RESUMEN
SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Similar to ubiquitylation, a SUMO-activating enzyme links to the SUMO carboxyl-terminal Gly in a thioester bond, and a SUMO-conjugating enzyme accepts activated SUMO and can transfer it to substrates. Unlike ubiquitylation, this transfer can also occur, in an unspecified number of cases, in the absence of ligase-like enzymes. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four, and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95% identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can be transferred to substrates as single moieties, but can also form SUMO chains, a process enhanced by chain-forming ligases. By combined action with SUMO chain recognizing ubiquitin ligases, chains can channel substrates into the ubiquitin-dependent degradation pathway.A method is described to sumoylate substrates and to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.
Asunto(s)
Arabidopsis , Sumoilación , Humanos , Escherichia coli/metabolismo , Arabidopsis/metabolismo , Ligasas/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/genética , Cisteína Endopeptidasas/química , Regulación de la Expresión Génica de las Plantas , Mutación , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Especificidad por Sustrato , Sumoilación , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Conjugation of the small ubiquitin-related modifier (SUMO) to protein substrates has an impact on stress responses and on development. We analyzed the proteome and phosphoproteome of mutants in this pathway. The mutants chosen had defects in SUMO ligase SIZ1, which catalyzes attachment of single SUMO moieties onto substrates, and in ligases PIAL1 and PIAL2, which are known to form SUMO chains. A total of 2657 proteins and 550 phosphopeptides were identified and quantified. Approximately 40% of the proteins and 20% of the phosphopeptides showed differences in abundance in at least one of the analyzed genotypes, demonstrating the influence of SUMO conjugation on protein abundance and phosphorylation. The data show that PIAL1 and PIAL2 are integral parts of the SUMO conjugation system with an impact on stress response, and confirm the involvement of SIZ1 in plant defense. We find a high abundance of predicted SUMO attachment sites in phosphoproteins (70% versus 40% in the total proteome), suggesting convergence of phosphorylation and sumoylation signals onto a set of common targets.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosforilación/fisiología , Sumoilación/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Ligasas/genética , Ligasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/genética , Proteoma/análisis , Proteoma/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Sumoilación/genéticaRESUMEN
SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95 % identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can form chains, even though they do not possess a consensus SUMOylation motif. The surprising finding that plants have dedicated enzymes for chain synthesis implies a specific role for SUMO chains in plants. By the cooperative action with SUMO chain recognizing ubiquitin ligases, chains might channel substrates into the ubiquitin-dependent degradation pathway.A method is described to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.
Asunto(s)
Proteínas de Arabidopsis/química , Biología Molecular/métodos , Ubiquitina/química , Secuencia de Aminoácidos/genética , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Escherichia coli/genética , Humanos , Isoformas de Proteínas , Sumoilación/genética , Ubiquitina/genéticaRESUMEN
The Arabidopsis thaliana genes PROTEIN INHIBITOR OF ACTIVATED STAT LIKE1 (PIAL1) and PIAL2 encode proteins with SP-RING domains, which occur in many ligases of the small ubiquitin-related modifier (SUMO) conjugation pathway. We show that PIAL1 and PIAL2 function as SUMO ligases capable of SUMO chain formation and require the SUMO-modified SUMO-conjugating enzyme SCE1 for optimal activity. Mutant analysis indicates a role for PIAL1 and 2 in salt stress and osmotic stress responses, whereas under standard conditions, the mutants show close to normal growth. Mutations in PIAL1 and 2 also lead to altered sulfur metabolism. We propose that, together with SUMO chain binding ubiquitin ligases, these enzymes establish a pathway for proteolytic removal of sumoylation substrates.
