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Molecular & Cellular Proteomics 4:246-254, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,¶
From the Department of Cell Biology and Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Boston, MA 02115
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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12 kDa in yeast) that forms an isopeptide bond with the 
-amine group of lysine residues in target substrates (2). Frequently, this modification is known to occur at a consensus motif in substrate proteins, defined by the amino acid sequence 
KxE/D where is a large hydrophobic amino acid (most typically valine, isoleucine, or leucine) and x is any amino acid. However, sumoylation can also occur at lysines that do not reside in this motif, as seen for the modification of K164 in proliferating cell nuclear antigen (5). A second feature the SUMO pathway shares with ubiquitination is that sumoylation of target proteins requires the sequential action of an E1-activating, E2-conjugating, and, at least in some cases, an E3-ligating enzyme. Specifically, in yeast, SUMO is first activated in an ATP-dependent reaction in which its most C-terminal glycine residue forms a high-energy thioester bond with a cysteine residue in the active site of the E1 heterodimer, Aos1/Uba2 (6). This activated SUMO moiety is subsequently transferred in a transesterification reaction to the E2 enzyme of the SUMO cascade, Ubc9 (7, 8). Ultimately, SUMO is transferred to a substrate lysine residue by the combined activity of Ubc9 and one of various SUMO E3 enzymes such as the yeast proteins Siz1 and Siz2 (9, 10).
A final feature of the SUMO pathway that mirrors ubiquitination is the reversible nature of the process. Multiple SUMO-specific proteases, such as the yeast proteins Ulp1 and Ulp2, are known to reside within cells. These polypeptides can actively cleave SUMO from substrate proteins (11, 12). Therefore, sumoylation is a highly dynamic process in which its steady-state levels are tightly regulated by the complex interplay of the SUMO E1, E2, E3, and proteolytic enzymes.
However, despite these three main mechanistic parallels between the two pathways, it is becoming increasingly apparent from preliminary studies that the functional outcomes signaled by SUMO modifications are very different from those directed by Ub. SUMO modification has not been found in any cases to date to signal protein degradation. In fact, several instances have been described in which SUMO competes with Ub for a given lysine acceptor site, thereby preventing its subsequent poly-ubiquitination and either degradation by the proteasome or other fates induced by ubiquitination (5, 13). A number of additional roles have also been proposed for sumoylation, including affecting protein function, protein subcellular localization, and/or multi-protein complex formation (4, 5, 14–16).
Although these insights have aided our understanding of sumoylation, details into the biological effects of SUMO are just beginning to be unraveled. In recent years, there has been a surge in interest in the functional outcomes of protein sumoylation. This is largely attributable to the fact that sumoylation is known to affect a broad array of cellular processes. For example, it has been implicated in processes ranging from transcriptional regulation to DNA damage repair to the immune response (2, 5, 13, 17). Interest in protein sumoylation is also largely driven by the knowledge that several components of the SUMO pathways play roles in a number of different diseases, including Alzheimer’s and Huntington’s diseases (18, 19). Clearly, a key step in gaining insight into a given biological pathway is to first define all of its components. Toward this end, our laboratory recently reported the large-scale identification of over 1,000 substrates of the ubiquitination pathway (20). Here, a similar proteomic undertaking is reported for substrates post-translationally modified by SUMO that should shed light on the crucial process of protein sumoylation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2.00. These cells were harvested by centrifugation, resuspended in lysis buffer (8 M Urea, 50 mM Tris, pH 8.0) containing 10 µg/ml each of aprotinin, leupeptin, and pepstatin A and 1 mM PMSF, and lysed with a French press. The resulting mixture was centrifuged at 4 °C for 30 min at 17,000 rpm in a Sorval SS34 rotor. The supernatant was incubated with a 3-ml slurry of Ni-NTA resin (Qiagen, Valencia, CA) for 2 h at 4 °C and then loaded onto a column. Bound proteins were washed with 50 ml of wash buffer (lysis buffer containing 5 mM imidazole and 300 mM NaCl) and eluted in buffer containing 300 mM imidazole, 1 M urea, and 50 mM Tris, pH 8.0. Fractions containing protein were combined and diluted 10-fold with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) containing 0.1% SDS, 1 mM PMSF, and 1 mM ß-mercaptoethanol. The mixture was then incubated with 0.4 ml of anti-FLAG Sepharose (Sigma, St. Louis, MO) at 4 °C overnight. The next day, the beads were pelleted by centrifugation, transferred to 1.5-ml centrifuge tubes, and washed six times with 1-ml aliquots of RIPA buffer containing 0.1% SDS. Proteins were eluted with buffer containing 100 mM glycine, pH 2.2, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS, and the pH of these elutions was then immediately adjusted with NaOH to pH 7.5. The control preparation for this experiment was prepared in the exact same manner, except the yeast strain SUB61 in which SUMO is not tagged was utilized.
Mass Spectrometry Analysis—
Isolated SUMO conjugates were loaded onto one lane of an 8.5% acrylamide gel and separated according to their molecular masses. This same protocol was also followed for the control preparation. The resulting gels were cut into 12 horizontal strips and digested with trypsin as previously described (21). Individual peptide fractions were loaded onto 75-µm inner diameter fused silica columns packed in-house with C18 resin (Michrom Bioresources, Inc., Auburn, CA) and separated using a 35-min gradient from 2.5 to 97.4% ACN. Eluting peptides directly entered an LTQ FT mass spectrometer (Thermo Electron, San Jose, CA) where the instrument alternated between acquiring a full FT-MS scan (m/z range of 300 to 1,400) and then 10 subsequent MS/MS spectra of the 10 most abundant precursor ions using the LTQ as the detector. Data were analyzed using the yeast NCBI database in conjunction with the Sequest search algorithm (22). Searches allowed for the following two potential modifications: +15.9949 Da on methionines and 484.2282 Da on lysines (SUMO remnant at sumoylation sites). Furthermore, a dCN value of 0.08 was required for matches and the following XCorr cutoff values for 1+, 2+, and 3+ peptides, respectively, were used: 2, 2.1, and 3. Following the initial filtering of identified peptides, in the case of one and two peptide hits, accurate mass measurements were then calculated with in-house software (VISTA; Bakalarski et al., manuscript in preparation). Figures and tables of the identified proteins were made using information from both the Saccharomyces cerevisiae Genome Database (SGD) (www.yeastgenome.org) and Bioknowledge Retriever (proteome.incyte.com). Finally, the sequences of identified proteins were analyzed to locate potential sumoylation consensus motifs.
Western Blotting and Silver Staining Analyses—
Aliquots of wild-type lysate (either 5 or 20 µl; prepared from the SUB592 strain) and both the SUMO and control preparations (either 10 or 30 µl) were quenched by the addition of SDS loading buffer to a final concentration of 1x (63 mM Tris, pH 6.8, 2% glycerol, 2.3% SDS, 100 mM DTT, and 0.05% bromphenol blue). These three samples were separated on NuPAGE Novex 4–12% Bis-Tris Gels (Invitrogen, San Diego, CA) using 1x NuPAGE MOPS SDS running buffer and then transferred to nitrocellulose membrane (GE Osmonics Labstore, Minnetonka, MN) at 100 V for 2 h. Subsequent Western blot analysis was then performed with primary antibodies raised against either the FLAG-epitope, Tup1, Rap1, Gin4, Reb1, Rsc2, Spt7, Sir4, Sir3, or Sir2 and either anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL). Results were visualized with the ECL detection system (Amersham Biosciences, Piscataway, NJ). Silver staining was performed with the SilverQuest silver staining kit (Invitrogen).
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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An aliquot of the SUMO preparation isolated in this manner was subsequently separated by SDS-PAGE and visualized by Western blot analysis performed with an anti-FLAG antibody, as displayed in Fig. 1. As evident from this immunoblot (A), a number of sumoylated candidates were successfully isolated in this double-affinity purification procedure, as indicated by their interaction with the FLAG antibody. Further support for the presence of SUMO-modified proteins in this mixture was obtained by separating samples of both the SUMO and control preparations by SDS-PAGE and performing subsequent silver staining analysis (B). While a multitude of proteins were clearly isolated from the (His)6-FLAG-SUMO strain (lane 2), only six bands were present in the control preparation (lane 3), and the two most predominant of these bands were found to correspond to antibody light and heavy chains, remnants from the FLAG purification.
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Identification of Sumoylated Candidate Proteins by Liquid Chromatography-Tandem Mass Spectrometry—
After concluding that indeed a significantly pure preparation of SUMO conjugates had been obtained, the identity of the various proteins was addressed. To date, a significant hurdle in identifying sumoylated substrates is believed to be their low abundance because in many cases only a small fraction of a substrate is thought to be modified at a given time. Therefore, we reasoned that a protocol centered around an LTQ FT mass spectrometer (Thermo Electron) would be well-suited for the purposes at hand, due to the sub-femtomole sensitivity of the instrument. Furthermore, it was anticipated that the exceptional mass accuracy of the instrument would greatly aid the confirmation process for proteins identified by only one or two peptides.
To implement this approach, SUMO conjugates were first separated in a single gel lane on a polyacrylamide gel. The gel was then divided into 12 different regions and in-gel trypsin digestions were performed. The resulting peptides were extracted, loaded onto a reverse-phase column, and eluted with increasing acetonitrile concentrations directly into the mass spectrometer. In the mass spectrometer, both accurate mass (MS) and sequence (MS/MS) information was acquired for eluting peptides. This information was used by the SEQUEST algorithm to identify the peptides in subsequent database searches performed against the yeast database. It is important to note that peptides identified in this way were subjected to stringent search criteria including high Xcorr values and mass accuracies of less than 10 ppm from expected masses for peptides with two or fewer matching peptides. This same protocol was performed in its entirety for the control sample. All proteins that were unique to the SUMO preparation and that were identified by two or more peptides are found in Table I (159 proteins) and are considered to be true candidates for sumoylation.
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It should be noted that if a particular protein detected in the SUMO preparation was also identified in the control preparation by one or more peptides that met the above-stated search criteria, it was not included in either of these two tables of sumoylation candidates. Instead, these proteins, along with all proteins unique to the mock purification, are tabulated in Supplemental Table III. As predicted from the above-mentioned silver-stained analysis and as evident in this table, very little overlap was observed between the datasets of the SUMO and control purifications. In fact, more proteins than expected appeared to be unique to the control purification. If proteins present in the control sample truly stemmed from nonspecific interactions with the affinity matrix, one might predict that all of these same proteins would have also been present in the SUMO purification. It seems highly likely that more of these proteins were in fact present in the SUMO preparation but were not identified in the analysis due to the complexity of the sample and the fact that the limited sequencing time in the mass spectrometer was instead spent analyzing more abundant, sumoylated species.
Western Blot Confirmation of Sumoylated Substrates—
Importantly, in the above analysis, most proteins known to be sumoylated in yeast were identified. In fact, five of these proteins, Top2, Cdc3, Cdc11, Pol30, and Shs1 were identified by more than 10 peptides each (5, 16, 24). Another protein, Ycs4, was also identified by the high mass accuracy of one peptide, leaving only one known yeast-sumoylated protein (Pds5) not detected in our study (25, 26). These findings lend great support to the value of the dataset obtained.
To further validate our proteomics strategy, two additional means of confirming the sumoylation of identified proteins were sought. The first approach was to validate the sumoylation of numerous candidate proteins by immunoblotting. Specifically, samples containing aliquots of either wild-type cell extract, the double-affinity purified SUMO preparation, or the control preparation were separated by SDS-PAGE. Western blot analyses were then performed for sumoylated protein candidates that specific antibodies could readily be obtained for. This included the following eight candidate substrates: Tup1, Rap1, Gin4, Reb1, Sir4, Rsc2, Sir3, and Spt7. As indicated in Fig. 2, in all eight of these cases examined, the proteins were found to migrate significantly slower in the SUMO preparation (lane 2) than their predominant species in the yeast extract (lane 1), suggesting that indeed these proteins are post-translationally modified by SUMO. In fact, for six of the proteins studied, several such bands were observed, suggesting that these proteins are either modified by chains of SUMO, on various different acceptor sites and/or by both SUMO and Ub, a protein also identified in the MS analysis. The nature of these modifications is supported by the absence of these proteins in the control purification (lane 3). Furthermore, an additional control experiment was performed in which this same Western blot analysis was performed for Sir2, a protein not identified in the MS analysis. As expected, a strong signal was seen for this protein in the wild-type cell extract. However, this protein was not detected in either the SUMO or control preparations. The proteins examined by immunoblotting are believed to be a reasonable test for the dataset obtained because the proteins examined vary significantly in the number of peptides by which they were identified, ranging from 0 (Sir2) to 4 (Sir3) to 31 (Rsc2).
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Analysis and Categorization of Identified Proteins—
In an attempt to gain insights into the 159 potential SUMO substrates identified, the S. cerevisiae Genome (SGD) and the Bioknowledge Retriever databases were used to group these proteins according to their molecular environments (A) and functional categories (B); this information is graphically displayed in Fig. 4. Furthermore, this information, along with the molecular functions of these polypeptides, is tabulated in Supplemental Table I. From these analyses, several conclusions can be drawn, many of which are also supported by other recent proteomic studies of sumoylation (23, 27, 28). First, sumoylation has been traditionally viewed as a predominantly nuclear process. Indeed, 43% of the proteins identified in this work are reported to be nuclear proteins, found either directly or indirectly associated with DNA. However, it is interesting to note that a large percent of cytosolic, membrane, and/or other subcellular organelle proteins were also detected. For example, 31% of the proteins localized in soluble fractions, while 11 and 5% are, respectively, ribosome- and actin-associated. These findings seem to suggest that sumoylation is a less centralized process than previously thought. An important caveat to these conclusions is that it is possible that sumoylation of these substrates normally found in these various subcellular compartments targets them for the nucleus. Distinguishing between these two different scenarios will require further study.
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A third trend apparent from these data is that multiple members of the same protein complexes appear to be sumoylated, a trend also recently noted by Wohlschlegel et al. (23). Due to the highly denaturing conditions (8 M urea) employed in the purification protocol, it is not believed that a significant fraction of nonsumoylated members of a protein complex were isolated via their association with a sumoylated protein. As previously reported in the literature, a number of septins known to be sumoylated in the bud neck of dividing yeast were found to be sumoylated in the current work, such as Shs1, Cdc3, and Cdc11 (16). Further examples of this trend are provided by numerous members of the Rsc chromatin remodeling complex, such as Rsc1, Rsc2, Rsc8, Rsc58, and Sth1, and also by protein component of complexes present at silent-mating type loci, such as Sir3, Sir4, Rap1, and Abf1. The biological significance of this redundancy in the regulation of a given complex is currently unknown and will be an interesting phenomenon to address in future works.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A second important feature of our proteomic strategy was the use of the LTQ mass spectrometer for the collection of MS/MS spectra. This was critical because, in many cases, only a small fraction of a protein is believed to be sumoylated at a given time. The high sensitivity of the instrument presumably enabled the identification of significantly more SUMO substrates than found in recent studies utilizing less-sensitive instrumentation (27, 28). Furthermore, the excellent mass accuracy was exploited by the detection of all full MS spectra in the FT-ICR cell, greatly engendering confidence in the proteins detected, in particular those identified by only one or two peptides.
The value of the dataset obtained in this manner is underscored by several different lines of evidence: i) a number of known SUMO targets were identified in this work; ii) the sumoylation of multiple candidates was confirmed by Western blotting procedures; and iii) for several proteins, precise sites of sumoylation were identified by MS. However, despite these facts, it is important to note that, as seen in any proteomic study, presumably some fraction of these candidate proteins are not true SUMO substrates. Despite the careful controls, it is still possible that a small percent of proteins were carried nonspecifically through the double-affinity purification. In particular, it seems highly likely that some or all of the GAL proteins identified are artifacts because galactose media was used to induce the GAL promoter regulating SUMO expression. We also suspect that additional proteins identified that are known to be highly abundant species, such as some of the ribosomal and heat shock factors, might also be false positives. Furthermore, despite the highly denaturing purification conditions employed, it is still possible that some nonsumoylated proteins present in very stable complexes were carried through the purification via their association with true sumoylated substrates. Addressing these issues in such instances will require further experiments, such as more immunoblotting studies and/or sumoylation site identifications, performed on a case-by-case basis.
As mentioned previously, due to its functional significance, protein sumoylation is an area of intense research. Therefore, a number of proteomic studies have been performed recently to identify the substrates of sumoylation both in yeast and higher eukaryotes (23, 27–31). In the current work, a significant fraction of the proteins identified are novel potential substrates of the SUMO pathway. However, equally important, a large number of the proteins detected have also been identified in other recent works. Therefore, in addition to providing new insights, this study also provides confirmation in many instances to these previous reports. To aid the complementary nature of these experiments, we have tabulated the proteins identified, along with the number of peptides with which these identifications were made, in each of the four such works performed to date in S. cerevisiae (23, 27, 28). This tabulation can be found in Supplemental Table IV. This comparative list should help compile the wealth of information obtained in recent works and help direct more focused studies in the future that will ultimately result in a better understanding of protein sumoylation.
Sumoylation is known to be a highly dynamic process and the profile of sumoylated substrates is known to vary dramatically throughout the cell cycle and in response to various stimuli (16, 27). Therefore, a particular line of future work that should be aided by this information involves using quantitative MS tools, such as isotope-coded affinity tags (ICAT), stable isotope labeling with amino acids in cell culture (SILAC), and/or absolute quantification (AQUA) technologies, to identify how the modifications of these SUMO substrates change in various cellular conditions and genetic backgrounds (32–34). Furthermore, the use of proteomic approaches involving double-affinity purification procedures and LTQ FT mass spectrometry as described here should be applicable to future studies aimed at increasing the number of sumoylated substrates identified to date in higher eukaryotes. Finally, this approach should prove to be a valuable strategy for studying the substrates of various other Ub-like proteins, including Ned8, Atg12, and ISG15.
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ACKNOWLEDGMENTS |
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-RSC2, Fred Winston (Harvard Medical School) for -Spt7, Doug Kellogg (University of California at Santa Cruz) for -Gin4, David Shore (Unige) for -Rap1, and Jonathan Warner (Einstein College of Medicine) for -REB1. |
FOOTNOTES |
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Published, MCP Papers in Press, November 12, 2004, DOI 10.1074/mcp.M400154-MCP200
1 The abbreviations used are: Ub, ubiquitin; SUMO, small ubiquitin-like modifier. 
* This work was supported by National Institutes of Health Grants HG00041 and GM67945 (to S. P. G.) and the Jane Coffin Childs Memorial Fund for Medical Research (to A. D. R.).
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
¶ To whom correspondence should be addressed: Department of Cell Biology and Taplin Biological Mass Spectrometry Facility, 240 Longwood Avenue, Harvard Medical School, Boston, MA 02115. Tel.: 617-432-3155; Fax: 617-432-1144; E-mail: steven_gygi{at}hms.harvard.edu
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B inhibits NF-B activation.
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