Sumo Proteins

Small Ubiquitin-related Modifier (SUMO) Proteins

Sumoylation, Desumoylation and Function

Assignment for Genomics and Proteomics

Done By: 5041 Priyanka Singh 5042 Priyanka Singh 5043 Purushottam Grover 5044 Radha Patel 5045 Rahul Pratap Singh 5046 Rajeev Singh Rajput 5048 Reshu Goel 5049 Ritika Sharma 5050 Rohit Chadha 5051 Roshni Nair 5052 Ruchika Gupta

Small Ubiquitin-related Modifier or SUMO proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off. It has been shown that the SUMO conjugation system operates in plants, through a characterization of the Arabidopsis SUMO pathway. An eight-gene family encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation, and release. A diverse array of conjugates were detected, some of which appeared to be SUMO isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3 conjugates increased substantially following exposure of seedlings to stress conditions, including heat shock, H2O2, ethanol, and the amino acid analog canavanine. The heat-induced accumulation could be detected within 2 min from the start of a temperature upshift, suggesting that SUMO1/2 conjugation is one of the early plant responses to heat stress. Overexpression of SUMO2 enhanced both the steady state levels of SUMO2 conjugates under normal growth conditions and the subsequent heat shock-induced accumulation. This accumulation was dampened in an Arabidopsis line engineered for increased thermotolerance by overexpressing the cytosolic isoform of the HSP70 chaperonin. Structure schematic of human SUMO1 protein made with iMol and based on PDB file 1A5R, an NMR structure; the backbone of the protein is represented as a ribbon, highlighting secondary structure; N-terminus in blue, C-terminus in red

The same structure represented with atoms represented as spheres to show the shape of the protein; human SUMO1, PDB file 1A5R

Structure Sumo proteins are small proteins; most are around 100 amino acids in length and 12 kDa in mass. The exact length and mass varies between Sumo family members and depends on which organism the protein comes from. For example, human SUMO1, also shown in the figures, is 101 residues long and has a mass of 11.6 kDa. Its homologues in rat and mice are also 101 residues long, while the presumed relative in C. elegans has only 91 amino acids.

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SUMO Conjugation SUMO conjugation to its target is analogous to that of Ubiquitin (as it is for the other Ubiquitin-like proteins such as NEDD 8). A C-terminal peptide is cleaved from SUMO by a protease (in human these are the SENP proteases or Ulp1 in yeast) using ATP to reveal a diglycine motif. SUMO then becomes bound to an E1 enzyme (SUMO Activating Enzyme (SAE)) which is a heterodimer. It is then passed to an E2 which is a conjugating enzyme (Ubc9). Finally, one of a small number of E3 ligating proteins attaches it to the protein. In yeast, there are two SUMO E3 proteins, Siz1 and Siz2. Whilst in ubiquitination an E3 is essential to add ubiquitin to its target evidence suggests that the conjugator is sufficient in Sumoylation as long as the consensus sequence is present. It is thought that the E3 ligase aids enhancement of Sumoylation and facilitates attachment when this consensus sequence is absent. The B-K-x-D/E motif is not an absolute requirement for SUMO binding. E3 ligases are more abundant than E1 and E2 for SUMO There are also the HECT proteins and E3's are known to arise from a complex of proteins such as with RanGap. Recent evidence has shown that PIAS-gamma is required for the sumoylation of the transcription factor yy1 but it is independent of the zinc-RING finger (identified as the functional domain of the E3 ligases). It is as yet unknown whether there is another dimension to SUMO conjugation or if this is specific to yy1. Sumoylation is reversible and is removed from targets by a protease in an ATP dependant manner. The Ulp2 protease is found bound at the nuclear pore and maybe very important in regulating the localisation of proteins to the nucleous; a known role of SUMO. SUMO has also been shown to form chains. It is thought that these preassemble on the conjugator and are then passed to the target. The biological significance of these is yet unknown.

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Enzymology and Regulation of SUMO Conjugation and Deconjugation Three different ubiquitous SUMO-related proteins have been identified in mammalian cells, SUMO-1, SUMO-2, and SUMO-3, with SUMO-2 and SUMO-3 having greater sequence relatedness with each other than with SUMO-1 (3, 4). Recently a tissue-specific SUMO-4 has been identified in human kidney with homology to SUMO-2/3, which raises the possibility that some SUMO proteins could have tissue-dependent functions (9). SUMO modification occurs on the lysine in the consensus sequence KXE (where represents a hydrophobic amino acid, and X represents any amino acid) (2, 3). The mechanism involved in maturation and transfer of SUMO to target substrates is very similar to that seen with ubiquitination and other ubiquitin-like proteins (3, 4). This process involves four enzymatic steps: maturation, activation, conjugation, and ligation (Fig.). In the first step the SUMO protein is cleaved by SUMO-specific carboxyl-terminal hydrolase to produce a carboxyl-terminal diglycine motif. This process of maturation is identical with all three mammalian SUMO forms. After maturation, SUMO proteins are able to be utilized for conjugation to proteins. The SUMO-activating (E1) enzyme is a heterodimer consisting of Aos1 and Uba2 (also known as SAE1/SAE2 or Sua1/hUba2 in humans). Activation of SUMO by the E1 is an ATP-dependent process and results in the formation of a thioester bond between SUMO and the Uba2 subunit of the E1-activating enzyme. Activation is followed by transfer of SUMO from the E1 enzyme to a conserved cysteine in the conjugating (E2) enzyme, Ubc9. This single E2 enzyme identified so far for the sumoylation pathway contrasts with the multiple E2 enzymes involved in attaching ubiquitin to proteins (4, 10).

FIG. The SUMO conjugation pathway. SUMO is cleaved into its mature form by the SUMO protease Ulp1. After this step it is activated in an ATP-dependent manner by conjugation to the Uba2 subunit of the E1-activating heterodimer Aos1/Uba2. Following activation SUMO is transferred to the E2-conjugating enzyme Ubc9. In the final step SUMO is transferred in a ligation reaction to substrate proteins forming an isopeptide bond between the terminal glycine on SUMO and the -amino group of a lysine in the target protein to be modified. This ligation reaction is aided by SUMO ligase E3 proteins (E3), which can directly interact with target proteins or the E2 enzyme.

The final step of sumoylation involves ligation of SUMO to the target protein. Until recently there was speculation as to whether SUMO ligation to target proteins involved E3 ligase-like proteins such as are required for ubiquitination. However, it is now clear that such E3 ligases do exist for the SUMO-1 modification pathway and that they play important roles in

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modulating the efficiency of SUMO attachment to target proteins (2). As with the ubiquitin system, SUMO E3 proteins are defined by three characteristics: binding to the substrate protein either directly or indirectly, binding to the E2 conjugation enzyme, and the ability to stimulate transfer of the modifier to the substrate or to another modifier in the case of modifier chain formation. Three different general types of SUMO E3 ligases have been described (11–16). The first E3 group comprises the PIAS family of proteins. In yeast only two E3 proteins have been identified (Siz1 and Siz2) which have sequence similarity to mammalian PIAS proteins, of which at least five members have SUMO E3 activity (11, 12, 14, 17). These proteins share a common RING finger-like structure and bind directly to the Ubc9 E2 enzyme and some SUMO protein targets. This RING finger motif has also been identified in some of the ubiquitin E3 ligases (18). A second type of SUMO E3 protein found in mammalian systems is RanBP2, which is part of the nuclear pore complex (15). RanBP2 differs from the PIAS proteins in that it does not have a RING finger domain or homology to ubiquitin E3 proteins. However, it interacts with Ubc9 although not the sumoylation target protein (15). The final E3 protein type (Pc2) belongs to the Poly-comb protein family and stimulates sumoylation of C terminus binding protein (16). In some cases sumoylation can exist in the form of polymeric chains because the SUMO-1 paralogs SUMO-2 and SUMO-3 have internal SUMO modification consensus sites that allow the formation of polymeric SUMO chains on modified proteins (19). Other post-translational modifications can regulate the SUMO modification of a protein. Phosphorylation can also act positively, as in the case of the transcription factor HSF1 where sumoylation is stimulated by phosphorylation of serine residues near the SUMO modification site (20, 21). As with other post-translational modifications, SUMO groups can be removed from proteins in a reaction catalyzed by SUMO-specific proteases (2, 3). Some of these proteases have dual functionality in that they both process SUMO to its mature diglycine form and also cleave the isopeptide bond between SUMO and its target proteins (2). In yeast, two SUMO proteases have been identified, Ulpl and Ulp2/Smt4, both of which are specific for SUMO and display compartmentalization, with Ulp1 being present at the nuclear pore complex and Upl2/Smt4 being present in the nucleoplasm. In mammals, several SUMO proteases have been confirmed with the possibility of many more being present due to alternative splice variants (2). As with yeast, many of the mammalian SUMO proteases are localized to different cellular compartments, which may function to regulate the balance of protein sumoylation in these compartments.

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Function SUMO modification of proteins has many functions. Among the most frequent and best studied are protein stability, nuclear-cytosolic transport, transcriptional regulation (mostly transcriptional repression). As opposed to ubiquitin modification which targets proteins for degradation, SUMOylation increases a protein's lifetime. It can also change a protein's location in the cell. For example, the Sumo modification of hNinein leads to its movement from the centrosome to the nucleus. In most cases Sumo attachment to transcriptional regulators correlates with inhibition of transcriptio. There are many more proposed functions. Refer to the GeneRIFs of the Sumo proteins, e.g. human SUMO1, to find out more. SUMO-1 is the main SUMO in human cells and is the one that organisms like yeast show the most similarity to. However, there are a further 3 isoforms in humans. SUMO-2/3 show high similarity to each other more so then to SUMO-1. On stress the free SUMO-2/3 pool disappears and a range of specific SUMO-2/3 modifications occur. They seem to be involved specifically in the stress response. SUMO-4 shows similarity to -2/3. Until recently it was thought SUMO-4 was either tissue specific (pancreas) or a pseudo gene. Evidence is now indicating it is the former and SUMO-4 defects may be involved in Type-1 and -2 diabetes. Sumoylation and Subcellular Localization Depending on the target protein, sumoylation can occur in the cytoplasm or nucleus, and this modification is involved in regulating the subcellular localization of a number of substrate proteins. RanGAP1 was the first identified SUMO substrate and plays an important role in the regulation of transport of ribonucleoproteins and proteins across the NPC (22, 23). Unmodified RanGAP1 resides predominantly in the cytoplasm and upon conjugation with SUMO associates with the cytoplasmic fibers of the NPC (22, 24). SUMO modification directs RanGAP1 to the NPC by an interaction with RanBP2/Nup358, possibly mediated by sumoylation-induced formation of a binding interface for interaction of these two proteins (25). Analysis of nuclear localization signal mutants of a protein called Smad4, a factor with a major role in the TGF- signal transduction pathway, indicates that nuclear import of this protein is required for it to be sumoylated (26). Smad4 moves to the nucleus in response to TGF- stimulation, and immunofluorescence analysis of TGF-induced cells that were SUMO-1 transfected demonstrated an increase in the nuclear localization of Smad4 (26). Nuclei contain a number of distinct bodies that are defined, at least in part, by the proteins contained in them. For example, the PML and Sp100 proteins are major components of PML nuclear bodies (PML NBs), also called ND10. Sumoylation has been found to be required for the subcellular localization of some, but not all, proteins found in bodies such as ND10. For example, the sumoylated forms of PML and Sp100 are found exclusively in the nucleus (27). SUMO conjugation was determined to be essential for PML protein localization in ND10, whereas the targeting and accumulation of Sp100 in these bodies was not sumoylation-dependent (27, 28). This appears to be true for other SUMO substrates as well (p53, LEF1, Daxx, and SRF1) which will localize to ND10 even after mutation of their target lysine (3). An exciting development in understanding both the subcellular sites of sumoylation and the role of this modification in regulating subcellular localization of proteins has been the discovery that components of the sumoylation machinery are localized at the

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nuclear pore complex (32, 33). This localization suggests that sumoylation of at least some proteins occurring as they enter the nucleus could be involved in nuclear import itself or perhaps retention of these proteins in the nucleus. For example, Nup358, a nuclear pore protein demonstrated to have SUMO E3 ligase activity, localizes predominantly to the cytoplasmic filaments of the NPC and regulates targeting of RanGAP1 to the NPC (2, 25). Other SUMO enzymes, Ubc9 and SENP2, have also been found to localize at the nuclear pore. Ubc9, the E2 conjugating enzyme for SUMO, localizes to the cytoplasmic and nucleoplasmic faces of the NPC as visualized by immunogold analysis of the nuclear envelope (32). Ubc9 interacts with Nup358 as well as RanGAP1/SUMO-1, and a model has been proposed in which these three proteins interact to form a stable trimeric complex (15, 32). This model is strengthened by the observation that RanGAP1 is protected from SENP2 (SUMO protease) degradation when found in this complex (15, 32). SENP2/Axam itself associates with the nucleoplasmic face of the NPC via its NH2terminal domain (32, 34), and loss of this domain results in relocalization of this enyzme and increased capacity for deconjugation of substrates (34). SENP2 also associates with Nup153, a component of the nuclear basket in humans. Ulp1, the yeast homologue of the vertebrate SUMO isopeptidase SENP2, is required for progression through the cell cycle and also localizes to the NPC via the NH2-terminal domain, which appears to be necessary for localization as well as enzymatic specificity (35). Further establishing the connection between sumoylation and nuclear import are data showing that yeast Ulp1 and Ulp2 mutants, deficient in SUMO conjugation, display an impairment of classical nuclear localization signal-mediated protein import (36). Sumoylation is most often implicated in promoting localization of proteins to the nucleus and in some cases to nuclear bodies. However, there is evidence that SUMO modification could also function to regulate nuclear export of some substrates. For example, nuclear sumoylation of Dictyostelium Mek1 is responsible for its movement to the cytoplasm (37), and mutation of lysine 99 of the TEL protein leads to increased levels of this protein in the nucleus, suggesting a possible role for sumoylation in its nuclear export (38). In addition, sumoylation of heterogeneous nuclear ribonucleoproteins M and C has been proposed to function as a regulator of conformational changes that may influence nucleocytoplasmic transport of these protein complexes (39). SUMO and Transcription Regulation The sumoylation of transcription activators, repressors, coactivators, corepressors, and components of PML NBs is involved in the regulation of gene expression (3, 6, 8). The activities of many transcription factors are regulated by association with PML NBs, and assembly of PML NBs requires sumoylation of the PML protein (3, 7). Thus, alteration of PML sumoylation has broad effects on transcription (1, 7). For example, sumoylation of PML recruits corepressor Daxx to PML NBs, thereby relieving Daxxmediated repression of these genes. Similarly, sumoylation of PML directs p53 to PML NBs and could then trigger some modification, such as acetylation and sumoylation, which stimulates the transcriptional activity of p53. Also, sumoylation of PML recruits another sumoylated nuclear body-associated protein, Sp100. Sumoylation of other transcription factors has also been found to regulate their localization, including Drosophila Dorsal, Bicoid, p73 , and Pdx1 (1). Similar to what is observed for PML, corepressor HIPK2 and repressor TEL and TEL-AML1 localize to nuclear dots in a SUMO-dependent manner. Whether sumoylation alters the repressive function of these transcription factors is unclear.

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The sumoylation of transcription factors has been reported to have different effects on their activities in various pathways including those involving cytokines, WNT, steroid hormone, and AP-1 (3, 6, 8). In most cases, SUMO modification plays a negative role in transcription regulation. The transcription factors that are inhibited by SUMO modification include STAT1, catenin-TCF/LEF, c-Jun, Ah receptor nuclear translocator (ARNT), CEBP , c-Myb, Sp3, IRF-1, SREBPs, SRF, Elk, AP1, AP2, androgen receptor (AR), glucocorticoid receptor (GR), and progesterone receptor (PR), as well as huntingtin (3, 6, 40). The KXE sumoylation site motifs of some factors such as GR, Sp3, c-Myb, C/EBP, and the SREBPs are located within an inhibitory or negative regulatory domain or the so-called "synergy control" motifs that can transrepress transcriptional activity. Mutation of sumoylation sites in transcription factors has been found to increase their transcriptional activity, for example, transcription factors Elk-1, Sp-3, SREBPs, STAT-1, SRF, c-Myb, C/EBPs, AR, p300, c-Jun, GR, and peroxisome proliferatoractivated receptor that may reflect a role for SUMO-1 modification as a negative regulator of transactivation domains (3, 6, 41). Consistent with this idea, overexpression of free SUMO-1 can suppress AP2 and AP2-mediated transcription (8). Direct evidence for repression of transcriptional activity by sumoylation is that fusion of SUMO to GAL4 drastically reduces its activity in reporter gene assays (42). Furthermore, SUMO is also able to inhibit transcription in trans as demonstrated by SUMO-dependent trans-repression of the VP-16 activation domain (43). The effects of SUMO on transcriptional activity may be complicated by the finding that a number of transcription co-factors, such as GRIP1, SRC-1, and histone deacetylases (HDAC) 1 and 4, are also sumoylated (1, 3, 6, 8). Sumoylation might be involved in modulating the functions of proteins as co-activators (GRIP1, SRC-1) or co-repressors (HDAC1, HDAC4) but is not essential (8). Several observations reveal that the PIAS/SUMO system may modulate the assembly of coactivator or corepressor complexes that regulate transcription (40). Other findings indicate further links between the SUMO system and class I and class II HDACs that mediate transcription repression. For example, sumoylation of p300 can mediate repression of gene activity by recruitment of the corepressor HDAC6 (44). Although sumoylation of most transcription factors results in repression, SUMO modification appears to have positive effects on transcriptional activation by the heat shock factors HSF1 and HSF2 and the -catenin-activated factor Tcf-4. Sumoylation of HSF1 and HSF2, which is stress-induced in the case of HSF1 but can be observed for HSF2 present in non-stressed cells, is correlated with their localization to PML NBs (31, 49). For both HSFs, in vitro sumoylation leads to increased DNA-binding activity, but in the case of HSF1, mutation of the sumoylation site did not appear to block stress-induced DNA-binding activity in cells (21, 31, 49). One possibility is that, because of the critical importance of inducing heat shock protein expression in response to cellular stress, cells may have evolved multiple independent pathways for activating HSF1 DNA binding, of which sumoylation is only one. Tcf-4-dependent transcription is activated by coexpression of -catenin and PIASy, and this activation is reduced when Tcf-4 lacks SUMO attachment sites, suggesting that sumoylation activates Tcf-4 (50). Role of SUMO in Genomic Integrity and Chromosomes Since its discovery, sumoylation has held an important role in cell biology that extends into fields such as chromosome cohesion and kinetochore assembly. During mitosis, the proper distribution of chromosomes into replicated cells is a highly

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ordered and complex process that is dependent upon the proper timing of sister chromatid assembly and separation. Misregulation of this process was one of the first phenotypes described in SUMO-1 (Smt3/Pmt3) mutants in yeast, characterized by aberrant mitosis and defects in chromosomal segregation (51, 52). Other components of the sumoylation machinery, such as the E2 conjugating enzyme and SUMO isopeptidases, have also been shown to be important for this process. For example, the SUMO-1 isopeptidase in budding yeast (Smt4) acts as a key regulator of centromeric chromatid cohesion (53). The same study also suggested that the yeast homologue of topoisomerase II (Top2) was the SUMO-1 substrate important for this process, because topoisomerase II mutants lacking SUMO-1 modifications sites can rescue defects in the isopeptidase mutants (53). In support of this idea, topoisomerase II was found to be the major high molecular weight, chromatin-dependent sumoylated protein in mitotic Xenopus extracts (54). Interestingly, this effect does not appear to be mediated via sumoylation-induced changes in topoisomerase II activity, because a dominant negative mutation in the Ubc9 (SUMO E2 conjugation enzyme) prevented sister chromatid cohesion at the metaphase to anaphase transition but did not alter topoisomerase II activity (54). Sumoylation of other proteins such as the Psds5 protein has also been implicated in sister chromatid cohesion. In the case of Psds5, which is a non-essential regulator of cohesion maintenance in yeast, results suggest that the desumoylation of this protein promotes sister chromatid dissolution by rendering the cohesin complex, which acts as the molecular glue between sister chromatids, accessible to other factors that promote dissolution (55). The role of SUMO-1 in mitotic chromosome structure is not limited to the cohesive properties of centromeres, as sumoylation has also been implicated in the maintenance and recruitment of proteins to the kinetochore, the protein complex that forms at the centromere and recruits microtubules for anaphase separation. In Saccharomyces cerevisiae, SUMO-1 has been shown to be a suppressor of a temperature-sensitive MIF2 (yeast homologue of CENP-C), a protein which links satellite-containing centromeric DNA to the proteins of the inner kinetochore plate (56) . Interestingly, neither CENP-C nor MIF2 is sumoylated, and thus the role of sumoylation is thought to be indirect (57). In addition to modifying several central kinetochore/centromere proteins, sumoylation is also thought to target various proteins to this region during mitosis for reasons which remain unclear. For example, during mitosis SUMO-1 targets RanGAP1 to the mitotic spindles and kinetochores, and at the kinetochores specifically, RanBP2/Nup358 colocalizes specifically at the kinetochore (58). In addition to the apparent role of sumoylation in chromosome maintenance and kinetochore assembly/disassembly, the sumoylation modification of a number of critical tumor suppressor and repair proteins implicated SUMO-1 as an integral player in maintaining genomic stability. p53 and Mdm2 are both targets of sumoylation, which has functional effects on the activities of these proteins (3, 6, 7, 48). Evidence indicates that components of the Wnt signaling cascade (e.g. axin, -catenin, LEF/Tcf-4) are also targets of sumoylation, which may regulate the system at multiple vertical and horizontal steps (3, 6, 7). Although sumoylation apparently affects cell cycle and developmental proteins, the integrity of DNA itself may also rely on the sumoylation status of various proteins, particularly those involved in DNA repair. Both UBL1 (SUMO-1) and UBE2I (Ubc9) have been identified to interact with RAD51/52, proteins well known for their role in homologous recombination and repairing double-stranded breaks in cells (59, 60). Sumoylation has also been implicated in the repair of the DNA damage mediated by topoisomerase II (61, 62), which is of clinical relevance because topoisomerase is a target of numerous anti-cancer therapies. More recent evidence suggests that the Rad6 postreplicative repair pathway

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is apparently sumoylation-dependent, particularly with respect to PCNA, the activity of which varies by which molecular tag (e.g. SUMO-1 or ubiquitin) it carries (63, 64). The balance between sumoylation and ubiquitination of PCNA, which determines which specific repair pathway the cell utilizes, illustrates the critical role of sumoylation in this important cell function. SUMO and Viral Proteins Two major-immediate-early (MIE) proteins critical for propagation of the human cytomegalovirus and herpesvirus, IE1 (IE1-p72 or IE72) and IE2 (IE2-p86 or IE86), have been found to be sumoylated. In the case of the IE1 protein, which is known to interact with PML, blocking this modification does not disrupt targeting of this protein to ND10 nuclear bodies nor does it affect ND10 organization, suggesting that sumoylation is not a prerequisite for the derepressive activities of IE1 (65, 66). However, IE1 does modulate the sumoylation of PML and is important for effects on nuclear body dynamics (67). Targeting of IE2 to ND10 is not affected by mutations of its target lysine residues, but sumoylation appears to be critical for IE2-mediated transactivation (68). Recent studies have also shown that PIAS1 can increase the levels of IE2 sumoylation which leads to enhanced IE2-mediated transactivation (69). The BZLF1 (Z) protein of Epstein-Barr virus (EBV) is SUMO-1-modified at lysine 12 within the transactivation domain, and results suggest that BZLF1 sumoylation decreases PML SUMO modification by competing for limiting amounts of free SUMO protein (70). The immediate early protein E1 of bovine papillomavirus was found to interact both in vivo and in vitro with Ubc9 using a yeast two-hybrid screen and to be sumoylated at lysine residue 514 (71). Mutations within E1 that prevent SUMO-1 modification do not affect intracellular stability but do disrupt nuclear import and accumulation leading to the decreased ability of E1 to replicate the viral genome (71, 72). A number of adenoviral proteins important for viral replication have also been shown to be sumoylated. Adenoviral E1B is modified at SUMO-1 at lysine 104, which is important for the ability of this protein to transform primary cells and inhibit p53mediated transactivation, and also appears to mediate E1B localization to the nucleus (73) . Expression of adenoviral Gam1 leads to a global reduction in sumoylation including the reduced sumoylation of HDAC1, dispersal of PML-containing nuclear bodies, and delocalization of SUMO-1 (74). The adenovirus E1A protein has been shown to physically interact with the SUMO E2 Ubc9 protein although no clear role has been identified (75). In addition, yeast two-hybrid analysis indicated that the geminivirus TYLCSV protein Rep interacts with a Ubc9 homologue from Nicotiana benthamiana (NbSCE1), but it is not clear if Rep is a substrate for sumoylation (76).

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Perspectives Investigation of the regulation and function of SUMO modification of proteins is an exciting and rapidly growing field. The recent use of broad proteomics approaches to identify large numbers of new putative sumoylated proteins will only add to the already rapid pace of advance (77–81). Key areas requiring further investigation include understanding the underlying molecular and biochemical mechanisms by which this modification plays its critical roles in regulating subcellular localization, transcription, chromosome function, and genomic integrity, to name a few, and to understand how sumoylation leads to different effects in different proteins. Further investigation of the mechanisms and function of nuclear poreassociated sumoylation, including identification of additional proteins that are sumoylated at this cellular site, should also yield interesting new results and better understanding of the role of this important protein modification.

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