The modification of substrate proteins by covalent linkage to the small ubiquitin-related modifier, SUMO, occurs by a dedicated set of enzymes. The process is mechanistically similar to the conjugation of ubiquitin, the most prominent representative of a family of small protein modifiers with conserved structure (Hochstrasser, 2009). SUMO is synthesized as a pre-protein that needs to be processed by SUMO proteases to expose a carboxyl-terminal diglycine motif. In a two-step reaction, the heterodimeric SUMO activating enzyme, SAE, forms a thioester between SUMO’s terminal glycine (Gly) residue and an active site cysteine (Cys) of the enzyme. The process starts with the formation of an AMP–SUMO linkage from ATP and SUMO’s carboxyl-terminal Gly residue. Under release of AMP, the activated SUMO carboxyl terminus is then transferred onto the active site Cys of SAE to form a thioester. The whole process involves dramatic conformational changes of the enzyme (Olsen et al., 2010). Subsequently, activated SUMO is transferred to the active site Cys of the SUMO conjugating enzyme (SCE; also called UBC9 in some animals and fungi). SCE can conjugate SUMO to substrate proteins, resulting in an isopeptide linkage formed between the carboxyl-terminal Gly of SUMO and the ε-amino group of a lysine (Lys) residue within the substrate. So far, conjugation has been observed exclusively to ε-amino groups of Lys residues. This differs from the more complex ubiquitin conjugation machinery, where transfer to α-amino groups, or to substrate Cys residues, has also been documented (cf. Vosper et al., 2009, and references therein). In vitro, and probably also in vivo, SCE can modify many substrates in the absence of substrate specificity factors (SUMO ligases). Direct substrate interaction and modification by SCE usually depend on the presence of the short sumoylation consensus motif, consisting of a hydrophobic aliphatic amino acid, followed by the Lys residue to be modified, any amino acid and an acidic residue (ΨKxD/E in one-letter code). Nonetheless, SUMO ligases play important roles in vivo to determine the substrate range and extent of sumoylation. Figure 1 summarizes the reaction steps of the SUMO conjugation cycle.
Figure 1. The small ubiquitin-related modifier (SUMO) conjugation cycle. Each plant has several SUMO isoforms, all of which are translated as precursor proteins. SUMO-specific proteases cleave off a carboxyl-terminal peptide to expose a glycine (Gly) residue at the carboxyl terminus, which extends from the globular body of SUMO (step 1). SUMO’s terminal Gly is activated by forming a thioester with the active site cysteine (Cys) residue of SUMO activating enzyme SAE (step 2). From there, SUMO is transferred to a Cys residue of SUMO conjugating enzyme SCE (step 3). SCE can transfer SUMO directly to substrates (step 4), provided that these contain a binding motif, usually the ‘sumoylation consensus sequence’ (see text). Proteins devoid of an SCE interaction motif require SUMO ligase assistance for modification. Substrate release (step 5) results usually in a monosumoylated protein. In an unspecified number of cases, however, a SUMO chain is attached to the substrate. The reversibility of SUMO conjugation results from the hydrolysis of the isopeptide bond by SUMO-specific proteases to release SUMO for further conjugation cycles (step 6). In a separate modification cascade, SUMO chains serve as the signal for the attachment of a ubiquitin chain (yellow dots) by a dedicated ubiquitin ligase complex (step 7), which results in proteasomal degradation of the substrate (steps summarized as arrow number 8).
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Functional studies in plants, as well as the characterization of sumoylation enzymes, have so far been restricted largely to Arabidopsis thaliana (for recent reviews, see Miura et al., 2007a; Lois, 2010; Miura & Hasegawa, 2010; H. J. Park et al., 2011). More recently, the first experimental data for the monocot plant rice were published (Park et al., 2010; Thangasamy et al., 2011; Wang et al., 2011). SUMO conjugation has been shown to be essential in Arabidopsis (Saracco et al., 2007), and work by several groups has demonstrated its importance for the integration of environmental inputs and for adequate reaction to stress conditions (Yoo et al., 2006; Catala et al., 2007; Conti et al., 2008; Jin et al., 2008; Chen et al., 2011; Miura et al., 2011). A significant number of plant-specific sumoylation substrates have been identified recently (Budhiraja et al., 2009; Elrouby & Coupland, 2010; Miller et al., 2010). With an impressive body of information available for Arabidopsis, but relatively little insight into SUMO conjugation in other plants, we wanted to understand which of the components identified in Arabidopsis are conserved in other plants, and which genes point to more divergent features of the pathway. The increasing number of sequenced plant genomes (for review, see Feuillet et al., 2011) and plant gene databases (Martinez, 2011) provide promising tools for the characterization of complete pathways. For comparison with Arabidopsis, we used the assembled genome data of tomato (Solanum lycopersicum, genome size c. 800 Mb), grapevine (Vitis vinifera, genome size 487 Mb), poplar (Populus trichocarpa, genome size 550 Mb), rice (Oryza sativa; genome size 466 Mb), Brachypodium distachyon (genome size 270 Mb), Sorghum bicolor (genome size 697 Mb) and maize (Zea mays; genome size c. 2800 Mb). In the latter case, we expected to find genes with high similarity to the Sorghum homologs, but, as a result of a recent genome duplication, the number should be twice that of Sorghum. Surprisingly, we did not find this situation in most instances, which we ascribe to incomplete sequence availability/annotation in maize. Generally, annotation of the Arabidopsis and rice genomes is most advanced, whereas we found some genes not annotated in their full length in other species. We are nonetheless convinced that the survey provides a valid overview over the set of SUMO conjugation and deconjugation enzymes in plants.