RESUMEN
Plant SUMO conjugation is an essential posttranslational modification involved in plant development and responses to environmental stress. Most likely, this biological diversification is supported by a functional specialization of the different isoforms of the SUMO conjugation machinery. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display higher conjugation rate than SUMO3 and 5, which are not essential, linking their specific biochemical properties to their biological role. To study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Sumoilación , Cinética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2UFDCt domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2UFDCt highly divergent across kingdoms. Transgenic plants expressing the SAE2UFDCt domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/microbiología , Enfermedades de las Plantas/microbiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Ascomicetos/fisiología , Botrytis/fisiología , Regulación de la Expresión Génica de las Plantas , Mutagénesis , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente , Dominios Proteicos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Plants display a high diversification degree of the SUMO conjugation machinery, which could confer a biological specialization of the different isoforms. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display the highest conjugation rate when compared to SUMO3 and 5, suggesting that their specific biochemical properties may be linked to their biological specialization. In order to study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.
Asunto(s)
Proteínas de Arabidopsis/química , Biología Molecular/métodos , Isoformas de Proteínas/química , Sumoilación/genética , Secuencia de Aminoácidos/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cinética , Isoformas de Proteínas/genéticaRESUMEN
Sumoylation is an essential posttranslational modification that participates in many biological processes including stress responses. However, little is known about the mechanisms that control Small Ubiquitin-like MOdifier (SUMO) conjugation in vivo. We have evaluated the regulatory role of the heterodimeric E1 activating enzyme, which catalyzes the first step in SUMO conjugation. We have established that the E1 large SAE2 and small SAE1 subunits are encoded by one and three genes, respectively, in the Arabidopsis genome. The three paralogs genes SAE1a, SAE1b1, and SAE1b2 are the result of two independent duplication events. Since SAE1b1 and SAE1b2 correspond to two identical copies, only two E1 small subunit isoforms are present in vivo: SAE1a and SAE1b. The E1 heterodimer nuclear localization is modulated by the C-terminal tail of the SAE2 subunit. In vitro, SUMO conjugation rate is dependent on the SAE1 isoform contained in the E1 holoenzyme and our results suggest that downstream steps to SUMO-E1 thioester bond formation are affected. In vivo, SAE1a isoform deletion in T-DNA insertion mutant plants conferred sumoylation defects upon abiotic stress, consistent with a sumoylation defective phenotype. Our results support previous data pointing to a regulatory role of the E1 activating enzyme during SUMO conjugation and provide a novel mechanism to control sumoylation in vivo by diversification of the E1 small subunit.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Evolución Molecular , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Enzimas Activadoras de Ubiquitina/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Teorema de Bayes , Secuencia Conservada , Genes de Plantas/genética , Cinética , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrés Fisiológico , Fracciones Subcelulares/enzimología , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
Protein modification by SUMO (small ubiquitin-related modifier) has emerged as an essential regulatory mechanism in eukaryotes. Even though the molecular mechanisms of SUMO conjugation/deconjugation are conserved, the number of SUMO machinery components and their degree of conservation are specific to each organism. In the present paper, we show data contributing to the notion that the four expressed Arabidopsis SUMO paralogues, AtSUMO1, 2, 3 and 5, have functionally diverged to a higher extent than their human orthologues. We have explored the degree of conservation of these paralogues and found that the surfaces involved in E1-activating enzyme recognition, and E2-conjugating enzyme and SIM (SUMO-interacting motif) non-covalent interactions are well conserved in AtSUMO1/2 isoforms, whereas AtSUMO3 shows a lower degree of conservation, and AtSUMO5 is the most divergent isoform. These differences are functionally relevant, since AtSUMO3 and 5 are deficient in establishing E2 non-covalent interactions, which has not been reported for any naturally occurring SUMO orthologue. In addition, AtSUMO3 is less efficiently conjugated than AtSUMO1/2, and AtSUMO5 shows the lowest conjugation level. A mutagenesis analysis revealed that decreases in conjugation rate and thioester-bond formation are the result of the non-conserved residues involved in E1-activating enzyme recognition that are present in AtSUMO3 and 5. The results of the present study support a role for the E1-activating enzyme in SUMO paralogue discrimination, providing a new mechanism to favour conjugation of the essential AtSUMO1/2 paralogues.
