Analysis of Small Ubiquitin-Like Modifier (SUMO) Targets Reflects the Essential Nature of Protein SUMOylation and Provides Insight to Elucidate the Role of SUMO in Plant Development

Abstract

Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) has received much attention, reflected by a flood of recent studies implicating SUMO in a wide range of cellular and molecular activities, many of which are conserved throughout eukaryotes. Whereas most of these studies were performed in vitro or in single cells, plants provide an excellent system to study the role of SUMO at the developmental level. Consistent with its essential roles during plant development, mutations of the basic SUMOylation machinery in Arabidopsis (Arabidopsis thaliana) cause embryo stage arrest or major developmental defects due to perturbation of the dynamics of target SUMOylation. Efforts to identify SUMO protein targets in Arabidopsis have been modest; however, recent success in identifying thousands of human SUMO targets using unique experimental designs can potentially help identify plant SUMO targets more efficiently. Here, known Arabidopsis SUMO targets are reevaluated, and potential approaches to dissect the roles of SUMO in plant development are discussed.

Figure 1. The SUMO conjugation and deconjugation system. SUMO is produced as a precursor protein with a C-terminal extension. SUMO proteases cleave off the C-terminal tail to expose the reactive carboxyl group of the C-terminal Gly. The SAE (or El) with its two subunits (SAE1 and SAE2) forms a thioester bond with this Gly residue to prepare for its transfer to the SCE (or E2). In addition to the thioester bond, the SCE binds SUMO noncovalently as well and eventually transfers SUMO to a target protein, usually with the aid of a third enzyme (E3), the SUMO ligase such as SIZ1. Targets, now covalently modified by SUMO through an isopeptide bond, perform specific functions, which are subsequently terminated by either removing SUMO from the target protein (deconjugation) or by regulated proteolysis. SUMO proteases with isopeptidase activity specifically and precisely hydrolyze the isopeptide bond, releasing free SUMO and target protein. Alternatively, a polySUMO chain forms through the activity of SUMO ligases (E4) such as PIAL1 and PIAL2, and this chain recruits STUbLs, which ubiquitinate both SUMO and the target protein and target them for degradation by the 26S proteasome. All gene models are derived from terminology in Arabidopsis. S, SUMO; Ub, ubiquitin; UPS, ubiquitin-proteasome system.

Figure 2.

SUMO is involved in protein complex assembly. A, PML protein (red) forms nuclear bodies called PML-NBs that assemble in normal mammalian nuclei and are important for DNA repair, apoptosis, and viral resistance. They include, in addition to the PML protein, additional accessory proteins (gray and purple). Complex assembly relies heavily on covalent and noncovalent interactions with SUMO (blue). B to E, SUMO is also important in the assembly of transcriptional regulator complexes. B, SUMOylation of transcriptional factors (TFs; red) may be important for their activity and/or assembly on chromatin by recruiting other transcriptional factors containing SIMs. C and D, SUMOylated transcription factors may also recruit chromatin-modifying proteins (e.g. demethylases or methyltransferases) by covalent or noncovalent interactions, leading to transcriptional activation or repression. E, STUbLs may also be recruited to polySUMOylated proteins such as transcriptional factors, and this leads to their degradation by the ubiquitin-proteasome system, introducing another layer of transcriptional regulation. Depiction of protein complex assembly on chromatin is based on data from yeast, metazoans, and Arabidopsis. Specific examples are indicated in the text.

Figure 3.

Characterization of SUMO targets in Arabidopsis. A, Identification of 653 SUMO targets by proteomic (Budhiraja et al., 2009; Miller et al., 2010; Park et al., 2011; López-Torrejón et al., 2013) and yeast two-hybrid (Elrouby and Coupland, 2010) approaches. Numbers indicate the number of targets identified by each approach. B to D, Gene Ontology analyses of Arabidopsis SUMO targets. Proteins shown in A were used to identify molecular (B), cellular component (C), and KEGG term (D) categories in which SUMO targets (SUMOylome) are enriched compared with the whole proteome. Only terms with statistically significant differences are presented and shown as the ratio of representation in the SUMOylome to that of the whole proteome. SUMO targets collected from the five main studies were compared for overlap, pooled, and then used in term enrichments. For this purpose, FatiGo (Al-Shahrour et al., 2007) was used as part of the Babelomics suit of programs (http://v4.babelomics.org). TCA, Tricarboxylic acid.

Figure 4.

Recent technological advances to identify SUMO targets and SUMO attachment lysines, mostly from human cells, provide a blueprint that could be used to identify SUMO targets in plants. A, Strategy to reduce the size of SUMO footprint produced by Trypsin digestion as a prerequisite to identifying SUMO targets and attachment lysines by mass spectrometry (MS). Introducing an Arg residue (yellow) a couple of residues upstream of the C-terminal di-Gly of SUMO (in blue) allows shortening of the SUMO footprint while being distinct from footprints produced by ubiquitin and other ubiquitin-like modifiers. B, The Arg could be introduced immediately upstream of SUMO’s di-Gly, and purified proteins labeled with different stable isotopes to distinguish between the different protein modifiers (using the stable isotope labeling by amino acids in cell culture [SILAC] method) are then enriched using anti-di-Gly monoclonal antibody (anti-KεGG). C, Using a mutant form of SUMO (K0-SUMO) in which all lysines are converted to a different amino acid allows the use of Lys-C endopeptidase, which cleaves purified SUMO targets at Lys residues. Intact SUMO attached to a short target peptide is subsequently trypsinized, allowing precise detection of target protein identity and SASs by mass spectrometry. D, A Lys residue can also be introduced immediately upstream of SUMO’s di-Gly, and purified SUMO targets are first treated with Lys-C, prior to their enrichment with anti-KεGG antibody and mass spectrometric characterization.