Arabidopsis FUSCA5 encodes a novel phosphoprotein that is a component of the COP9 complex.

The COP9 complex is a regulator essential for repression of light-mediated development in Arabidopsis. Using partial amino acid sequence data generated from purified COP9 complexes, we cloned the Arabidopsis cDNA encoding the 27-kD subunit of the COP9 complex and showed that it is encoded by the previously identified FUSCA5 (FUS5) locus. fus5 mutants exhibit constitutive photomorphogenic phenotypes similar to those of cop9 and fus6. Point mutations in FUS5 that led to a loss of FUS5 protein were detected in four fus5 allelic strains. FUS5 contains the PCI/PINT and mitogen-activated protein kinase kinase activation loop motifs and is highly conserved with the mammalian COP9 complex subunit 7 and the Aspergillus nidulans AcoB proteins. FUS5 is present in both complex and monomeric forms. In the COP9 complex, FUS5 may interact directly with FUS6 and COP9. Mutations in FUS6 and COP9 result in a shift in the electrophoretic mobility of FUS5. This shift can be mimicked by in vitro phosphorylation of FUS5 by plant extracts. These findings further support the hypothesis that the COP9 complex is a central and common regulator that may interact with multiple signaling pathways.     

Making sense of the COP9 signalosome. A regulatory protein complex conserved from Arabidopsis to human

The COP9 signalosome, once defined as a repressor complex of light-activated development in Arabidopsis, has recently been found in humans and is probably present in most multicellular organisms. The COP9 signalosome is closely related to the lid sub-complex of the 26S proteasome in structural composition and probably shares a common evolutionary ancestor. A multifaceted role of the COP9 signalosome in cell-signaling processes is hinted at by its associated novel kinase activity, as well as the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. The molecular genetic studies in Arabidopsis suggest that the complex functions as part of a highly conserved regulatory network, whose physiological role in animals remains to be determined.     

Isolation and mapping of rFUS6, a rice orthologue of Arabidopsis thaliana FUS6.

COP9 complex is one of the most important components that act in repressing photomorphogenesis in Arabidopsis thaliana. FUS6 has been identified as one of eight subunits of the COP9 complex in Arabidopsis. Using Arabidopsis Fus6 cDNA as a probe, we screened a rice root cDNA library and a rice genomic library. A 1730-bp cDNA was obtained, which has an open reading frame corresponding to 441-amino-acid. This 441 amino acids putative protein has 67% identity with Arabidopsis COP11/FUS6 (AtFUS6) and 40% identity with human GPS1, an AtFUS6 orthologue. So we designated this novel gene as rFUS6. The 6.2-kb genomic sequence of rFUS6 was also obtained. Sequence comparison showed that the rFUS6 gene had six exons and five introns. Sequence inspection of the 5'-flanking region revealed the presence of some potential light-regulated cis-elements such as a G-box, GT-1 binding sites, and a TGACG motif. Southern hybridization with rice total DNA showed that rFUS6 was perhaps a single copy gene. The rFUS6 locus was mapped by hybridization with a rice BAC library membrane and the results showed that rFUS6 had a locus at 16.3 cM of chromosome 1.     

The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 A crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits.     

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.     

Molecular Characterization of Subunit 6 of the COP9 Signalosome and Its Role in Multifaceted Developmental Processes in Arabidopsis

The COP9 signalosome is a highly conserved protein complex initially identified as a repressor of photomorphogenesis. Here, we report that subunit 6 of the Arabidopsis COP9 signalosome is encoded by a family of two genes (CSN6A and CSN6B) located on chromosomes V and IV, respectively. The CSN6A and CSN6B proteins share 87% amino acid identity and contain a MPR1p and PAD1p N-terminal (MPN) domain at the N-terminal region. The CSN6 proteins share homology with CSN5 and belong to the Mov34 superfamily of proteins. CSN6 proteins present only in the complex form and coimmunoprecipitate with other known subunits of the COP9 signalosome. Partial loss-of-function strains of the COP9 signalosome created by antisense and cosuppression with CSN6A exhibit diverse developmental defects, including homeotic organ transformation, symmetric body organization, and organ boundary definition. Protein blot analysis revealed that the defective plants accumulate significant amounts of ubiquitinated proteins, supporting the conclusion that the COP9 signalosome regulates multifaceted developmental processes through its involvement in ubiquitin/proteasome-mediated protein degradation.     

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 SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), 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.     

Regulation of the anaphase-promoting complex by the COP9 signalosome

The COP9 complex (signalosome) is a known regulator of the proteasome/ubiquitin pathway. Furthermore it regulates the activity of the cullin-RING ligase (CRL) families of ubiquitin E3-complexes. Besides the CRL family, the anaphase-promoting complex (APC/C) is a major regulator of the cell cycle. To investigate a possible connection between both complexes we assessed interacting partners of COP9 using an in vivo protein-protein interaction assay. Hereby, we were able to show for the first time that CSN2, a subunit of the COP9 signalosome, interacts physically with APC/C. Furthermore, we detected a functional influence of the COP9 complex regarding the stability of several targets of the APC/C. Consistent with these data we showed a genetic instability of cells over-expressing CSN2.     

Molecular evolution of ACTIN RELATED PROTEIN 6, a component of SWR1 complex in Arabidopsis

To date, it has been assumed that the evolution of a protein complex is different from that of other proteins. However, there have been few evidences to support this assumption. To understand how protein complexes evolve, we analyzed the evolutionary constraints on ACTIN RELATED PROTEIN 6 (ARP6), a component of the SWR1 complex. Interspecies complementation experiments using transgenic plants that ectopically express transARP6s (ARP6s from other organisms) showed that the function of ARP6s is conserved in plants. In addition, a yeast two-hybrid analysis revealed that this functional conservation depends on its ability to bind with both PIE1 and AtSWC6. ARP6 consists of 4 domains similar to actin. Functional analysis of chimericARP6s (domain-swapped ARP6s between Arabidopsis and mouse) demonstrated that each domain of ARP6s imposes differential evolutionary constraints. Domains 1 and 3 of ARP6 were found to interact with SWC6 and PIE1, respectively, and domain 4 provides a nuclear localization signal. Moreover, domains 1 and 3 showed a slower evolution rate than domain 4, indicating that the interacting domains have higher evolutionary constraints than non-interacting domains do. These findings suggest that the components of this protein complex have evolved coordinately to preserve their interactions.     

Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation

Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development.     

JAB1/CSN5 and the COP9 signalosome. A complex situation

The Jun activating binding protein (JAB1) specifically stabilizes complexes of c-Jun or JunD with AP-1 sites, increasing the specificity of target gene activation by AP-1 proteins. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). Over the past year, JAB1/CSN5 has been implicated in numerous signaling pathways including those that regulate light signaling in plants, larval development in Drosophila, and integrin signaling, cell cycle control, and steroid hormone signaling in a number of systems. However, the general role of the CSN complex, and the specific role of JAB1/CSN5, still remain obscure. This review attempts to integrate the available data to help explain the role of JAB1/CSN5 and the COP9 signalosome in regulating multiple pathways (in this review, both JAB1 and CSN5 terminologies are used interchangeably, depending on the source material).     

Evidence for filaggrin as a component of the cell envelope of the newborn rat.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.     

Arabidopsis COP8, COP10, and COP11 genes are involved in repression of photomorphogenic development in darkness.

Wild-type Arabidopsis seedlings are capable of following two developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. Screening of Arabidopsis mutants for constitutive photomorphogenic development in darkness resulted in the identification of three new loci designated COP8, COP10, and COP11. Detailed examination of the temporal morphological and cellular differentiation patterns of wild-type and mutant seedlings revealed that in darkness, seedlings homozygous for recessive mutations in COP8, COP10, and COP11 failed to suppress the photomorphogenic developmental pathway and were unable to initiate skotomorphogenesis. As a consequence, the mutant seedlings grown in the dark had short hypocotyls and open and expanded cotyledons, with characteristic photomorphogenic cellular differentiation patterns and elevated levels of light-inducible gene expression. In addition, plastids of dark-grown mutants were defective in etioplast differentiation. Similar to cop1 and cop9, and in contrast to det1 (deetiolated), these new mutants lacked dark-adaptive change of light-regulated gene expression and retained normal phytochrome control of seed germination. Epistatic analyses with the long hypocotyl hy1, hy2, hy3, hy4, and hy5 mutations suggested that these three loci, similar to COP1 and COP9, act downstream of both phytochromes and a blue light receptor, and probably HY5 as well. Further, cop8-1, cop10-1, and cop11-1 mutants accumulated higher levels of COP1, a feature similar to the cop9-1 mutant. These results suggested that COP8, COP10, and COP11, together with COP1, COP9, and DET1, function to suppress the photomorphogenic developmental program and to promote skotomorphogenesis in darkness. The identical phenotypes resulting from mutations in COP8, COP9, COP10, and COP11 imply that their encoded products function in close proximity, possibly with some of them as a complex, in the same signal transduction pathway.     

A gain-of-function phenotype conferred by over-expression of functional subunits of the COP9 signalosome in Arabidopsis

The COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild-type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6-1 mutant phenotype. Moreover, light-grown rFUS6 over-expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild-type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long-hypocotyl phenotype was also observed in transgenic seedlings over-expressing Arabidopsis COP9. This finding indicates that over-expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain-of-function phenotype relative to the complex. Human GPS1, when expressed in the fus6-1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low-abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.     

The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.