Patent Application
20130017554
Not applicable.
The field of the invention relates to a specific affinity tag for SUMO, as well as applications for use of the affinity tag, including purification and detection.
Ubiquitin and SUMO belong to a conserved family of post-translational modifiers that become covalently attached to specific proteins in a reversible manner (Kerscher et al. (2006) Annual Review of Cell and Developmental Biology 22, 159-180). Ubiquitin is best known for its role in the targeted destruction of proteins including key cell-cycle regulators, but also holds many non-proteolytic functions (Chen et al. (2009) Molecular Cell 33, 275-286). Sumoylation, on the other hand, does not directly target proteins for proteasomal 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 formation of nuclear bodies (Wang et al. (2009) Journal of Cell Science 122, 4249-4252). These functional distinctions between ubiquitin and SUMO have been further blurred by the recent discovery of SUMO-targeted Ubiquitin Ligases (STUbLs) that enable SUMO to play an indirect role in proteasome-mediated degradation (Perry et al. (2008) Trends in Biochemical Sciences 33, 201-208).
SUMO proteins are highly conserved from yeast to humans. Yeast cells express one SUMO protein, Smt3, while vertebrates express three isoforms, SUMO1, SUMO2, and SUMO3 (Wilkinson et al. (2010) Biochemical Journal 428, 133-145). 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 3 chains (Matic et al. (2008) Mol Cell Proteomics 7, 132-144). 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, as reported by Hannich et al. ((2005) Journal of Biological Chemistry 280, 4102-4110) and Wykoff et al. ((2005) Mol Cell Proteomics 4, 73-83).
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, as reported by Baldwin et al. ((2009) Cell Cycle 8, 3406-3419), Bylebyl et al. ((2003) Journal of Biological Chemistry 278, 44113-44120), Kroetz et al. ((2009) Molecular Biology of the Cell 20, 2196-2206), Mukhopadhyay et al. ((2006) Journal of Cell Biology 174, 939-949), and Uzunova et al. ((2007) Journal of Biological Chemistry 282, 34167-34175). 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 (Li et al. (1999) Nature 398, 246-251). Therefore, impairment of Ulp1 results in the accumulation of SUMO conjugates and the inability to carry out de novo sumoylation.
Ulp1 and several other SUMO proteases play important roles in mitosis. In budding yeast, Li et al. ((1999) Nature 398, 246-251) report that 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.
One set of cytosolic substrates of the Ulp1 SUMO protease are the septins, as reported by Makhnevych et al. ((2007) Journal of Cell Biology 177, 39-49) and Takahashi et al. ((2000) Journal of Biochemistry 128, 723-725). 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 (Spiliotis, E. T. (2010) Cytoskeleton 67, 339-345). The septins Cdc3, Cdc11, and Shs1 are subject to sumoylation.
It would be useful to identify features of Ulp1 required for substrate-targeting in vivo and in vitro, and to identify and analyze distinct mutations in Ulp1 that affect the targeting and retention to sumoylated target proteins at the bud-neck of dividing cells.
In particular, it would be useful to provide Ulpl fragments that bind tightly to SUMO and can be used for a range of applications including, for example, purification and detection of sumoylated proteins.
A new SUMO-specific affinity tag is described herein. This affinity tag is based on the amino acid sequence from 403-621 of the SUMO protease Ulp1 and requires a protease-inactivating mutation C580S. This affinity tag, referred to herein as “Ulp1(3)(C580S)” (or simply “U-tag”), interacts strongly with SUMO, SUMO chains, and sumoylated proteins. Ulp1(3)(C580S) is particularly useful for a range of applications including but not limited to: affinity purification of sumoylated proteins from cell extracts using an immobilized Ulp1(3)(C580S) or matrix-bound Ulp1(3)(C580S), dimerization of SUMO-tagged proteins with Ulp1(3)(C580S) fusion proteins, in vitro detection of sumoylated proteins using a fluorescently labeled Ulp1(3)(C580S), and association of purified SUMO chains to a Ulp1(3)(C580S) fusion protein for downstream applications including in vitro ubiquitylation assays with SUMO-targeted Ubiquitin ligases.
Functionally equivalent versions of the Ulp1(3)(C580S) protein are also contemplated, including truncated and lengthened versions, provided said functionally equivalent versions (1) contain the C580S mutant, and (2) have at least 80% amino acid homology with the Ulp1(3)(C580S) polypeptide sequence as set forth in SEQ ID NO: 1. Amino acid residue 178 of SEQ ID NO: 1 comprises the crucial substitution of serine for cysteine.
In another aspect, a composition is described comprising DNA isolates encoding for Ulp1(3)(C580S) proteins.
In another aspect, a composition is described comprising a recombinant expression vector including the DNA encoding for Ulp1(3)(C580S) proteins.
It is an object of the present invention to provide a method for affinity purification of sumoylated proteins.
It is an object of the invention to provide a method for dimerization of SUMO-tagged proteins.
It is an object of the invention to provide a method for detection of sumoylated proteins in vitro.
It is an object of the invention to provide a method for detecting a SUMO-tagged protein comprising the sequential steps of: covalently bonding a fluorescent tag to the Ulp1(3)(C580S) polypeptide to produce a labeled Ulp1(3)(C580S) polypeptide, mixing a SUMO-tagged protein with said labeled Ulp1(3)(C580S) polypeptide, and determining the extent of labeled Ulp1(3)(C580S) polypeptide that is bound to SUMO-tagged proteins.
The summary above, and the following detailed description, will be better understood in view of the drawings that depict details of preferred embodiments.
As used herein, abbreviations and names for proteins are consistent with terms generally used in the art. The term “Ulp1” refers to the Ubiquitin-Like Protein, systematic name YPL020C. SUMO (Small Ubiquitin-like Modifier) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. The term “C580S” means that the cysteine residue corresponding to position 580 in Ulp1 has been replaced with serine. In the 219 amino acid Ulp1(3)(C580S) polypeptide described herein (see, for example, SEQ ID NO: 1 and
A new SUMO-specific affinity tag is described herein. This affinity tag is based on the amino acid sequence from 403-621 of the SUMO protease Ulp1 and requires a protease-inactivating mutation C580S. This affinity tag interacts strongly with SUMO, SUMO chains, and sumoylated proteins, and is particularly useful for a range of applications including but not limited to: affinity purification of sumoylated proteins from cell extracts using an immobilized Ulp1(3)(C580S) or matrix-bound Ulp1(3)(C580S), dimerization of SUMO-tagged proteins with Ulp1(3)(C580S) fusion proteins, in vitro detection of sumoylated proteins using a fluorescently labeled Ulp1(3)(C580S), and association of purified SUMO chains to a Ulp1(3)(C580S) fusion protein for downstream applications including in vitro ubiquitylation assays with SUMO-targeted Ubiquitin ligases.
Functionally equivalent versions of the Ulp1(3)(C580S) protein are also contemplated, including truncated and lengthened versions, provided said functionally equivalent versions contain the C580S mutant and have at least 80% amino acid homology with the Ulp1(3)(C580S) polypeptide sequence as set forth in SEQ ID NO: 1. Amino acid residue 178 of SEQ ID NO: 1 comprises the crucial substitution of serine for cysteine. In some embodiments, functionally equivalent versions of the Ulp1(3)(C580S) protein have between 1 and 10 amino acid residue modifications relative to the Ulp1(3)(C580S) polypeptide sequence as set forth in SEQ ID NO: 1.
In another aspect of the invention, a composition is described comprising DNA isolates encoding for Ulp1(3)(C580S) proteins, or encoding for functionally equivalent versions of the Ulp1(3)(C580S) protein. In another aspect, a composition is described comprising a recombinant expression vector including the DNA encoding for Ulp1(3)(C580S) proteins, or for functionally equivalent variants of the Ulp1(3)(C580S) protein.
Methods are described herein for affinity purification of sumoylated proteins, and also for detection of sumoylated proteins by binding them to fluorescently labeled Ulp1(3)(C580S) protein, or functionally equivalent versions thereof. For example, one method for detecting a SUMO-tagged protein comprises the sequential steps of: covalently bonding a fluorescent tag to the Ulp1(3)(C580S) polypeptide to produce a labeled Ulp1(3)(C580S) polypeptide, mixing a SUMO-tagged protein with said labeled Ulp1(3)(C580S) polypeptide, and determining the extent of labeled Ulp1(3)(C580S) polypeptide that is bound to SUMO-tagged proteins.
The methods of the invention also contemplate dimerization of SUMO-tagged proteins.
Fusion protein comprising Ulp1(3)(C580S) can only be produced according to the methods of the invention. For example, a fusion protein can be synthesized comprising a Ulp1(3)(C580S) protein fused at the C terminus with a protein or peptide capable of being fused to Ulp1(3)(C580S). Alternatively, a fusion protein can be synthesized comprising a Ulp1(3)(C580S) protein fused at the N terminus with a protein or peptide capable of being fused to Ulp1(3)(C580S).
Exemplary investigations supporting the compositions and methods of the present invention are presented below. The examples that follow are intended in no way to limit the scope of this invention, but are provided to illustrate representative embodiments of the present invention. Many other embodiments of this invention will be apparent to one skilled in the art.
Experimental Procedures.
Yeast strains, media and plasmids. Yeast strains and plasmids used in this study are listed in Table 1. Yeast media preparation and manipulation of yeast cells was performed as previously published (see Guthrie, C., and Fink, G. R. (1991) Guide to yeast genetics and molecular biology, Academic Press, San Diego). 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 (44). Primer pairs used for full-length Ulp1 amplification were OOK2 (ULP1 (−310 to −294)) and OOK3 (ULP1 (+1842 to +1863)). To prepare truncated and mutated Ulp1-GFP constructs listed in Table 1 below, Quikchange XL Site-Directed Mutagenesis (Stratagene) and Phusion Site-Directed Mutagenesis kits (Finnzyme) were used according to manufacturers' instruction. Primer sequence information for the construction of individual mutants and truncations are available upon request. All constructs were sequenced verified and/or confirmed in complementation assays. For two-hybrid constructs, ORFS of the indicated genes were PCR-amplified and recombined into gapped pOAD and pOBD2 vectors (Yeast Resource Center, WA). To overexpress and purify Ulp1 truncations from bacteria, the respective Ulp1 fragments were PCR-amplified and cloned into pMALc-HT (obtained as a gift from Sean Prigge, JHSOM), thereby adding an in-frame maltose-binding protein (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 containing a pRIL plasmid.
Yeast Two-Hybrid Assays- 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.
Pulldown Assays, Affinity Purification, and Protein extracts—Frozen bacterial cell pellets from 200 ml of IPTG-induced BL21 Star (DE3) cells were thawed on ice and re-suspended in 2 ml 1× phosphate buffered saline (PBS) containing 1× Halt Protease Inhibitor Cocktail (Pierce Cat. # 78430). Ice-cold cells were sonicated using a Branson Sonifier, and extracts were cleared by centrifugation at 15k RPM for 8 minutes at 4° C. Cleared bacterial extracts were added to 15 mL conical tubes and diluted using 4 mL 1× PBS containing the protease inhibitor cocktail. MBP-tagged proteins (MBP-Ulp1(3), Ulp1(3)(C580S), or Ulp1(3)(C580S)ΔSBS) were bound to 5 ml columns containing 300 μl amylose resin (New England Biolabs) 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), Ulp1(3)(C580S), or Ulp1(3)(C580S)ΔSBS were eluted with 100 mM maltose or SDS-PAGE sample buffer. Yeast cell protein extracts were generated by bead-beating ˜50 ODs of yeast cell pellets in 1× Cell Lysis buffer (#9803—Cell Signaling Technology, MA) containing 25 mM N-Ethylmaleimide (NEM). For SUMO pulldown experiments, recombinant MBP-Ulp1(3)(C580S) or MBP-Ulp1(3) was incubated with SUMO-1 or SUMO-2 agarose (Boston Biochem) in 1 ml of 1× PBS with protease inhibitors (Thermo Scientific). 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 NOVEX 4-12% BIS-TRIS gradient gels (Invitrogen #NP0321) using MOPS-SDS running buffer (Invitrogen #NP0001).
Fluorescent Microscopy—Unless otherwise noted, cells were grown in rich media, arrested in G2/M using nocodazole (15 μg/ml/3h/30° C.), washed in 2% dextrose, and harvested by centrifugation. Images of live cells were collected using a Zeiss Axioskop fitted with a Retiga SRV camera (Q-imaging), i-Vision software (BioVision Technologies), and a Uniblitz shutter assembly (Rochester, N.Y.). Pertinent filter sets for the above applications include CZ909 (GFP), XF114-2 (CFP), XF104-2 (YFP) (Chroma Technology Group). Images were normalized using i-Vision software and pseudo-colored and adjusted using Adobe Photoshop software (Adobe Systems Inc.).
In vitro ubiquitylation reactions, recombinant proteins, and antibodies—Enzymes and substrates used in our in vitro ubiquitylation assays were quantified using a Protein 230 kit on the Agilent 2100 Bioanalyzer according to the manufacturer's instructions. 10× ubiquitylation buffer, E1 enzyme (Uba1), ATP, and 20× ubiquitin were provided in a commercial ubiquitylation kit (Enzo # BML-UW0400). Ubiquitylation buffer, IPP (100 U/ml), DTT (50 μM), E1 (Uba1), E2 (Ubc4), and E3 enzymes (RNF4) were combined with purified SUMO2 chains (#ULC-210—Boston Biochem, MA) and ubiquitin. Reactions totaled 27 μL and were incubated at 30° C. for three hours. Reactions were stopped by adding an equal volume of SUTEB sample buffer (0.01% bromophenol blue, 10 mM EDTA, 1% SDS, 50 mM Tris at pH 6.8, 8 M Urea) containing DTT (5 μL of 1 M DTT/1 mL SUTEB sample buffer). Protein products were boiled in a 65° C. heat block for ten minutes and analyzed by Western blot with anti-human SUMO2 antibody. Antibody sourcing was a follows: anti-human SUMO2 # BML-PW0510-0025 (ENZO Life sciences, PA), anti-GFP: JL8 # 632380 (Clontech, CA), anti-FLAG(M2) #F3165 (Sigma-Aldrich, MO), anti-PGK: 22C5 # 459250 (Invitrogen, CA), anti Cdc11 (y415):sc-7170 (Santa Cruz Biotechnology, CA).
The localization of green-fluorescent protein (GFP)-tagged versions of both the full-length wildtype Ulp1 (WT) and a catalytically inactive mutant of Ulp1 (Ulp1(C580S) in G2/M-arrested yeast cells was analyzed (see materials and methods). 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 fluorescent microscope. Shown in
Referring to
The next step was to determine 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), along with a wildtype control strain (WT). 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 wildtype strain. In our analyses, we found that in both the absence of SUMO chains (in the R11,15,19 mutant) and improperly formed SUMO chains (in the smt3-331 mutant), the localization of Ulp1C580S to the septin ring was reduced but not abolished in frequency and intensity (
In summary, Smt3 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 suggests 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 wildtype Ulp1 (WT) 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-Kap60-binding domain with a role in NPC anchoring (region 2), a coiled-coil domain harboring a nuclear export signal (CC), and the catalytic ubiquitin-like protease domain (UD) (region 3) (25-27). Depictions and images of these domains and their subcellular localizations are shown in
We demonstrated that the Ulp1 protein lacking region 2, (Δ2) localized to the septin ring in the majority of large-budded, arrested cells (27). 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 (
Aspartate 451 (D451) in Ulp1 is required to form an essential salt-bridge with arginine 64 of Smt3. Therefore, we introduced a D451 N mutation into Ulp1C580S and found that it abolished the accumulation of the full-length Ulp1 double mutant (D451 N, C580S) 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 (
The previously published co-crystal structure of Ulp1 with Smt3 (MMDB database # 13315) reveals that amino acids 418-447 of region 3 make extensive contact with Smt3 and constitute an exposed SUMO-binding surface (“SBS”). 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 to all non-redundant protein sequences in the NCBI database for seven iterations and limited the output to the top 250 matches. Our results contained 81 different 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 “other” (crystal structures, unanalyzed sequence, etc.). The alignment of these sequences allowed us to identify areas of strong conservation.
We also investigated the effect of deleting the entire SBS domain on the localization of Ulp1(3)(C580S). A Ulp1(3)(C580S)SBSΔ construct does not localize to the septin ring in the majority of cells (96%). We also cloned and expressed the SBS domain as a fusion with GFP (SBS-GFP). This construct distributed diffusely throughout the cell and failed to localize to the septin ring. 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 conserved asparagine 450 (N450) residue that resides immediately next to the SBS domain. N450 is mutated in region 3 of the temperature-sensitive ulp1ts-333 allele which arrests in mitosis and accumulates unprocessed SUMO precursor and sumoylated proteins (Li et al. (1999) Nature 398, 246-251). Our ulp1ts construct of region 3, Ulp1(3)ts, contains three mutations (I435V, N450S, I504T), and introduction of C580S into Ulp1(3)ts greatly reduced the incident and intensity of septin ring localization (compare panels in
In conclusion, we find that several features (N450, D451, and C580S) in region 3 of Ulp1, beyond the previously identified SBS domain, are required for targeting and retention at the septin ring.
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, Li et al. ((2003) Journal of Cell Biology 160, 1069-1081) 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 wildtype Ulp1, full-length Ulp1C580S, and Ulp1(3)(C580S). Kap121ts cells were transformed with plasmids expressing GFP-tagged wildtype (WT) Ulp1, Ulp1(C580S), and Ulp1(3)(C580S) under the control of the Ulp1 promoter (YOK 1487, YOK 1488, YOK 1944). Shown in
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 nuclear envelope and septin ring of G2/M-arrested cells. After a shift to the non-permissive temperature, however, Ulp1C580S could no longer be detected at the septin ring (
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 (
The finding that a single amino-acid change in the catalytic domain of Ulp1 yielded 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). Referring to
Full-length wildtype Ulp1, the full-length catalytically inactive Ulp1C580S mutant, the Ulp1 Kap121-interacting domain (region 1), and the Ulp1 Kap60/Kap95-interacting domain (region 2), all failed to interact with Smt3-BD (
We focused on the important residues near the SBS domain (see
The observation that the ts mutations in Ulp1(3)ts weakened or disrupted the interactions with Smt3 suggests that these mutations could help explain the unexpectedly diminished levels of Smt3 interaction with the Ulp1(3)(C580S) mutant. We reasoned that Ulp1(3)(C580S)failed to score strongly with Smt3 because it was avidly interacting with free Smt3 or was sequestered by sumoylated proteins in the cell and, therefore, failed to interact with the BD-Smt3 fusion. Introduction of the ts mutations in Ulp1(3)ts could weaken the substrate-trapping phenotype of Ulp1(3)(C580S), allowing it to regain the interaction with the BD-Smt3 fusion. Indeed, we found that combining these mutations in the Ulp1(3)ts (C580S) construct reestablished the interaction with Smt3. This unique observation provides 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. In order 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. 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).
As shown in
Additionally, we probed extracts and eluted proteins shown in
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 the GFP-tagged Ulp1(3), Ulp1(3)(C580S), or the Ulp1(3)(C580S)-ΔSBS (see
We also analyzed if immobilized Ulp1(3)(C580S) could be used to purify SUMO chains. For 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 analyzed by Western blotting with an anti-SUMO2 antibody. SUMO2 chains could clearly be detected in the input (FIG. 6D—lane 2) and bound to the MBP-Ulp1(3)(C580S) (lane 4), but not to the resin-only control (FIG. 6D—lane 3). Both lower and higher molecular weight adducts of SUMO2 were purified with preference for higher molecular weight chains (5-7 mers). These data suggest that the 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.
SUMO-targeted ubiquitin ligase proteins (STUbLs) (e.g., the yeast Slx5/Slx8 heterodimer and the human RNF4 protein) efficiently ubiquitylate proteins modified with SUMO chains (51,52). These proteins interact with their respective sumoylated ubiquitylation targets through SIMs. STUbL reactions have been reconstituted in vitro, but the purification of target proteins modified with SUMO chains has been technically difficult and/or prohibitively expensive. The ability of Ulp1(3)(C580S) to interact with SUMO can provide a simple way to purify a SUMO-chain-modified STUbL target of choice.
To demonstrate that 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 ubiquitylation reaction containing recombinant RNF4 (Fryrear and Kerscher, unpublished reagents). Proteins in the STUbL-mediated ubiquitylation assay were separated on SDS-PAGE gels and analyzed by Western blotting with an anti-SUMO antibody. Referring to
YOK428, aulp1::KANmx deletion strain, carries plasmids expressing both temperature sensitive ulp1ts under control of the native ULP1 promoter as well as FLAG-tagged conjugation competent SUMO (SUMOgg-FLAG) under control of the yeast GPD promoter. Two liters of YOK428 cells were grown to late log-phase in media containing G418 and lacking leucine. Cells were then harvested by centrifugation at 4° C. and pellets were washed once with 1 L ice cold wash buffer (50 mM HEPES, 3 mM DTT, 2% dextrose). Cells were then re-suspended in a volume of extrusion buffer (50 mM HEPES 7.8, 325 mM NaCl, 14 mM BME, 5 mM MgCl2, protease inhibitors) equal to that of the packed cell volume. Cells were spun down and most of the supernatant was discarded. The remaining cell paste was scraped into a 10 mL syringe and snap-frozen by extruding the paste into a 50 mL centrifuge tube containing liquid nitrogen, resulting in high cell density yeast noodles. Cells were lysed in a cryogenic tissue grinder, and the resulting powder was placed at −80° C. to allow the dry ice to sublimate. Powdered yeast lysate was dissolved in 10 ml extraction buffer (50 mM HEPES 7.8, 325 mM NaCl, 14 mM BME, 5 mM MgCl2, 10% glycerol, protease inhibitors·) and snap frozen in liquid nitrogen. Protein extracts were thawed on ice and insoluble cellular components were spun out of solution. 10 mM NEM was added to inhibit desumoylating enzymes and a 30 μl aliquot was taken as a whole cell extract control. The remaining volume was gravity-fed through a 1 ml bed of either washed, equilibrated resin-bound MBP-Ulp1(3)(C580S) (“U-tag resin”) or an amylose control resin. The U-Tag and amylose resins were washed twice with 5 bed volumes of column wash buffer (50 mM HEPES, 325 mM NaCl, 1% Triton x-100) each. The beads were then removed to 1.7 mL eppendorf tubes and sumoylated proteins were eluted by processing with recombinant Ulp1 protease. Specifically, SUMO bound resin was incubated with 10U of SUMO-HIS6x protease for 6 hours in 1× SUMO protease buffer with salt. After processing, beads were collected by brief centrifugation at <800 xg and the supernatant containing SUMO substrates was put through a PrepEase (USB corporation) His affinity column to remove the HIS6x tagged Ulp1 SUMO protease. Samples of whole cell extracts and eluted proteins were analyzed by SDS-PAGE as shown in
Region 3 of Ulp1, the catalytic domain, contains critical information for the 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 a partial redistribution from the nuclear envelope to the septin ring of dividing yeast cells. The re-localization of Ulp1 depended on functional Smt3 and sumoylated proteins at the septin ring of dividing cells.
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 SUMO-binding surface (SBS) (amino acids 418-447) and two residues (N450 and D451) that reside near the carboxy-terminus of Smt3. D451 of Ulp1 contacts Smt3 through a salt bridge interaction. In contrast, N450, a residue that is mutated in the ulp1ts allele (N450S), does not seem to contact Smt3. Therefore, it is possible that perturbation of the D451 salt-bridge, due to the juxtaposed N450S mutation, results in the reduced ability to dock Smt3 in place once it has contacted the SBS domain.
The sole requirement for the enrichment of full-length Ulp1 at the septin ring was the catalytically inactivating C580S mutation in the catalytic domain of Ulp1. This finding has important implications for the targeting role played by the amino-terminal karyopherin binding domains of Ulp1. Additionally, catalysis of Smt3 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, while the catalytically active wildtype Ulp1(3) shows merely a diffuse staining throughout the cell. The C580S mutation may trap a bound Smt3 protein, allowing it to be observed in association with cellular desumoylation substrates. In support of this assessment, combining the D451 N with the C580S mutation abolished all visible bud-neck localization (
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.
Our research demonstrates for the first time that non-covalent interactions between Ulp1 and SUMO are not only important for SUMO binding, but also for the cytosolic targeting of this SUMO protease to the bud-neck and potentially sumoylated septins. Sumoylated proteins that accumulate or aggregate in the cytosol of yeast cells may be readily detectable by Ulp1(3)(C580S). Ulp1(3)(C580S) also provides a useful tool to purify these sumoylated proteins. In conclusion, 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.
One intriguing aspect of the above Examples is the 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 comprised of sumoylated septins in vivo. Second, the reduced interaction of Ulp1(3)(C580S)with Smt3 in a two-hybrid assay can be re-established by the introduction of mutations that weaken the interaction with Smt3. Third, the purified, recombinant Ulp1(3)(C580S) protein is a potent affinity-tag for the purification of Smt3 conjugates and SUMO-modified proteins. A related study involving the C603S mutant of the human SENP1 protease confirms our assessment of the substrate-trapping feature. The authors observe re-localization 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. For example, two-hybrid screens with Ulp1(3)(C580S) have already identified several novel cytosolic desumoylation targets.
All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes to the same extent as if each was so individually denoted.
Equivalents
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, a protein means one protein or more than one protein.
Any ranges cited herein are inclusive.
This invention was made with government support under Grant No. R15-GM085792, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61500434 | Jun 2011 | US |
