SUSP1 depletion causes redistribution of SUMO2 and -3 but not SUMO1

To examine the biological role of SUSP1, its expression was suppressed by siRNA-mediated gene silencing in human osteosarcoma-derived cells (U2OS cells) that stably express N-terminal EGFP fusions to SUMO1, -2, or -3 (Fig. 2 A). 48 h after transfection, cells that were treated with siRNAs against SUSP1 mRNA showed substantially lower levels of SUSP1 protein (<10%) than control cells that were treated with siRNAs directed against Lamin A/C mRNA (Fig. 2 B). In SUSP1-depleted cells, EGFP-SUMO1 distribution was indistinguishable from Lamin-depleted controls (Fig. 2 A). In contrast, EGFP-SUMO2 and -SUMO3 showed striking accumulation within nuclear foci in ~30% of the SUSP1-depleted cells, although this redistribution was not observed in the control cells. Although the overall spectrum of EGFP-SUMO2– or EGFP-SUMO3–conjugated targets detected by Western blotting was not grossly different after depletion of SUSP1, we observed a moderate but consistent increase in very high molecular weight GFP-containing species (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200510103/DC1). No comparable accumulation of high molecular weight GFP-containing species occurred in EGFP-SUMO1–expressing cells (unpublished data). With prolonged incubations after siRNA treatment, we observed a higher level of cell death in the SUSP1-depleted cells expressing EGFP-SUMO2 and -SUMO3 than in Lamin-depleted controls (unpublished data), possibly suggesting that the accumulation of such SUMO2/3-conjugated species is detrimental to cell survival.

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Figure 2.

Deficit of SUSP1 deconjugating activity causes focal concentration of EGFP-SUMO2 or -SUMO3. (A) U2OS cells stably expressing full-length EGFP-SUMO1, -SUMO2, or -SUMO3 were transfected with Lamin or SUSP1 siRNA. After 48 h, live cell images were acquired. Approximately 30% of EGFP-SUMO2– or EGFP-SUMO3–expressing cells showed the speckled phenotype after SUSP1 depletion. This phenomenon was never observed in the Lamin-depleted cells or in the EGFP-SUMO1–expressing cells. (B) Cells stably expressing EGFP–SUMO3 were transfected with a plasmid for the synthesis of SUSP1-RFP (pDSRed-N1-Mono-SUSP1), encoding by an mRNA with point mutations that rendered it impervious to RNAi. (a) Western blotting analysis with anti-SUSP1 antibodies (top). Lane 1 shows SUSP1 siRNA without rescue, lane 2 shows SUSP1 siRNA with rescue, lane 3 shows Lamin siRNA without rescue, and lane 4 shows Lamin siRNA with rescue. The same membrane was reprobed with anti-actin antibodies (bottom), to show protein loading. (b) A representative picture showing SUSP1-RFP in rescued cells. EGFP-SUMO3 is in green, SUSP1-RFP is in red, and DNA is in blue. (c) The percentage of cells showing the EGFP-SUMO3 speckles after SUSP1 siRNA and transfection with a control plasmid, pDSRed-N1 (−Rescue) or with pDSRed-N1-Mono-SUSP1 (+Rescue). Error bars represent SD for two separate experiments counting >200 total cells. n = 2. (C) U2OS cells stably expressing mature EGFP-SUMO2 or -SUMO3, ending in C-terminal diglycine motifs were transfected with Lamin siRNA or SUSP1 siRNA as in A. (D) U2OS cells stably expressing nonconjugatable EGFP-SUMO2 or -SUMO3, ending in a single C-terminal glycine were transfected with Lamin or SUSP1 siRNA, as in A. Bars, 10 μM.

We did not observe redistribution of endogenous SUMO2 and -3 after SUSP1 depletion by immunofluorescence in U2OS cells without EGFP-SUMO fusions (unpublished data). The most straightforward explanation for this difference is that there is sufficient SENP activity remaining in SUSP1-depleted cells, either from residual SUSP1 or from the redundant activity of other SENP/Ulps, to prevent the inappropriate accumulation of conjugated species when SUMO2 and -3 are present at physiological concentrations. In this case, the amount of SUSP1 activity may become limiting only when the concentration of conjugated species is elevated, as in cells expressing EGFP-SUMO2 or -SUMO3.

Importantly, redistribution of EGFP-SUMO2 and -SUMO3 was not observed when siRNAs were cotransfected with a plasmid that expresses a fusion between SUSP1 and the red fluorescent protein (RFP), encoded by a mutant mRNA that is not degraded by the siRNAs (Fig. 2 B). This finding substantiates the conclusion that altered EGFP-SUMO2 and -SUMO3 distributions are a direct result of SUSP1 depletion. Together, these findings indicate that SUSP1 plays a paralogue-specific role in the regulation of nucleoplasmic SUMO2/3.

SUSP1 acts as an isopeptidase for SUMO2/3 deconjugation

To determine whether the processing function of SUSP1 was crucial for the redistribution of EGFP-SUMO2 or -SUMO3, we repeated the siRNA experiment in U2OS-derived cell lines stably expressing the processed forms of EGFP-SUMO2 and -SUMO3, which terminate with the mature diglycine motif (Fig. 2 C). Similar to cells expressing the unprocessed forms, EGFP-SUMO2(GG) or -SUMO3(GG) became concentrated strongly into nuclear foci. This result implies that accumulation of EGFP-SUMO2 or -SUMO3 into foci after SUSP1 depletion results from the inability to deconjugate these fusion proteins from their substrates rather than insufficient processing capacity. Consistent with this conclusion, we observed no redistribution after siRNA of nonconjugatable EGFP-SUMO2 or -SUMO3 that had only a single C-terminal glycine (Fig. 2 D).

EGFP-SUMO2 and -SUMO3 accumulate in PML bodies after SUSP1 depletion

Together, our results suggested that insufficient levels of SUSP1 deconjugation activity caused the accumulation of EGFP-SUMO2 and -SUMO3 in nuclear foci. It was therefore of interest to characterize these structures. First, we examined the size of EGFP-SUMO2– or EGFP-SUMO3–labeled foci in cells depleted of SUSP1 or Lamin A/C (Fig. 3). We observed a bimodal distribution of foci size in the SUSP1-depleted cells, with a substantial peak of larger foci centered ~0.8 μm in diameter. There were very few foci of this size in the Lamin-depleted cells.

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Figure 3.

Large EGFP-SUMO3 speckles in SUSP1-depleted cells colocalize with PML. (A) U2OS cells expressing EGFP-SUMO3 (green) were treated with Lamin or SUSP1 siRNA as indicated. The cells were fixed, permeabilized, and stained with anti-PML antibodies (red). Merged images are shown on right. (B) The diameters of EGFP-SUMO3 foci in SUSP1-depleted (a) and Lamin-depleted (b) cells were measured. These distributions were compared with the size distribution of foci containing (yellow bars) or lacking (green bars) colocalized PML in SUSP1-depleted (c) and Lamin-depleted (d) cells. Bar, 10 μM.

We reasoned that these foci might correspond to nuclear subcompartments where SUMO2/3 normally play a physiological role. To test this idea, we stained SUSP1-depleted cells with a variety of antibodies that recognize antigens characteristic of splicing foci (SC35; Huang and Spector, 1991), pericentric heterochromatin (trimethyl-Histone H3 [Lys9]; Peters et al., 2003), and centromeres (CREST sera; Earnshaw and Rothfield, 1985). We did not observe colocalization of EGFP-SUMO2/3 with either SC35 or trimethyl-Histone H3 (unpublished data). We observed some colocalization with CREST sera staining, but the extent of this accumulation was not substantially different between SUSP1-depleted and control cells (unpublished data). We also stained the cells with antibodies directed against a variety of SUMO substrates, including Bloom's antigen (BLM), Wilms' tumor 1 (WT1), proliferating cell nuclear antigen (PCNA), p300, and PML (Johnson, 2004; Smolen et al., 2004). We did not find redistribution of BLM, WT1, PCNA, or p300, nor their accumulation within the EGFP-SUMO2/3 foci (unpublished data). However, we saw a substantial increase in the number of PML bodies after SUSP1 depletion and extensive colocalization of PML with EGFP-SUMO2 or -SUMO3 (Fig. 3 A).

Comparison of PML bodies versus EGFP-SUMO3 foci (Fig. 3 B) showed that the size distribution of PML bodies after SUSP1 depletion mirrored the change in EGFP-SUMO3 foci. Indeed, the larger EGFP-SUMO3 foci in SUSP1-depleted cells almost universally correlated to PML-containing structures (Fig. 3 A), suggesting that enlarged PML bodies result from insufficient SUMO2/3 deconjugation after SUSP1 depletion. The smaller, non–PML-containing bodies may analogously represent other structures to which SUMO2/3 conjugates associate under normal circumstances.

SUSP1 acts specifically as a SUMO2/3 protease in vitro

To directly determine the paralogue specificity of SUSP1, we used VS derivatives of tagged SUMO1 and -2 (HA-SU1-VS and HA-SU2-VS; Hemelaar et al., 2004). VS reagents derived from ubiquitin-like proteins covalently react with the nucleophilic active site residues of their respective modifying enzymes, showing considerable preference toward deconjugation enzymes over E1 and E2 enzymes. To test the reactivity of SUSP1 for SUMO1 and -2, extracts of control and SUSP1-depleted U2OS cells were incubated with HA-SU1-VS or HA-SU2-VS. Samples were taken at different times and subjected to SDS-PAGE and Western blotting with anti-HA antibodies (Fig. 4 A). We observed a band in HA-SU2-VS–treated control extracts that migrated with an apparent molecular mass of ~160 kD. This band was absent in the SUSP1-depleted extracts. Both the molecular mass of this band and its depletion through RNAi indicate that it was derived from a covalent attachment of HA-SU2-VS and SUSP1. Moreover, when we immunoblotted the same samples with anti-SUSP1 antibodies, we observed that SUSP1 was quantitatively shifted into a higher molecular mass band within 5 min of incubation with HA-SU2-VS, confirming this conclusion.

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Figure 4.

SUSP1 paralogue preference for SUMO2. Cell lysates from SUSP1- or Lamin-depleted cells were incubated with HA-SU1-VS or HA-SU2-VS for the indicated times. The reactions were terminated with SDS sample buffer and analyzed by Western blotting using anti-HA (A) or anti-SUSP1 antibodies (B).

Notably, an HA-reactive band of the same size was only weakly seen in the reaction containing HA-SU1-VS, suggesting that it has a significantly lower affinity for SUSP1. Although some SUSP1 migrated at a higher molecular weight after incubation with HA-SU1-VS, this conversion was significantly slower than in reactions containing HA-SU2-VS and did not proceed to completion within 30 min. Together, these results suggest that SUSP1 has a strong preference for SUMO2 over SUMO1. To test this conclusion using more stringent criterion, we preblocked U2OS cell lysates with a threefold molar excess (over the VSs) of untagged aldehyde derivatives of SUMO1 or -2, which bind reversibly to the active sites of SUMO proteases (Pickart and Rose, 1986). After blocking, the extracts were allowed to react with HA-SU2-VS and analyzed by Western blotting using anti-HA antibodies (Fig. 5). In these experiments, SUMO2 aldehyde, but not SUMO1 aldehyde, competed for the binding of HA-SU2-VS to SUSP1 and caused reduction in the intensity of the HA-reactive band at 160 kD, rigorously supporting the conclusion that SUSP1 has higher affinity for SUMO2/3 paralogues.

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Figure 5.

SUSP1 preferentially binds to SUMO2 aldehyde. U2OS cell lysates were incubated with 0.3 or 0.9 ng/μl of SUMO1 or SUMO2 aldehyde. The samples were then allowed to react with HA-SU1-VS or HA-SU2-VS, as indicated, for 5 min. The reactions were terminated with sample buffer and analyzed by Western blotting with anti-HA antibodies.

DNA constructs

pEGFP and pDsRed vectors were obtained from CLONTECH Laboratories, Inc. Restriction enzymes were purchased from New England Biolabs, Inc. DNA primers and Platinum Taq Hi Fi DNA polymerase were obtained from Invitrogen.

Full-length (unprocessed) human SUMO1, -2, and -3; mature SUMO1GG, SUMO2GG, and SUMO3GG (with diglycine at amino acid positions 96–97, 92–93, and 90–91, respectively); and nonconjugatable SUMO1G, SUMO2G, and SUMO3G (with a single C-terminal glycine at amino acid positions 96, 92, and 90, respectively) coding sequences were subcloned into HindIII and BamHI restriction sites of pEGFP-C3. Full-length SUSP1 was cloned into XhoI and BamHI sites of either pEGFP-NI or -C2 by RT-PCR from HeLa cell total RNA. Deletion fragments of SUSP1 were amplified by PCR using specific primers and cloned into XhoI and BamHI sites of pEGFP-N1.

The rescue SUSP1 plasmid was prepared by sense-antisense method of site-directed mutagenesis using the primer set 5′-GAAGAATCTGAAGGGGATACA-3′, 5′-TTCATGATCAAAGCTCCAATT-3′, which incorporates three sense point mutations at 124 bp (A to T), 125 bp (G to C), and 132 bp (A to G) from the start of the open reading frame and generates a BciVI restriction site. This was then subcloned into XhoI and BamHI restriction sites of pDSRed-Mono-N1 vector. The absence of any unintended mutations was confirmed for all constructs by DNA sequencing (SeqWright DNA Technology Services).

Cell culture

HeLa cells, U2OS cells, and their derivative stable lines were grown at 37°C in a humidified atmosphere of 5% CO₂ in DME (Biosource International) with 2 mM glutamine supplemented with 10% fetal bovine serum (Gemini Bio-Products), 100 U penicillin/ml, and 100 μg/ml streptomycin. Cells were transfected with plasmids using Effectene reagent (QIAGEN) according to the manufacturer's instructions.

siRNA-mediated depletion of SUSP1

For transient transfections, cells were grown to 50% confluency and Effectene reagent (QIAGEN) was used according to the manufacturer's instructions. Duplex siRNA directed against SUSP1 and Lamin A/C messages were obtained from QIAGEN. SUSP1 and Lamin A/C siRNA sequences were 5′-AAGAAAGTGAAGGAGATACAG-3′ and 5′-AACTGGACTTCCAGAAGAACA-3′, respectively, with standard dTdT 3′ extension.

siRNA experiments were performed in 24-well culture plates, coverslip-bottomed 4-well chambers (LabTekII; Fisher Scientific), or 6-cm dishes. Cells were allowed to attach overnight at 40% confluency and washed once with DME without antibiotics immediately before transfection of siRNA. 3 μl siRNA (20 μM) and 3 μl of oligofectamine (Invitrogen) were suspended in 50 and 12 μl of OptiMEM (Invitrogen), respectively, in separate tubes. The tubes were gently tapped and kept at RT for 5 min, and the contents of the two tubes were mixed, kept at RT for a further 20 min, and added to a single well of 24-well plate or a well of 4-well chamber containing 0.5 ml complete DME without antibiotics. For the 6-cm dish, the same procedure was followed but the reagents were scaled up to four times.

Production and maintenance of stable cell lines

For stable cell line selection, 0.5 mg/ml Geneticin was added to culture medium 24 h after transfection. Cells were incubated in Geneticin-containing culture medium that was refreshed daily for a period of 1 wk after resistant colonies were reseeded sparsely to culture single cell–derived colonies. Uniformly fluorescent colonies derived from single cells were marked and isolated under an inverted fluorescence microscope. Stably transgenic cells were maintained thereafter in medium containing 0.25 mg/ml Geneticin.

Immunofluorescence

Cells were grown either on poly-l-lysine–coated coverslips or LabTekII coverslip-bottomed chambers. For immunofluorescence, the cells were washed with PBS and fixed for 12 min at ambient temperature with 4% paraformaldehyde in PME buffer (PBS supplemented with 5 mM each of MgCl₂ and EGTA). The cells were permeabilized with 0.5% Triton X-100 for 10 min. After washing with PME, the cells were blocked for 10 min in 1% normal horse serum (Vector Laboratories) in PME and incubated for 1 h with anti-PML antibodies diluted 1:200 in blocking solution. The coverslips were washed and incubated for 45 min with Alexa Fluor 568–conjugated secondary antibodies (Invitrogen) diluted 1:400 in blocking solution. Unbound antibodies were removed by washing, and the cells were briefly incubated in 100 ng/ml Hoechst 33258 DNA stain. Finally, the coverslips were mounted in Vectashield mounting medium (Vector Laboratories).

Microscopy

Fluorescence microscopy was performed on a confocal microscope (LSM510 META; Carl Zeiss MicroImaging, Inc.) equipped with 40× Plan Neofluar objective. LabTekII coverslip-bottomed chambers were used for live analyses. Temperature (37°C) and CO₂ concentration (5%) were maintained using a humidified environmental chamber. Confocal microscopy software (SP2 version 3.2; Carl Zeiss MicroImaging, Inc.) was used for capturing images, which were then analyzed by Photoshop 7.0 (Adobe). In all figures, scale bars represent 10 μm.

Illumination of samples for fluorescent imaging

We used a 543-nm HeNe laser (5 mW output; detection LP560 nm) for detection of RFP-SUSP1-rescue signal and indirect immunolocalization of PML by Alexa Fluor 568–labeled antibodies. The 488-nm line of an Argon laser (25 mW nominal output; detection BP 505–530 nm) were used for analysis of GFP-conjugated proteins. Hoechst 33258 images were captured using the 364-nm line of an ion laser (Enterprise II ML UV [Coherent, Inc.]; 80 mW nominal output; detection BP 385–470 nm).

Synthesis of SUMO derivatives

Expression of SUMO-intein-CBD fusion proteins in E. coli was as described in Hemelaar et al., (2004). Bacterial lysates were prepared as described previously (Hemelaar et al., 2004) and bound to chitin bead columns (New England Biolabs, Inc.). The columns were washed with five volumes of lysis buffer (50 mM Hepes, 100 mM sodium acetate, pH 6.5, and 50 μM PMSF), followed by three volumes of lysis buffer containing 50 mM β-mercaptoethanesulfonic acid (MESNa; Sigma-Aldrich). The column was incubated overnight at 37°C in the buffer with MESNa to allow on-column cleavage. The SUMO-MESNa thiolesters were eluted with 1.5 column volumes of lysis buffer, and the fractions containing SUMO-MESNa products were concentrated on a Centriprep (3,000-molecular-weight cutoff; Millipore). To convert SUMO-MESNa products to their VS derivatives, a large excess of Glycine VS (final concentration 0.25 M) was added to concentrated SUMO-MESNa (1–3 mg/ml; 500 μl), followed by addition of 75 μl of 2 M N-hydroxysuccinimide and 30 μl of 2 M NaOH (provided by H. Ovaa, Netherlands Cancer Institute, Amsterdam, Netherlands; Hemelaar et al., 2004). The mixture was incubated for 1–2 h at 37°C, and the reaction was terminated by the addition of 30 μl of 2 M HCl.

To obtain SUMO1 and -2 aldehydes, SUMO1 and -2 acetals were synthesized by reacting SUMO-MESNa thiolesters (1–3 mg/ml; 500 μl) with 0.2 ml of 4 M aminoacetaldehyde diethyl acetal, pH 8.5, and 0.14 ml of N-hydroxysuccinimide, pH 7.0, at 37°C for 2 h. To obtain SUMO1 and -2 aldehydes, SUMO1 and -2 acetals were separately incubated with 0.15 M HCl at 37°C for 30 min. All steps of derivative syntheses were monitored by HPLC and by SDS-PAGE.

SUMO derivatives were purified by ion exchange chromatography, desalted on PD10 columns, and dialyzed into 50 mM Triethanolamine, pH 6.5. SUMO1 and -2 aldehydes were further purified by adsorbing on an anion exchange column (Mono Q HR; Pharmacia) equilibrated with 50 mM TEA, pH 6.5, and eluted with a linear gradient of NaCl from 0 to 0.5 M (flow rate of 0.75 ml/min; fraction size = 1 ml). For purification of HA-SUMO1-VS and HA-SUMO2-VS on Mono Q, 50 mM Bis-Tris, pH 5.5, was used, with a gradient from 0 to 0.15 M NaCl. In all cases, proteins were monitored by absorbance at 205 nm. Individual Mono Q fractions were assayed by HPLC, and appropriate fractions were pooled and stored at −80°C.

HA-SUMO-VS experiments

VS and aldehyde derivatives of SUMO1 and -2 were prepared as described previously (Hemelaar et al., 2004). 48 h after transfection with siRNAs directed against Lamin or SUSP1, as indicated, U2OS cells were harvested and washed twice in PBS containing 1 mM AEBSF. The cells were resuspended in reaction buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, and 25 μg/ml each of leupeptin, aprotinin, and pepstatin) and sonicated. Cell lysates were clarified by centrifugation at 16,000 g for 10 min. The protein concentration of the total cell lysate was maintained between 500 μg/ml and 2 mg/ml. SUMO-VS was added to a final concentration of 0.3 ng/μl and allowed to react for the indicated intervals. The reactions were terminated by the addition of SDS sample buffer and analyzed by Western blotting with the indicated antibodies.

SUMO deconjugation assay

SUMO1- and SUMO2-conjugated (His)₆-T7-RanGAP-C2 fragment were produced in bacteria (Uchimura et al., 2004) and purified using Ni-NTA beads (provided by Y. Uchimura and H. Saitoh, Kumamoto University, Kumamoto, Japan). SUSP1 was immunoprecipitated from HeLa cell lysate using antibody conjugated to N-hydroxysuccinimide–Sepharose (2 μg/ml of beads). 10 μl SUSP1 beads were incubated with 500 ng of SUMO-conjugated T7-RanGAP-C2 in a total reaction volume of 30 μl at 23°C for the indicated times. The reaction was terminated with sample buffer and analyzed by Western blotting with antibodies against T7, SUMO1, or SUMO2/3.

Preparation of SUMO processing substrates

Full-length SUMO1, -2, and -3 cDNAs were subcloned into pET28b expression plasmids that had been cut with NcoI–NdeI, NcoI–NdeI, and BspH1–NdeI restriction endonucleases, respectively. These plasmids were transfected into E. coli, and the expression of the fusion proteins was induced with 0.4 mM IPTG under standard conditions. The expressed C-terminally tagged SUMO-T7-His proteins were purified on a Ni-NTA column followed by Q-Sepharose and MonoQ columns to generate pure tagged substrate for the processing reactions.

SUMO processing assay

HeLa cells from confluent 15-cm dishes were harvested by trypsinization and washed twice with ice-cold PBS. The cells were snap frozen in liquid nitrogen and then sonicated in 500 μl lysis buffer (10 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM AEBSF, and 5 mg/ml each of leupeptin, pepstatin, and aprotinin). The cell lysates were centrifuged at 120,000 rpm for 5 min at 2°C. The cleared supernatants were incubated with 5 μg of rabbit IgG, affinity-purified anti-SENP1 antibody, or affinity-purified anti-SUSP1 antibody for 1 h at 4°C. 50 μl preblocked protein A beads were added to the lysates and incubated for another hour and subsequently washed three times in lysis buffer. The beads were reacted with 0.3 ng/μl HA-SUMO2-VS in lysis buffer containing 100 μg/ml BSA for 15 min at RT, and the reactions were terminated with sample buffer. Using the different dilutions of the HA-SUMO-VS adducts thus obtained, beads having an equivalent enzymatic activity for SENP1 and SUSP1 were determined by anti-HA Western blot. Beads having equivalent amount of HA-SUMO2-VS reactivity and control beads were then incubated with 40 ng/μl SUMO1, -2 or -3 T7 for different time points at 23°C in 20 μl reaction volume. The reactions were stopped by the addition of sample buffer, and Western blots were performed using anti-T7, -SUMO1, or -SUMO2/3 antibodies.

Evolutionary relationship of SENP/Ulp family members

The eukaryotic genome databases of the National Center for Biotechnology Information were searched using psi-BLAST using human Senp protein sequence and query with e-value inclusion cutoff of 0.001, for 6–10 cycles. Whenever any specific genome of interest failed to give any hit, the nucleotide genome sequence of corresponding organism was searched using TBLASTN with human Senp protein sequences as query. The collected protein sequences were aligned using MUSCLE (Edgar, 2004) with 100 iterations. The generated multiple alignments were manually corrected using the conserved C48 peptidase domain as anchor. Phylogenetic analyses were performed using minimum evolution (least-square) method as implemented in MEGA3.1 (Kumar et al., 2004), with Poisson correction model and pairwise deletion of gaps and 1,000 bootstrap replicates.

We would like to thank Dr. M.K. Basu (National Library of Medicine, National Institutes of Health) for his help in construction of the SENP evolutionary tree. We would like to thank Dr. M. Matunis for providing anti-BLM antibody and H. Ovaa (Netherlands Cancer Institute) for a gift of gylcyl VS tosylate. We would like to thank Drs. Y. Uchimura and H. Saitoh (Kumamoto University) for providing constructs and strains required for production of substrates used in Fig. 7.

D. Mukhopadhyay, F. Ayaydin, S.-H. Tan, and M. Dasso were supported throughout this work by National Institute of Child Health and Human Development Intramural funds. T. Anan was the recipient of a fellowship from the Japan Society for the Promotion of Science. K.D. Wilkinson and N. Kolli were supported by National Institutes of Health grant GM66355. Studies on PML were initiated with support from Human Frontier Science Program research grant RG0229/1999-M.