targeting full-length ulp to the septin ring shown in figure additionally, it may indicate that d is required for targeting of sumoylated proteins and the c580s mutation is required for retention of ulp at the septin ring. most intriguingly, we found that a truncation consisting only of region with the c580s mutation ) displayed robust septin ring localization in 59% of cells. in stark contrast, regions and and wt region lacking the c580s mutation, failed to localize to the septin ring. however, in strains with diffuse ulp truncations, the septin ring stays intact. therefore, necessary and sufficient sumo-dependent targeting information is contained in region of ulp but not in regions and the latter conclusion is confirmed by two-hybrid assays with smt the previously published cocrystal structure of ulp with smt reveals that amino acids to of region make extensive contact with smt and constitute an exposed sbs. the sbs is situated next to, but does not include, the critical d residue that contacts smt. additionally, deletion of this sbs in region of ulp 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 ulp 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 matches. our results contained different species. among these species, 61% of the sequences were identified as verified or predicted sentrin/sumo protease/ulp genes, 24% were identified as unnamed protein products or hypothetical genes and 15% were classified as "other". the alignment of these sequences allowed us to identify areas of strong conservation. using this approach, we identified several highly conserved residues in the sbs. however, these amino acids did not contact smt in the published cocrystal structure and likely play structural roles in ulp folding. we investigated the effect of deleting the entire sbs domain on the localization of ulp a ulp1sbsΔ construct did not localize to the septin ring in the majority of cells. these results match those obtained by li and hochstrasser using a wt ulp1Δsbs construct. we confirmed that sbsΔ and other ulp 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. this construct was distributed diffusely throughout the cell and failed to localize to the septin ring. these data suggest that the sbs domain of region may be required for the initial interaction with sumoylated substrates, but additional features of ulp are required for targeting and retention of this sumo protease at the septin ring. next we directed our attention to the temperature-sensitive ulp1ts- allele. this mutant allele causes cells to arrest in mitosis and accumulates unprocessed sumo precursor and sumoylated proteins. our ulp1ts construct of region ulp1ts, contains three mutations, and introduction of c580s into ulp1ts showed a greatly reduced incidence and intensity of septin ring localization. we noted that the mutation in the ts construct was located next to the salt bridge-forming residue d described above and that both residues were highly conserved in the consensus sequence of ulp1-like molecules. this suggests that residues altered in ulp1ts- specifically n may contribute to smt interaction and possibly substrate targeting. it is possible that n450s perturbs the salt-bridge interaction formed between d of ulp and r of smt thus reducing the interaction with smt and contributing to the temperature-sensitive phenotype. in support of this hypothesis, correction of the n450s mutation in ulp1ts partially rescued the slow growth defect of a ulp1Δ strain at 30°c and 37°c. the effect of the ulp1ts mutation on ulp1's ability to interact with smt is explored in more detail below. we tested which domains of ulp are required for targeting and retention at the septin ring in vivo. using our region and region gfp-tagged constructs, we show that septin-targeting information is not contained in the domains that are known to interact with karyopherins. the Δ construct recapitulates the previous finding that kap121-binding to region regulates access of ulp to the septin ring. the full-length ulp1c580s mutant reveals that a single substrate-trapping mutation in ulp suffices to enrich ulp at the septin ring. to show that an smt interaction is required for the septin localization of ulp1c580s, we created the double-mutant. the d451n mutation is known to destroy an essential salt bridge formed between smt and ulp next, using the ulp1c580s construct, we show that the septin-targeting information is limited to region of ulp. further truncating ulp1c580s revealed that a previously identified sbs domain in ulp1c580s is also involved in septin targeting and retention. to test whether mutations found in region of the ulp1ts mutant play a role in septin localization, we introduced three additional mutations, i435v, n450s and i504t, into ulp1c580s. this ulp1ts c580s construct showed a reduced ability to enrich at the septin ring, suggesting that its ability to interact with sumoylated septins may be reduced but not abolished. kap121-independent sumo-targeting information resides in the catalytic domain of ulp1 in the preceding sections, we described our identification of necessary and sufficient substrate-targeting information in the catalytic domain of ulp however, region of ulp may not be the only domain involved in targeting to the septin ring. region of ulp the kap121-binding domain, has previously been implicated in septin targeting. specifically, it has been reported that kap is required for targeting ulp to the septin ring during mitosis. therefore, we decided to assess the role of kap in the substrate-targeting of ulp specifically, we used a kap121ts mutant to assess the septin ring-targeting of wt ulp full-length ulp1c580s and ulp in our analysis, we found that full-length ulp1c580s required kap function for targeting to the septin ring. at the permissive temperature, 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. surprisingly, the ulp truncation was localized to the septin ring at the permissive and nonpermissive temperatures in a kap121ts strain. as shown herein, ulp resided both inside the nucleus and at the septin ring at 30°c and 37°c. our data suggest that ulp contains both kap121-dependent and kap121-independent septin ring-targeting information. the only requirement to detect full-length ulp at the septin ring is the c580s mutation and functional kap. in contrast ulp which lacks all domains required for npc interaction through kap kap and kap 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 of ulp multiple features in the catalytic domain of ulp affect sumo interactions our finding that a single amino acid change in the catalytic domain of ulp results in greatly enhanced, sumo-dependent localization to the septin ring also prompted us to investigate the two-hybrid interactions of ulp with budding yeast sumo. full-length wt ulp the full-length catalytically inactive ulp1c580s mutant, the ulp kap121-interacting domain, the ulp kap60/kap95-interacting domain and the catalytic domain failed to interact with smt3-bd. however, the catalytically inactive ulp truncation interacted reproducibly and above background with smt. ulp appears to interact only weakly with our smt two-hybrid bait construct, as indicated by fewer colonies on the reporter media. however, such an interpretation assumes an equally available pool of both bait and prey. one possible explanation for this result is that the ulp two-hybrid prey construct interacts with a number of available substrates in the cell, and, as a result, this sequestration is no longer available to the smt 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 ulp making more of the pool available to engage the smt bait construct. consistent with this model, when we introduced the ulp1ts mutations into the substrate-trapping ulp prey construct, we observed a more robust two-hybrid interaction with the smt bait. 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 ulp to interact with smt next we focused on the d451n mutant of ulp that prevents the interaction of ulp with sumo. as shown above, d451n, when introduced into ulp prevents localization of this construct to the septin ring. correspondingly, we found that introduction of the d451n mutation into ulp completely abolished the two-hybrid interaction with smt. these observations provide evidence that the targeting of ulp to sumoylated substrates is a closely balanced act involving both smt targeting and retention. ulp truncation binds sumo and sumo-modified proteins we hypothesized that if ulp were to interact avidly with smt this mutated moiety of ulp could efficiently interact with sumo adducts in vitro. therefore, to test the direct interaction of ulp with sumo, we fused this domain to the carboxy terminus of maltose-binding protein and expressed the recombinant fusion protein in bacteria. subsequently, the mbp-ulp fusion protein was purified from bacterial extracts and bound to amylose resin. as a control to assess the ability of mbp-ulp to interact with sumoylated proteins, we also purified a second mbp-fused ulp construct lacking the sbs domain Δsbs). first we determined the ability of mbp-ulp to affinity-purify sumoylated proteins from crude yeast cell extracts. ulp1ts- cells expressing flag-tagged smt were grown to log phase prior to preparation of yeast cell extracts. these extracts were then incubated with resin-bound mbp-ulp mbp-ulp1-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 could clearly be detected bound to mbp-ulp but not to the mbp-ulp1-Δsbs control protein. we identified both unconjugated flag-smt proteins as well as several higher-molecular-weight adducts. these data suggest that ulp can efficiently bind and enrich sumoylated proteins from crude yeast cell extracts. to demonstrate the versatility of ulp1-aided smt purification, we also purified monomeric and conjugated gfp-smt from yeast cells. additionally, we probed the extracts and eluted proteins shown in figure 6b with an anti-cdc antibody, which revealed the specific copurification of cdc with immobilized ulp. in the reciprocal experiment, we tested whether a gfp-tagged ulp construct expressed in yeast cells could bind immobilized sumo which is highly conserved to yeast smt in this experiment, yeast cells expressing cen-plasmid levels of gfp-tagged ulp ulp or the ulp1-Δsbs were grown to log phase prior to preparation of yeast cell extracts. individual extracts were then incubated with sumo immobilized on agarose beads. 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 ulp could be detected in the wces and bound to the sumo agarose. in contrast, neither the wt catalytic domain of ulp ) nor ulp was bound to sumo agarose. similarly, the ulp could also be purified on sumo agarose. we also tested whether immobilized ulp could be used to purify sumo chains. in this experiment, we incubated purified sumo chains with our immobilized ulp or the unbound amylose resin. after washing, bound sumo chains were eluted, separated on sds-page gels and examined by western blot analysis with an anti-sumo antibody. sumo chains could clearly be detected in the input and bound the mbp-ulp, but not the resin-only control. both lower- and higher-molecular-weight adducts of sumo were purified with a preference for higher-molecular-weight chains. these data suggest that ulp can efficiently bind and enrich sumo chains in vitro and that the mbp fusion of ulp may also be useful for the purification of sumoylated proteins from mammalian cells. a sumo2-binding platform for substrate ubiquitination stubls such as the yeast slx5/slx heterodimer and the human rnf protein efficiently ubiquitinated proteins modified with sumo chains. 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 ulp to interact with sumo may therefore provide a simple way to purify a sumo chain-modified stubl target of choice. to test whether ulp can serve as a platform to modify a purified protein with sumo chains, we incubated the immobilized mbp-ulp with sumo chains. unbound sumo chains were removed by washing. the mbp-ulp sumo chain complex was then eluted and added into a stubl in vitro ubiquitinated reaction containing recombinant rnf. proteins in the stubl-mediated ubiquitination assay were separated on sds-page gels and examined by western blot analysis with an anti-sumo antibody. consistent with previous observations, we were able to detect ubiquitinated sumo chains after the stubl reaction. this ubiquitination was dependent on rnf and sumo chains. on the basis of these results, we propose that ulp may provide a useful, widely applicable tool for the study of sumoylated proteins and stubl targets. discussion in this study, we have demonstrated that region of ulp the catalytic domain, contains critical information for subcellular targeting to sumoylated substrates, including the septin cdc to determine how ulp is targeted to its substrates, we took advantage of a catalytically inactive ulp mutant that exhibited partial redistribution from the ne to the septin ring of dividing yeast cells. we found that the relocalization of ulp depended on functional smt and sumoylated proteins at the septin ring of dividing cells. importantly, using this novel ulp in vivo septin-ring localization assay, we traced the critical targeting information to two features in region of ulp a previously identified sbs and a sumo-contacting residue that resides near the carboxy terminus. d of ulp has previously been shown to contact smt through a salt-bridge interaction. indeed, in our analysis, we provide evidence that the salt bridge-disrupting d451n mutation abolishes the sumo-dependent targeting of ulp to septins and prevents the two-hybrid interaction of the catalytic domain of ulp with smt. the sole requirements for the enrichment of full-length ulp at the septin ring were the catalytically inactivating c580s mutation in the catalytic domain of ulp and functional kap this finding has important implications for the targeting role played by the amino-terminal karyopherin-binding domains of ulp. additionally, smt processing appears to be required for substrate release. the catalytically inactive ulp mutant is predominantly localized to the septin ring and nucleus of dividing yeast cells, whereas the catalytically active wt ulp shows merely diffuse staining throughout the cell. this is not due to differential expression or stability of either gfp fusion, as similar amounts of proteins were detected in cell extracts. 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. therefore, we propose that ulp first targets and docks smt through the sbs domain and the salt bridge-forming d residue, then traps it in place because of its inability to cleave after the di-glycine motif of smt we can assume that a trapped substrate prevents further catalysis or interactions with other smt molecules, an assessment that is borne by our finding that, despite its septin-targeting and sumo-binding properties, ulp interacts only weakly with smt in a two-hybrid assay. a better understanding of how the sumoylated substrate is trapped by ulp 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 ulp with smt relies on multiple hydrophobic and salt-bridge interactions between the catalytic domain of ulp and the carboxy-terminal extension of smt by making multiple contacts with smt ulp is particularly well-suited to interact with a wide variety of sumoylated substrates. other sumo proteases, ulp and several senp proteins, are believed to interact noncovalently with their sumoylated substrates through dedicated sims. on the basis of our structure-function analysis of region ulp seems to employ a unique mode of interaction with smt and sumoylated substrates. ulp does not appear to contain bona fide canonical sims, and neither of the amino-terminal domains of ulp interact with smt or become enriched at the septin ring. this assessment is also underscored by the arrangement of smt and ulp in the cocrystal structure. the hydrophobic groove of smt that would interact with a sim-containing protein is turned away from the domains of ulp that interact with smt interestingly, this may suggest that ulp 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 ulp 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 ulp and are arguably the most prevalent; therefore, they may be readily scored in our septin ring targeting assay. we predict that ulp is also targeted to other cytosolic and septin-bound sumoylated substrate proteins, such as the karyogamy protein kar. 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 ulp as detailed below, ulp also provides a useful tool with which to purify these sumoylated proteins, and such studies are underway. 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 ulp our alignments of the sbs domain and the juxtaposed salt bridge-forming d residue reveal that this mode of targeting may also be conserved in other metazoan ulp1-like sumo proteases. though we clearly show herein that ulp becomes enriched at the septin ring, we do not yet fully understand how ulp arrives at this subcellular localization. our findings support the previous observation that kap plays an important role in promoting ulp targeting to the septin ring. similar to a previously described ulp mutant that lacks the kap60/kap binding domain, the septin ring localization of the full-length ulp protein described herein is dependent on functional kap it is unlikely that the association with kap shuttles ulp to the septins. rather, in mitosis, kap becomes associated with a transport inhibitory nucleoporin, nup and may thus exclude ulp access to the inner phase of the npc. this suggests that, in the absence of kap121-binding, a fraction of ulp is free to associate with sumoylated septins. our studies confirm that ulp lacking the kap60/kap95-binding domain is enriched at the npc and the septin ring. we extend these observations by showing that the ability to target sumoylated septins resides in the catalytic domain of ulp we found that a ulp mutant, but not wt ulp is enriched at the septin ring in the absence of kap these data suggest that both the ulp mutant and wt ulp can interact with sumoylated septins, but, unlike the subtrate-trapping ulp mutant, the catalytically active wt ulp may be quickly released after desumoylation of the target protein, giving it a diffuse appearance in the cell. how kap helps ulp1-Δ to be retained at the septin ring in the absence of the c580s mutation is currently unclear. kap may theoretically promote the interaction with a septin ring-localized protein. however, the localization of kap to septins has not previously been reported. one intriguing aspect of our study is the results of our analysis of the substrate-trapping ulp construct. three lines of evidence reveal the avid interaction of ulp 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, ulp1c580s interacts with smt in a two-hybrid assay, whereas wt ulp does not. third, the purified recombinant ulp protein is a potent affinity-tag for the purification of smt conjugates and sumo-modified proteins. furthermore, in preliminary biacore analyses, we determined the kd for the interaction between ulp and sumo to be nm, which is about 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 senp protease confirms our assessment of the substrate-trapping feature. those authors observed relocalization of their senp mutant in vivo to pml nuclear bodies and domains of the hdac 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 ulp in the laboratory have already identified several novel cytosolic desumoylation targets. we are also exploring the ability of ulp to act as a sumo chain-binding tag that can be used to promote the interaction of putative stubl target proteins with rnf and other stubls. how ulp and other sumo proteases target specific mitotic substrates for desumoylation remains unknown. our analysis of sumo-dependent ulp 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 ulp construct may prove useful for the identification of clinically relevant targets of conserved ulp1-like sumo proteases in yeast and human cells.  ulp 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 ulp an npc-associated sumo protease, targets mitotic desumoylation targets in the cytosol. the current study of ulp reveals for the first time that specific determinants of ulp1's catalytic domain are utilized to target the septins at the bud-neck of dividing cells, ulp requires sumo and intact sumoylation for correct cytosolic targeting, ulp unlike ulp relies on specific salt bridge-forming residues for substrate targeting, septin-targeting information in the catalytic domain of ulp is independent of kap and the substrate-targeting domain of ulp can be modified to identify and purify sumoylated substrates of ulp in genetic screens and in biochemical analyses. 
to aid our studies, we took advantage of a catalytically inactive mutant of ulp that is greatly enriched at the septin ring of dividing yeast cells. in the yeast saccharomyces cerevisiae, the essential small ubiquitin-like modifier protease ulp is responsible for both removing sumo/smt from specific target proteins and for processing precursor sumo into its conjugation-competent form. furthermore, we found that a substrate-trapping ulp interacts robustly with human sumo sumo and sumo chains, making it a potentially useful tool for the analysis and purification of sumo-modified proteins. we also used the targeting and sumo-binding properties of ulp to purify smt3-modified proteins from cell extracts. our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of ulp ulp that is necessary and sufficient for septin localization. we found that the localization of ulp to the septins requires both sumo and specific structural features of ulp1's catalytic domain.
 in the yeast saccharomyces cerevisiae, the essential small ubiquitin-like modifier protease ulp is responsible for both removing sumo/smt from specific target proteins and for processing precursor sumo into its conjugation-competent form. ulp localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. how ulp 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 ulp that are required for substrate targeting. to aid our studies, we took advantage of a catalytically inactive mutant of ulp that is greatly enriched at the septin ring of dividing yeast cells. we found that the localization of ulp 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 ulp ulp that is necessary and sufficient for septin localization. we also used the targeting and sumo-binding properties of ulp to purify smt3-modified proteins from cell extracts.  our study provides novel insights into how the ulp sumo protease is actively targeted to its substrates in vivo and in vitro. furthermore, we found that a substrate-trapping ulp interacts robustly with human sumo sumo and sumo chains, making it a potentially useful tool for the analysis and purification of sumo-modified proteins. 