SUMOYLATION

SUMO is an acronym for small ubiquitin-like modifier and describes an evolutionarily conserved and ancient polypeptide consisting of 101 amino acids, which is posttranslationally attached to its target proteins by a system analogous to but distinct from ubiquitylation. The sequence identity between SUMO and ubiquitin is only 18%, but these proteins share a similar three-dimensional structure (Bayer et al.

1998).

Figure 4: Pathway of SUMOylation (modified from (Seeler and Dejean 2003))

SUMO is translated as an immature protein, which undergoes truncation of the last three amino acids by Ulp1 (Ubiquitin-like protease), so that the C-terminal glycine residue is exposed. With ATP consumption, SUMO is bound to the dimeric activating E1-enzyme Uba2/Aos1 as a thioester. In a transesterification reaction, SUMO is passed over to the conjugating E2-enzyme Ubc9 at its active site Cys 93, from which it is ligated to a lysine residue of the target protein. Recent studies (Johnson and Gupta 2001; Takahashi et al. 2001) (Pichler et al. 2002) revealed the existence of several E3-ligating Siz/PIAS-RING-finger proteins. SUMOylation is a reversible process as SUMO can be cleaved off by isopeptidases, providing an important regulatory mechanism to control both the amount of free mature SUMO protein and the modified substrates.

Budding yeast has a single SUMO gene, known as the essential SMT3 gene, whereas three members of the SUMO family have been identified in vertebrates (for review see (Kim et al. 2002)). The pathway of SUMO conjugation, termed SUMOylation, is similar to ubiquitylation, but requires different enzymes (see Figure 4).

SUMO is first bound to a heterodimeric E1 composed of Aos1 and Uba2. It is then transferred to Ubc9, the only known SUMO-conjugating enzyme, which in turn catalyses the formation of an isopeptide bond between the C-terminus of SUMO and a ε-lysine residue of a target protein. Ubc9 shares structural similarities with ubiquitin-conjugating enzymes (Johnson and Blobel 1997) but unlike these E2s, Ubc9 contains a positively charged surface. SUMO is connected to a ε-lysine residue within the ΨKXE (Ψ stands for a large hydrophobic acid) consensus motif of the target protein (Bayer et al. 1998).

By analogy with ubiquitylation, E3 SUMO ligases were recently identified in yeast and mammals (Johnson and Gupta 2001); (Takahashi et al. 2001); (Pichler et al.

2002). None of the known three types of E3-SUMO ligases (Siz/PIAS, RanBP2, Pc2) forms a thioester with its substrate as the E3-ubiquitin ligases do (Jackson 2001).

Thus, the E3-SUMO-ligating enzymes seem to function as additional platforms that bring the charged E2 to the substrate protein. As all types of SUMO ligases show association with specific components of the cellular structure (e.g. septins), these E3-ligases are thought to confer the substrate specificity in their ancestral subcellular compartment (Seeler and Dejean 2003).

The large variety of SUMO ligases seems to allow SUMO´s diverse functions as in contrast to ubiquitylation; the modification of proteins with SUMO does not result in their proteolytic degradation. Instead, multiple other effects of SUMOylation have been described, for example the modulation of the subcellular localization of proteins, of protein-protein interactions or of the activity of transcription factors (Lehembre et al. 2001); (Wilson and Rangasamy 2001b).

Table 1: Selected Targets for SUMO

Process Modif.Protein Function of SUMOylation

RanGAP1 Translocation of RanGAP1 to nuclear pore complex (Matunis et al. 1996) or mitotic spindles/kinetochores (Joseph et al. 2002)

E1B Enable nucleocytoplasmic transport (Endter et al.

2001) Protein Targeting

Mek1 Enable nuclear export (Sobko et al. 2002)

GRIP1 Enhance transcriptional co-activation (Kotaja et al.

2002)

Myb, IRF etc. Represses transcriptional activity (Bies et al. 2002;

Nakagawa and Yokosawa 2002)

p53 Enhances transcriptional activation (Megidish et al.

2002) Transcription

IB Antagonizes SUMOylation prevents degradation (Hay et al. 1999)

PCNA Regulates DNA repair (Hoege et al. 2002)

WRN Unknown (Enomoto 2001)

Genomic integrity

TopI Unknown (Mao et al. 2000a; Mao et al. 2000b) Chromatid segregation TopII Prevents premature chromatid separation (Bachant et

al. 2002)

Table 1 shows a selection of various proteins in which SUMO influences the conformational structure, antagonizes ubiquitylation or affects the stability of large complexes. Intensively studied SUMO substrates are, for example, mammalian RanGAP1, a factor required for nucleocytoplasmic transport, and the tumor suppressor p53 (Gostissa et al. 1999). SUMO targets RanGAP1 to nuclear pores and stimulates the transcriptional and apoptotic activities of p53 (Rodriguez et al. 1999).

Previous studies have also revealed a functional link between SUMOylation and

ubiquitylation. In the case of IB, an inhibitor of the NF-κb transcription factor, SUMOylation was shown to antagonize ubiquitylation, thus preventing the degradation of the protein by the ubiquitin pathway (Hay et al. 1999).

SUMO´s only conjugating enzyme UBC9 was first described when Seufert and co-workers investigated mutants defective in the degradation of B-type cyclins caused by a temperature-sensitive mutation in the UBC9 gene (Seufert et al. 1995). Due to structure homology and its assumed function in protein degradation, they baptized the identified gene UBC9 (ubiquitin-conjugating enzyme). Two years later, several reports (Johnson and Blobel 1997) (Desterro et al. 1997) (Schwarz et al. 1998) clarified that Ubc9 exclusively connects SUMO and not ubiquitin to its target proteins. But a link between SUMOylation and ubiquitin-mediated proteolysis remained in the stabilization of cyclins.

The structure of Ubc9 shows important differences compared with the structures of known ubiquitin-conjugating enzymes (Giraud et al. 1998). At the N-terminal helix, the structural and sequence alignments identify one amino acid giving Ubc9 a different recognition surface compared to ubiquitin-conjugating enzymes. This is accompanied by a profound change in the electrostatic surface of the molecular interface. A high-resolution co-crystal structure of Ubc9 bound to its substrate RanGAP1 gave insights in how Ubc9 recruits its target proteins (Hochstrasser 2002) and passes the cysteine-bound SUMO on. Ubc9 binds directly to the ΨKXE-motif of its target, bringing it in close proximity to the Ubc9 active site where the cysteine-bound SUMO is located. Because SUMO is cysteine-bound as an activated thioester with an electrophilic carbon center, the presence of a weakly nucleophile substrate ε-amino group is sufficient to form the peptide bond.

Up to now, several reports have implicated important functions for SUMO during M-phase of the cell cycle. Yeast cells depleted of UBC9 were impaired in mitosis, and temperature-sensitive smt3 mutants, defective in the yeast SUMO gene, were identified in a screen for mutants defective in chromosome segregation (Biggins et al.

2001). Schizosaccharomyces pombe cells lacking the SUMO gene pmt3 and the UBC9 homologue hus5 strains also displayed defects in chromosome segregation (al-Khodairy et al. 1995; Tanaka et al. 1999).