Giovanna Serino

Role of SCF Ubiquitin-Ligase and the COP9 Signalosome in the N Gene–Mediated Resistance Response to Tobacco mosaic virus

The tobacco N gene confers resistance to Tobacco mosaic virus (TMV) and encodes a toll–interleukin-1 receptor/nucleotide binding/Leu-rich repeat class protein. Recent evidence indicates that the Nicotiana benthamiana Rar1 gene (NbRar1), which encodes a protein with a zinc finger motif called CHORD (Cys- and His-rich domain), is required for the function of N. To investigate the role of NbRar1 in plant defense, we identified its interaction partners. We show that the NbRar1 protein interacts with NbSGT1, a highly conserved component of the SCF (Skp1/Cullin/F-box protein)-type E3 ubiquitin ligase complex involved in protein degradation. In addition, we show that NbSGT1 interacts with NbSKP1. Suppression of NbSGT1 and NbSKP1 shows that these genes play an important role in the N-mediated resistance response to TMV. Both NbRar1 and NbSGT1 associate with the COP9 signalosome, another multiprotein complex involved in protein degradation via the ubiquitin-proteasome pathway. Silencing of the NbCOP9 signalosome also compromises N-mediated resistance to TMV. Our results reveal new roles for SCF and the COP9 signalosome in plant defense signaling.

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations). We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways.

Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex that mediates the repression of photomorphogenesis in the dark in Arabidopsis through the degradation of transcription factors such as HY5 and HYH. CSN-mediated HY5 and HYH degradation also requires the activity of the putative E3 ubiquitin ligase (E3) component COP1 and the E2-conjugating enzyme variant COP10. Recently, it was shown that CSN also is required for auxin responses mediated by the SCF-type E3 SCFTIR1. To determine whether Arabidopsis CSN is required for E3-mediated processes in a more general manner, we generated plants with reduced E3 function by suppressing AtRBX1, an essential core subunit of SCF-type E3s. We observed that AtRBX1 transgenic plants share multiple phenotypes with CSN reduced-function plants, such as morphological defects and reduced responses to auxin, jasmonic acid, and cold stress, suggesting that CSN is required for multiple AtRBX1-mediated processes. Furthermore, we observed that mutants with defects in AXR1, a protein that had been described only as a regulator of SCFTIR1 function, also is required for other E3-mediated processes and for the COP1/COP10/CSN-mediated repression of photomorphogenesis in the dark. We conclude that CSN and AXR1 are of general importance for different pathways that are controlled by E3-mediated protein degradation.

Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory Interaction with Arabidopsis Cullin3-Based E3 Ligases

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex that regulates a variety of biological processes. Among its eight subunits, CSN5 and CSN6 contain a characteristic MPN (for Mpr1p and Pad1p N-terminal) domain and, in Arabidopsis thaliana, are each encoded by two genes: CSN5A, CSN5B and CSN6A, CSN6B, respectively. We characterized both MPN subunits using a series of single and double mutants within each gene family. Our results indicate that although CSN6A and CSN6B retain mostly redundant functions, CSN5A and CSN5B play unequal roles in the regulation of plant development. Complete depletion of either of the two MPN members results in CSN instability and the decay of various CSN components, along with the complete loss of CUL1, CUL3, and CUL4 derubylation. Furthermore, we demonstrate that CSN interacts with CUL3, in addition to CUL1 and CUL4, and that the lack of CSN activity differentially affects the stability of those three cullins. Interestingly, we also show that optimal CUL3 activity is required to maintain the cellular pool of CSN5, through a posttranscriptional mechanism. Our data suggest the existence of reciprocal regulation between CUL3 and CSN5 accumulation. This study thus completes the genetic analysis of all CSN subunits and confirms the structural interdependence between PCI and MPN subunits in functional CSN complex formation.

Arabidopsis cop8 and fus4 Mutations Define the Same Gene That Encodes Subunit 4 of the COP9 Signalosome

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome–COP9 complex–eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4–encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome.

Characterization of the Last Subunit of the Arabidopsis COP9 Signalosome: Implications for the Overall Structure and Origin of the Complex

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCFTIR1 subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCFTIR1, but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.