SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

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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of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations).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.

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

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 Nterminal) 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.

These authors contributed equally to this work. SUMMARYAbscisic acid (ABA) and gibberellins (GAs) are plant hormones which antagonistically mediate numerous physiological processes, and their optimal balance is essential for normal plant development. However, the molecular mechanism underlying ABA and GA antagonism still needs to be determined. Here, we report that ABA-INSENSITIVE 4 (ABI4) is a central factor in GA/ABA homeostasis and antagonism in post-germination stages. ABI4 overexpression in Arabidopsis (OE-ABI4) leads to developmental defects including a decrease in plant height and poor seed production. The transcription of a key ABA biosynthetic gene, NCED6, and of a key GA catabolic gene, GA2ox7, is significantly enhanced by ABI4 overexpression. ABI4 activates NCED6 and GA2ox7 transcription by directly binding to the promoters, and genetic analysis revealed that mutation in these two genes partially rescues the dwarf phenotype of ABI4 overexpressing plants. Consistently, ABI4 overexpressing seedlings have a lower GA/ABA ratio than the wild type. We further show that ABA induces GA2ox7 transcription while GA represses NCED6 expression in an ABI4-dependent manner; and that ABA stabilizes the ABI4 protein whereas GA promotes its degradation. Taken together, these results suggest that ABA and GA antagonize each other by oppositely acting on ABI4 transcript and protein levels.