Biomedical subjects

A G von Arnim

Publications and source records attributed to A G von Arnim.

15 recordsLinked to original sources

PubMed2001

A hitchhiker's guide to the proteasome.

Regulated degradation of proteins is essential for viability and is involved in the control of many signal transduction pathways. von Arnim discusses a new model for how substrates destined for degradation by the 26S proteasome may be presented to the proteasome through a physical interaction between the proteasome and a complex consisting of the substrate and a ubiquitin-ligase. The new model suggests that the SCF (Skp1/cullin/F-box) protein complex may physically associate with the proteasome and that this interaction may be regulated by posttranslational modifications, such as phosphorylation or the covalent attachment of the Nedd8 protein, called neddylation. Additionally, other proteins may compete with the SCF complexes for binding to the proteasome and thus present another layer of regulation for controlling substrate targeting for ubiquitin-mediated degradation.

Adenosine Triphosphatases↗

PubMed2001

Arabidopsis eIF3e (INT-6) associates with both eIF3c and the COP9 signalosome subunit CSN7.

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

Animals↗

PubMed2001

PCI complexes: pretty complex interactions in diverse signaling pathways.

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

Animals↗

PubMed2000

Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta.

The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness. Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis. The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function. In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression. The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1. Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1. Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.

Arabidopsis↗

PubMed1999

A novel motif mediates the targeting of the Arabidopsis COP1 protein to subnuclear foci.

The constitutive photomorphogenesis 1 (COP1) protein of Arabidopsis thaliana accumulates in discrete subnuclear foci. To better understand the role of subnuclear architecture in COP1-mediated gene expression, we investigated the structural motifs of COP1 that mediate its localization to subnuclear foci using mutational analysis with green fluorescent protein as a reporter. In a transient expression assay, a subnuclear localization signal consisting of 58 residues between amino acids 120 and 177 of COP1 was able to confer speckled localization onto the heterologous nuclear NIa protein from tobacco etch virus. The subnuclear localization signal overlaps two previously characterized motifs, a cytoplasmic localization signal and a putative alpha-helical coiled-coil domain that has been implicated in COP1 dimerization. Moreover, phenotypically lethal mutations in the carboxyl-terminal WD-40 repeats inhibited localization to subnuclear foci, consistent with a functional role for the accumulation of COP1 at subnuclear sites.

Amino Acid Sequence↗

PubMed1999

Discrete domains mediate the light-responsive nuclear and cytoplasmic localization of Arabidopsis COP1.

The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) protein plays a critical role in the repression of photomorphogenesis during Arabidopsis seedling development. We investigated the control of COP1 partitioning between nucleus and cytoplasm, which has been implicated in the regulation of COP1 activity, by using fusion proteins between COP1 and beta-glucuronidase or the green fluorescent protein. Transient expression assays using onion epidermal cells and data from hypocotyl cells of stably transformed Arabidopsis demonstrated that COP1 carries a single, bipartite nuclear localization signal that functions independently of light. Nuclear exclusion was mediated by a novel and distinct signal, bordering the zinc-finger and coiled-coil motifs, that was able to redirect a heterologous nuclear protein to the cytoplasm. The cytoplasmic localization signal functioned in a light-independent manner. Light regulation of nuclear localization was reconstituted by combining the individual domains containing the nuclear localization signal and the cytoplasmic localization signal; the WD-40 repeat domain of COP1 was not required. However, phenotypic analysis of transgenic seedlings suggested that the constitutively nuclear-localized WD-40 repeat domain was able to mimic aspects of COP1 function, as indicated by exaggerated hypocotyl elongation under light conditions.

Amino Acid Sequence↗

PubMed1998

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants.

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP-S65T and GFP-Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.

Arabidopsis↗

PubMed1997

Genetic and developmental control of nuclear accumulation of COP1, a repressor of photomorphogenesis in Arabidopsis.

Using a beta-glucuronidase (GUS) reporter-COP1 fusion transgene, it was shown previously that Arabidopsis COP1 acts within the nucleus as a repressor of seedling photomorphogenic development and that high inactivation of COP1 was accompanied by a reduction of COP1 nuclear abundance (A.G. von Arnim, X.-W. Deng [1994] Cell 79: 1035-1045). Here we report that the GUS-COP1 fusion transgene can completely rescue the defect of cop1 mutations and thus is fully functional during seedling development. The kinetics of GUS-COP1 relocalization in a cop1 null mutant background during dark/light transitions imply that the regulation of the functional nuclear COP1 level plays a role in stably maintaining a committed seedling's developmental fate rather than in causing such a commitment. Analysis of GUS-COP1 cellular localization in mutant hypocotyls of all pleiotropic COP/DET/FUS loci revealed that nuclear localization of GUS-COP1 was diminished under both dark and light conditions in all mutants tested, whereas nuclear localization was not affected in the less pleiotropic cop4 mutant. Using both the brassinosteroid-deficient mutant det2 and brassinosteroid treatment of wild-type seedlings, we have demonstrated that brassinosteroid does not control the hypocotyl cell elongation through regulation nuclear localization of COP1. The growth regulator cytokinin, which also dramatically reduced hypocotyl cell elongation in the absence of light, did not prevent GUS-COP1 nuclear localization in dark-grown seedlings. Our results suggest that all of the previously characterized pleiotropic COP/DET/FUS loci are required for the proper nuclear localization of the COP1 protein in the dark, whereas the less pleiotropic COP/DET loci or plant regulators tested are likely to act either downstream of COP1 or by independent pathways.

Arabidopsis↗

PubMed1996

The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch.

Arabidopsis COP9 is a component of a large protein complex that is essential for the light control of a developmental switch and whose conformation or size is modulated by light. The complex is acidic, binds heparin, and is localized within the nucleus. Biochemical purification of the complex to near homogeneity revealed that it contains 12 distinct subunits. One of the other subunits is COP11, mutations in which result in a phenotype identical to cop9 mutants. The COP9 complex may act to regulate the nuclear abundance of COP1, an established repressor of photomorphogenic development. During the biogenesis of the COP9 complex, a certain degree of prior subunit association is a prerequisite for proper nuclear translocation. Since both COP9 and COP11 have closely related human counterparts, the COP9 complex probably represents a conserved developmental regulator in higher eukaryotes.

Arabidopsis↗

PubMed1995

Arabidopsis COP1 protein specifically interacts in vitro with a cytoskeleton-associated protein, CIP1.

Arabidopsis COP1 acts inside the nucleus to suppress photomorphogenic cellular development, and light inactivation of COP1 may involve a specific control of its nuclear activity in hypocotyls and cotyledons, but not in roots, of developing seedlings. To understand the molecular mechanisms of COP1 action during light-mediated development, we initiated a screen for Arabidopsis cDNAs encoding proteins which interact directly with COP1 in vitro as a step to identify the cellular components involved. We report here the isolation and characterization of a cDNA clone encoding a protein designated CIP1 (COP1-interactive protein 1). CIP1 is predominantly alpha-helical and most likely involved in coiled-coil formation. It interacts specifically with the putative coiled-coil region of COP1 in vitro. Further, CIP1 is encoded by a single gene in Arabidopsis, and its mRNA and protein levels are not regulated by light. Immunofluorescent labeling of CIP1 in Arabidopsis seedling protoplasts demonstrated that CIP1 is part of, or associated with, a cytoskeletal structure in hypocotyl and cotyledon cells, but not in roots. Our results are consistent with a possible role of CIP1 in mediating light control of COP1 nuclear activity by regulating its nucleocytoplasmic partitioning.

Amino Acid Sequence↗

PubMed1994

Light inactivation of Arabidopsis photomorphogenic repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning.

Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate this suppressive action. COP1, when fused to beta-glucuronidase (GUS), is enriched in the nucleus in darkness, but not in the light, in hypocotyl cells of Arabidopsis seedlings and epidermal cells of onion bulbs. In Arabidopsis hypocotyl cells, the nuclear GUS-COP1 level changes in response to dark-light transitions and quantitatively correlates with the extent of repression of photomorphogenic development. In root cells, GUS-COP1 is constitutively nuclear, consistent with an established role of COP1 in suppressing root chloroplast development in both light and darkness. We conclude that COP1 acts inside the nucleus to suppress photomorphogenesis and that light inactivation of COP1 involves a cell type-specific control of its nucleocytoplasmic partitioning.

Allium↗

PubMed1994

Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis.

Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings. In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development. Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation. Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene. Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels. This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1. It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.

Arabidopsis↗

PubMed1994

Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.

The Arabidopsis protein COP1, encoded by the constitutive photomorphogenic locus 1, is an essential regulatory molecule that plays a role in the repression of photomorphogenic development in darkness and in the ability of light-grown plants to respond to photoperiod, end-of-day far-red treatment, and ratio of red/far-red light. The COP1 protein contains three recognizable structural domains: starting from the N terminus, they are the zinc binding motif, the putative coiled-coil region, and the domain with multiple WD-40 repeats homologous to the beta subunit of trimeric G-proteins (G beta). To understand the functional implications of these structural motifs, 17 recessive mutations of the COP1 gene have been isolated based on their constitutive photomorphogenic seedling development in darkness. These mutations define three phenotypic classes: weak, strong, and lethal. The mutations that fall into the lethal class are possible null mutations of COP1. Molecular analysis of the nine mutant alleles that accumulated mutated forms of COP1 protein revealed that disruption of the G beta-protein homology domain or removal of the very C-terminal 56 amino acids are both deleterious to COP1 function. In-frame deletions or insertions of short amino acid stretches between the putative coiled-coil and G beta-protein homology domains strongly compromised COP1 function. However, a mutation resulting in a COP1 protein with only the N-terminal 282 amino acids, including both the zinc binding and the coiled-coil domains, produced a weak phenotypic defect. These results indicated that the N-terminal half of COP1 alone retains some activity and a disrupted C-terminal domain masks this remaining activity.

Alleles↗

PubMed1994

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.

Arabidopsis↗

PubMed1993

Ring finger motif of Arabidopsis thaliana COP1 defines a new class of zinc-binding domain.

The COP1 gene of Arabidopsis thaliana encodes a protein mediating the switch between the two developmental pathways utilized in light and darkness. A cysteine-rich motif identified the COP1 protein as a member of a group of regulatory proteins which share the amino acid motif Cys-X-X-Cys-loop I-Cys-X-His-X-X-Cys-X-X-Cys-loop II-Cys-X-X-Cys (ring finger). Although this new class of cysteine-rich motifs has been proposed to bind metal ions, no direct evidence supporting this has been presented. By analyzing the COP1 protein expressed in Escherichia coli, we demonstrate here that each COP1 molecule can bind up to two zinc atoms. The two zinc ions are bound with different affinities. One is tightly bound and resistant to urea and EDTA, whereas the other one is labile under those conditions. It is further shown that deletion of the ring finger motif abolishes the metal-binding capacity of COP1. We conclude that the ring finger motif constitutes a zinc-coordinating element distinct from previously characterized zinc-binding domains.

Amino Acid Sequence↗