ABBREVIATIONS
-Arg0, 12 -!KxE/D, consensus sumoylation site where ! is V, L, I, M or F and x is any amino acid.
-ZF, zinc finger
INTRODUCTION
Conjugation of ubiquitin-like protein modifiers to target proteins regulates a wide variety of cellular processes (1) . The ubiquitin family includes Small Ubiquitin-like MOdifiers (SUMOs) that are similar in structure to ubiquitin (2) , but in contrast to polyubiquitination, sumoylation does not target proteins for degradation by the proteasome (3) (4) (5) . Sumoylation can regulate the function of proteins by affecting proteinprotein interactions, which in turn can influence subcellular localization. This was first demonstrated for RanGAP1, a protein that is targeted to the nuclear pore component
RanBP2 by sumoylation (6) (7) (8) (9) .
The conjugation pathway of SUMO is similar to the conjugation pathway of ubiquitin and consists of E1, E2 and E3 enzymes (3) (4) (5) . SUMO is activated by the SUMO activating enzyme 1/2 dimeric E1 enzyme and subsequently SUMO is transferred to target proteins by a single E2 enzyme designated Ubc9. Several E3-like factors have been identified, including RanBP2 and the PIAS family, that enhance SUMO conjugation to proteins (3) (4) (5) 10, 11) . Sumoylation is a reversible process; SUMO-specific proteases can remove SUMO from target proteins (12) . These SUMO proteases are also essential for SUMO maturation because SUMO precursor proteins require C-terminal cleavage to expose a di-glycine motif essential for conjugation. RNAi and genetic studies of several components of the sumoylation pathway have established that sumoylation is critical for eukaryotic cell viability (13) (14) (15) (16) (17) .
Many target proteins have been identified for Smt3, the single SUMO in budding yeast (18) (19) (20) (21) (22) (23) . These include transcription factors, replication factors, RNA binding and by on November 20, 2006 www.mcponline.org
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processing proteins, translation factors, transport factors, cytoskeleton components and metabolic enzymes, highlighting the broad impact of SUMO on cellular processes.
In contrast to the single SUMO found in S. cerevisae, C. elegans and D.
melanogaster, higher eukaryotes express multiple different SUMOs. A complex SUMO family has been identified in A. thaliana with up to eight members (24, 25) . Humans express three SUMO family members, SUMO-1, SUMO-2 and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical (~95% identity), but differ substantially from SUMO-1 (~50% identity) (26) (27) (28) . In addition to genes that encode functional SUMOs, extensive sets of SUMO pseudogenes exist (29) .
We have previously purified and identified a set of target proteins for human SUMO-2 (30) and other groups have identified target proteins for SUMO-1 and SUMO-3 Mass spectrometry and data analysis. Mass spectrometric analysis was performed by nanoscale liquid chromatography-tandem mass spectrometry (LC MS/MS) using a linear ion trap Fourier-transform ion-cyclotron resonance mass spectrometer (LTQ-FT-ICR, Thermo-Finnigan, Bremen). Eluates were analyzed by 1-dimensional gel electrophoresis.
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The two gel lanes used were cut in 10 slices and subjected to in-gel digestion with trypsin. The resulting peptides were extracted, concentrated, and then loaded onto a fused silica capillary with a 75 $m ID and an 8 $m tip opening (New Objective, Woburn, MA) filled with Reprosil 3 $m reverse phase material (Dr. Maisch, Ammerbuch, Germany).
Peptides were eluted with a 140 min linear gradient of 95% buffer A (0.5% acetic acid in H 2 O) to 50% buffer B (80% acetonitrile, 0.5% acetic acid in H 2 O). The LTQ-FT-ICR instrument was operated in the data dependent mode to acquire high-resolution precursor ion spectra (from m/z 300-1500, R=25,000, and ion accumulation to a target value of 10,000,000) in the ICR cell. The three most intense ions were sequentially isolated for accurate mass measurements by SIM scans (10 Da mass window, R=50,000, and a target accumulation value of 50,000). The ions were simultaneously fragmented in the linear ion trap with a normalized collision energy setting of 27 % and a target value of 2,000. (43) . Peptide antibody AV-SM23-0100 against SUMO-2/3 was generated in rabbit using the peptide MEDEDTIDVFQQQTG (Eurogentec) (30) . Peptide antibody 1607 against SART1 was also generated in rabbit by Eurogentec using peptides CSLSIEETNKLRAKLGLKPLEV and CNLDEEKQQQDFSASSTT as described previously (44 
The three SUMOs also differ in their ability to form SUMO chains. This occurs via an internal sumoylation site that is present in SUMO-2 and SUMO-3 but is missing in SUMO-1 (43) . SUMO-2 chains are formed on PML in vitro and SUMO-2 dimers have been found attached to HDAC4 (43) .
Evidence exists that the closely related SUMO-2 and SUMO-3 proteins also display functional differences. Although mature SUMO-2 and SUMO-3 are nearly identical, the precursor proteins differ substantially in their C-termini (43) . This could indicate that the processing of the precursor proteins occurs differently, or is mediated by different SUMO proteases. The SUMO protease SENP2 has indeed been shown to catalyze the maturation of pre-SUMO-2 and pre-SUMO-3 with strikingly different efficiencies and this difference can be attributed to the differences in C-termini of these proteins (52) . Whether these differences between SUMO-2 and SUMO-3 affect their conjugation to target proteins is currently unclear.
In summary, we have identified a set of novel potential SUMO target proteins and in addition confirmed several previously identified SUMO conjugates. Some of these proteins are preferentially conjugated to SUMO-1, other proteins are preferentially conjugated to SUMO-2 and a third set of proteins was found to be conjugated to both SUMO-1 and SUMO-2. This indicates that SUMO-1 and SUMO-2 have probably both redundant and non-redundant cellular functions. (A) His 6 -SUMO conjugates were purified from HeLa His6-SUMO-1 nuclei and HeLa
His6-SUMO-2 nuclei. Control purifications from HeLa nuclei were included in the experiment.
Proteins were size-separated by SDS-PAGE, transferred to membranes and probed using antibodies directed against endogenous SUMO target proteins. RanGAP1 is preferentially conjugated to SUMO-1, Sp100 is conjugated to SUMO-2 and SART1 is conjugated to both SUMOs at similar levels. (B) Preferential conjugation of endogenous target proteins to endogenous SUMO-1 or endogenous SUMO-2/3. The SUMO-1 target protein RanGAP1 and the SUMO-2 target protein Sp100 were immunoprecipitated from HeLa cell lysates, transferred to a membrane and probed using antibody 21C7 directed against SUMO-1 or antibody AV-SM23-0100 directed against SUMO-2/3. Table 1 : SUMO target proteins.
RESULTS
SUMO-1 and SUMO2/3 conjugation profiles.
To study the target protein profiles for the nearly identical human SUMO family members SUMO-2 and SUMO-3, a polyclonal antiserum was raised in a rabbit against a peptide from the identical C-terminal regions of both SUMO-2 and SUMO-3. This antiserum specifically recognizes SUMO-2/3, but not SUMO-1 as judged by immunoblotting experiments, whereas the commercially available monoclonal antibody 21C7 specifically recognizes SUMO-1 but not SUMO-2/3 ( Fig. 1A and B). Endogenous SUMO target protein profiles in HeLa lysates were studied by immunoblotting, using both this SUMO-2/3 specific antiserum and monoclonal antibody 21C7 ( Of the 53 SUMO target proteins selected, 9 have previously been found in SUMO target protein screens. These proteins are RanGAP1 (6, 7, 8, 9) , PML (47-50), Delta 1-pyrroline-5-carboxylate synthetase (35, 33) , 82kDa FMRP interacting protein, Ataxin-2-like protein, PP1 regulator, Hypothetical protein DKFZp434D1319 (33) and SART1 (31) (32) (33) (34) (35) . Three proteins were previously identified in our screen for SUMO-2 targets, SART1, Ataxin-2-like protein and a homolog of Zinc finger protein 106 (30) . The lowest ratio of heavy arginine to light arginine that we observed for peptides from these 9 known SUMO target proteins was 1.53 for the PP1 regulator. This confirms the validity of using 1.5 as the SILAC cutoff ratio to assess specificity. We recognize that using the relatively low cutoff ratio of 1.5 harbors the risk of including false positive SUMO targets in the table.
On the other hand, we feel this is justified to avoid rejecting genuine SUMO targets. The relatively low SILAC ratios observed are likely due to the low abundance of sumoylated proteins and the co-purification of contaminating, non-sumoylated proteins. have previously been found to be conjugated to SUMO via lysines that are not situated in sumoylation consensus sites (3, 4, 19, 21) .
A striking feature of many SUMO-1 target proteins is the presence of one or more zinc fingers (table 1) , in agreement with a previous study (32 We compared our data set with data from previous studies on the target proteins that were identified for yeast SUMO, Smt3 (19) (20) (21) (22) (23) Several examples of peptide spectra corresponding to SUMO-1 and/or SUMO-2 target proteins are given in Fig. 4 . Chromodomain Helicase DNA-binding protein 2 (CHD2) is an example of a preferential SUMO-1 target (Fig. 4B ) whereas
Chromodomain Helicase DNA-binding protein 1 (CHD1) is an example of a nonsumoylated protein (Fig. 4A) . Interestingly, CHD1 is missing 5 of the 8 consensus sites for sumoylation that are found in CHD2 ( to SUMO-1, but the data also indicate the existence of a SUMO-2 linked form (Fig. 4C ).
SART1 is a target for both SUMO family members (Fig. 4D ), whereas PML (Fig. 4E) and the Ataxin-2 Related Domain Protein (Fig. 4F ) are preferential SUMO-2 targets.
Confirmation of SUMO-1-and SUMO-2 target protein preferences by immunoblotting.
To confirm that SUMO-1 and SUMO-2 display target protein preferences and also share target proteins, immunoblotting experiments were performed (Fig. 5A ). His 6 -SUMO-1
and His 6 -SUMO-2 conjugated proteins were purified separately and control HeLa cells were included in the experiment. Proteins were size-separated by SDS-PAGE, blotted to membranes and SUMO target proteins were detected by specific antibodies. In line with the results obtained for RanGAP1, this protein is detected as a preferential target for SUMO-1, however, a SUMO-2 conjugated form is also detectable (Fig. 5A ). Note that SILAC was able to find small amounts of RanGAP1 in the negative control fraction, whereas no RanGAP1 could be found in the negative control by immunoblotting. SILAC is apparently more sensitive in detecting proteins in the negative control fraction than immunoblotting. Sp100 (50) is an example of a specific target for SUMO-2 and SART1 is conjugated to both SUMO family members at similar levels (Fig. 5A ).
These results were obtained using endogenous SUMO target proteins, but exogenous His 6 -SUMOs. To investigate also endogenous SUMOs conjugated to endogenous proteins, immunoprecipitation assays were employed ( We confirmed these results by immunoblotting experiments for a subset of SUMO target proteins.
The purification and identification of sumoylated proteins has been hampered by the low abundance of many SUMO targets, the finding that usually only a small fraction of a protein is sumoylated at any time and the high activity of SUMO proteases (3). We have chosen to deal with these serious technical challenges using the novel approach of combining immobilized metal affinity chromatography with stable isotope labeling.
Because it is essential to both enrich the sumoylated target proteins and to block the action of SUMO proteases by using denaturing buffers, this limits in practice the choice of the affinity tag that can be used. The 6His tag is compatible with the use of a denaturing 8M Urea buffer and significant enrichment of tagged proteins can be obtained using immobilized metal affinity chromatography. Nevertheless, the resulting purified fractions, while enriched, are never completely pure and inevitably contain a variety of contaminating, non-specific proteins. Such contaminants are always observed using this and related methodologies and arise for several reasons. For example, they include the proteins that interact with the immobilized metal Cobalt via internal histidine rich regions and other abundant, "sticky" proteins that bind via lower affinity ionic interactions.
SILAC is employed to discriminate between such inevitable contaminants and the bona fide SUMO targets by accurately and objectively quantitating the specific enrichment of proteins above background levels. In addition, SILAC enables the quantitation of proteins that are preferentially conjugated to either 6His-SUMO-1 or 6His-SUMO-2. As an example, figure 5A shows that a small amount of SART1 can interact in its nonsumoylated form with immobilized Cobalt. Sumoylated forms can in addition be purified from lysates of 6His-SUMO expressing cells and SILAC is able to detect the larger amounts of SART1 present in the heavy arginine labeled forms, corresponding to the 6His-SUMO-1 and 6His-SUMO-2 conjugated fractions. Importantly, our successful identification of a number of known SUMO target proteins provides a powerful positive control that further underlines the validity of our approach.
Several lines of evidence further support the notion that different SUMO family members display target protein preferences in vivo. Preferential conjugation of RanGAP1
to SUMO-1 was previously noted by Saitoh and Hinchey (28) . In two proteomics approaches using SUMO-1 and SUMO-3, the sets of identified target proteins were also only partially overlapping, but the interpretation of these results is more complicated due to the use of non-quantitative proteomics approaches (33, 35 the preferential usage of SUMO-2 over SUMO-1 (51). This is in line with our results demonstrating the preferential conjugation of Sp100 to SUMO-2. However, it is currently unclear whether RanBP2 also regulates SUMO-2 conjugation of PML and Sp100 in cells.
The elucidation of the cellular mechanism underlying target protein preferences for different SUMO family members is therefore an important future objective.
Previously, it has been shown that Sp100 can also be conjugated to SUMO-1 in an interferon-dependent manner (50) . This could indicate that SUMO target protein preferences can be stimulus dependent. A more detailed study of conditional sumoylation is required to obtain better insight in the target protein preferences of different SUMO family member upon activation of specific cellular signaling pathways.
In addition to differences in target protein preferences for SUMO-1 and SUMO-2/3, the relative amount of conjugated SUMO compared to free SUMO is also different between these SUMO family members. It has been shown that a large pool of free nonconjugated SUMO-2/3 exists in COS-7 cells compared to SUMO-1 that mainly exists in the protein-conjugated form (28) . The free SUMO-2/3 pool is conjugated to target proteins in a stress-dependent manner. In contrast to the situation in COS-7 cells, the pool of free SUMO-2/3 in the HeLa cells used by us appears to be small, although it is probably larger than the pool of free SUMO-1, and many proteins are SUMO-2/3 conjugated in a stress-independent manner (Fig. 1) . Thus, cell-type specific differences in conjugation-efficiencies of SUMO-2/3 appear to exist.
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TABLES
Proteins identified by at least two arginine-containing peptides with a minimum SILAC cutoff ratio of 1.5 are presented here and include 25 SUMO-1 targets (top part), 19
SUMO-2 targets (middle part) and 9 proteins conjugated to both SUMO family members 

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