RESUMEN
Lysine (K) 63-linked polyubiquitination plays important roles in cellular processes including DNA-damage tolerance (DDT), NF-κB signaling and endocytosis. Compared to yeast and mammals, little is known about K63-linked polyubiquitination in plants. To date, a Uev-Ubc13 complex is the only known Ub-conjugating enzyme to catalyze K63-linked polyubiquitination, in which Uev serves as a regulatory subunit. The Arabidopsis thaliana genome contains four UEV1 genes that can be classified into two subfamilies (UEV1A/B and UEV1C/D), in which Uev1A/B have a C-terminal extension. Database analysis reveals that all higher plant genomes contain both subfamily UEV1s, which were evolved as early as angiosperm plants. Interestingly, all C-terminal tails in the Uev1A/B subfamily contain a putative prenylation motif, CaaX. Combined experimental results using AtUev1B demonstrated that it is most likely farnesylated and that its C-terminal tail, particularly the catalytic Cys residue in the CaaX motif, plays critical roles in protein-protein interaction, nuclear exclusion and membrane association. Using AtUev1B as bait for a yeast-two-hybrid screen, we identified 14 interaction proteins in a prenylation-dependent manner. These results collectively imply that prenylation of AtUev1A/B plays a critical role in its functional differentiation from AtUev1C/D.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factores de Transcripción/genética , Enzimas Ubiquitina-Conjugadoras/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Prenilación , Dominios y Motivos de Interacción de Proteínas/genética , Alineación de Secuencia , Fracciones Subcelulares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Endoplasmic reticulum (ER)-associated degradation (ERAD) is part of the ER protein quality-control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N-terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N-terminal region. These observations collectively define AtOTU1 as an OTU domain-containing deubiquitinase involved in Arabidopsis ERAD.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico/fisiología , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
UBC13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity remain to be defined. Here we used genetic and pathological methods to evaluate roles of Arabidopsis UBC13 in response to pathogens and environmental stresses. Loss of UBC13 failed to activate the expression of numerous cold-responsive genes and resulted in hypersensitivity to low-temperature stress, indicating that UBC13 is involved in plant response to low-temperature stress. Furthermore, the ubc13 mutant displayed low-temperature-induced and salicylic acid-dependent lesion mimic phenotypes. Unlike typical lesion mimic mutants, ubc13 did not enhance disease resistance against virulent bacterial and fungal pathogens, but diminished hypersensitive response and compromised effector-triggered immunity against avirulent bacterial pathogens. UBC13 differently regulates two types of programmed cell death in response to low temperature and pathogen. The lesion mimic phenotype in the ubc13 mutant is partially dependent on SNC1. UBC13 interacts with an F-box protein CPR1 that regulates the homeostasis of SNC1. However, the SNC1 protein level was not altered in the ubc13 mutant, implying that UBC13 is not involved in CPR1-regulated SNC1 protein degradation. Taken together, our results revealed that UBC13 is a key regulator in plant response to low temperature and pathogens.
Asunto(s)
Apoptosis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Enfermedades de las Plantas/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Frío , Resistencia a la Enfermedad , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Fenotipo , Ácido Salicílico/metabolismo , Estrés Fisiológico , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
BACKGROUND: The error-free branch of the DNA-damage tolerance (DDT) pathway is orchestrated by Lys63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA), and this polyubiquitination is mediated by a Ubc13-Uev complex in yeast. We have previously cloned OsUBC13 from rice, whose product functions as an E2 to promote Lys63-linked ubiquitin chain assembly in the presence of yeast or human Uev. RESULTS: Here we identify four highly conserved UEV1 genes in rice whose products are able to form stable heterodimers with OsUbc13 and mediate Lys63-linked ubiquitin chain assembly. Expression of OsUEV1s is able to rescue the yeast mms2 mutant from death caused by DNA-damaging agents. Interestingly, OsUev1A contains a unique C-terminal tail with a conserved prenylation site not found in the other three OsUev1s, and this post-translational modification appears to be required for its unique subcellular distribution and association with the membrane. The analysis of OsUEV1 expression profiles obtained from the Genevestigator database indicates that these genes are differentially regulated. CONCLUSIONS: We speculate that different OsUev1s play distinct roles by serving as a regulatory subunit of the Ubc13-Uev1 complex to respond to diverse cellular, developmental and environmental signals.
Asunto(s)
Genes de Plantas , Oryza/genética , Poliubiquitina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
The COP9 signalosome (CSN) plays an important role in proteasome-mediated degradation by regulating CUL1 rubylation of the SCF ligase and is involved in many crucial biological processes. Here, we demonstrate a link between IDEF1 accumulation and the decline in COP9 derubylation activity in response to iron deficiency (-Fe) in rice (Oryza sativa). CSN6 expression is rapidly down-regulated during Fe depletion, contributing to reduced CSN activity, as judged by CSN5 and CUL1 expression, indicating CSN6 is involved in the early stage response of -Fe. In contrast to CSN6, the IDEF1 protein and expression of several iron uptake/utilisation-related genes are increased in response to -Fe. Thus, we constructed CSN6 transgenic sense and antisense lines and found that experimental depletion of CSN6 results in accumulation of the IDEF1 protein and up-regulation of several iron uptake/utilisation-related genes. Furthermore, IDEF1 can be decorated with K48-linked polyubiquitin and degraded via the 26S proteasome. Accumulated IDEF1 in antisense lines led to increased chlorophyll and Fe content in seedlings during -Fe. Collectively, the cellular CSN6 level is decreased during early stages of -Fe to ensure the rapid accumulation of IDEF1, which in turn up-regulates several iron uptake/utilisation-related genes to help overcome -Fe stress in rice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Complejo del Señalosoma COP9/genética , Hierro/metabolismo , Oryza/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejo del Señalosoma COP9/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cullin/genética , Regulación de la Expresión Génica de las Plantas , Deficiencias de Hierro , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Oryza/metabolismo , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Unlike conventional lysine (K) 48-linked polyubiquitination, K63-linked polyubiquitination plays signaling roles in yeast and animals. Thus far, UBC13 is the only known ubiquitin-conjugating enzyme (E2) specialized in K63-linked polyubiquitination. Previous identification of Arabidopsis genes encoding UBC13 as well as its interacting partner UEV1 indicates that the UBC13-mediated ubiquitination pathway is conserved in plants; however, little is known about functions and signaling mediated through K63-linked polyubiquitination in plants. To address the functions of UBC13-mediated ubiquitination in plants, we created Arabidopsis ubc13 null mutant lines in which the two UBC13 genes were disrupted. The double mutant displayed altered root development, including shorter primary root, fewer lateral roots and only a few short root hairs in comparison with the wild type and single mutant plants, indicating that UBC13 activity is critical for all major aspects of root development. The double mutant plants were insensitive to auxin treatments, suggesting that the strong root phenotypes do not simply result from a reduced level of auxin. Instead, the ubc13 mutant had a reduced auxin response, as indicated by the expression of an auxin-responsive DR5 promoter-GFP. Furthermore, both the enzymatic activity and protein level of an AXR3/IAA17-GUS reporter were greatly increased in the ubc13 mutant, whereas the induction of many auxin-responsive genes was suppressed. Collectively, these results suggest that Aux/IAA proteins accumulate in the ubc13 mutant, resulting in a reduced auxin response and defective root development. Hence, this study provides possible mechanistic links between UBC13-mediated protein ubiquitination, root development and auxin signaling.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Lisina/metabolismo , Mutación , Fenotipo , Raíces de Plantas/genética , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
While plant growth and reproduction is dependent on sunlight, UV irradiation from sunlight is one of the major genotoxic stresses that threaten plant survival and genome stability. In addition, many environmental chemicals can also damage the plant genome. In yeast and mammalian cells protection against the above genome instability is provided by an error-free DNA-damage tolerance (DDT) pathway, which is dependent on Ubc13-mediated K63-linked polyubiquitination of the proliferating cell nuclear antigen (PCNA). In this study, we isolated the UBC13 gene from rice and characterized its functions. Expression of OsUBC13 can protect a yeast ubc13 null mutant against spontaneous and environmental DNA damage. Furthermore, OsUbc13 physically interacts with human Ubc13 partners Mms2 and Uev1A, and catalyzes K63 polyubiquitination in vitro. These observations collectively suggest that the K63 polyubiquitination is conserved in rice, and that OsUBC13 may be involved in DDT and other cellular processes. In addition, OsUBC13 is constitutively expressed at a high level even under various stress conditions, suggesting that it is a housekeeping gene.