JNK activation is not required for Fas-mediated apoptosis.
(73/11716)
Fas ligation in the presence of cycloheximide induced Jun N-terminal kinase 1 (JNK1) and JNK2 phosphorylation, caspase activation and cell death in the IL-3-dependent cell line BAF3. Fas-mediated apoptosis was prevented by expression of dominant negative FADD but not inhibited by IL-3. To investigate the role of JNK activation in this process, we examined cells over-expressing a JNK-specific phosphatase M3/6. M3/6 prevented Fas stimulation of JNK, but did not affect Fas-mediated caspase activation or cell death, demonstrating that JNK activation is not required for these processes. (+info)
In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family.
(74/11716)
Disruption of the two thioredoxin genes in yeast dramatically affects cell viability and growth. Expression of Arabidopsis thioredoxin AtTRX3 in the Saccharomyces thioredoxin Delta strain EMY63 restores a wild-type cell cycle, the ability to grow on methionine sulfoxide, and H2O2 tolerance. In order to isolate thioredoxin targets related to these phenotypes, we prepared a C35S (Escherichia coli numbering) thioredoxin mutant to stabilize the intermediate disulfide bridged complex and we added a polyhistidine N-terminal extension in order to purify the complex rapidly. Expression of this mutant thioredoxin in the wild-type yeast induces a reduced tolerance to H2O2, but only limited change in the cell cycle and no change in methionine sulfoxide utilization. Expression in the Delta thioredoxin strain EMY63 allowed us to isolate a complex of the thioredoxin with YLR109, an abundant yeast protein related to PMP20, a peroxisomal protein of Candida. No function has so far been attributed to this protein or to the other numerous homologues described in plants, animals, fungi, and prokaryotes. On the basis of the complementation and of low similarity with peroxiredoxins, we produced YLR109 and one of its Arabidopsis homologues in E. coli to test their peroxiredoxins activity. We demonstrate that both recombinant proteins present a thioredoxin-dependent peroxidase activity in vitro. The possible functions of this new peroxiredoxin family are discussed. (+info)
Spectroscopic and molecular characterization of a long wavelength absorbing antenna of Ostreobium sp.
(75/11716)
One of the strains of the marine green alga Ostreobium sp. possesses an exceptionally large number of long wavelength absorbing chlorophylls (P. Haldall, Biol. Bull. 134, 1968, 411-424) as evident from a distinct shoulder in the absorption spectrum at around 710 nm while in the other strain this shoulder is absent. Therefore, Ostreobium offers a unique possibility to explore the origin of these red-shifted chlorophylls, because strains with and without these spectral forms can be compared. Here, we characterize these red forms spectroscopically by absorption, fluorescence and CD spectroscopy. In the CD spectra at least three spectroscopic red forms are identified which lead to an unusual room temperature fluorescence spectrum that peaks at 715 nm. The gel electrophoretic pattern from thylakoids of Ostreobium sp. shows an intense band at 22 kDa which correlates with the presence or absence of long wavelength absorbing pigments. By protein sequencing of the N-terminus of the 22-kDa polypeptide and sequence alignments, this was identified as an Lhca1-type light-harvesting complex. The abundance of this polypeptide - and a possibly co-migrating one - in Ostreobium sp. indicates an antenna size of approximately 340 chlorophyll molecules (Chl a and Chl b) per PS IIalpha reaction center, which is significantly larger than in higher plants ( approximately 240). The red forms are more abundant in the interior of the thalli where a 'shade-light' light field is expected than in the white-light exposed surface. This demonstrates that algae exist which may be able to up-regulate the synthesis of large amounts of LHCI and associated red forms under appropriate illumination conditions. (+info)
Kinase interaction domain of kinase-associated protein phosphatase, a phosphoprotein-binding domain.
(76/11716)
Kinase-associated protein phosphatase interacts specifically with plant receptor-like protein kinases. This interaction is thought to be a key step in signal perception and transduction. The minimal kinase interaction (KI) domain of kinase-associated protein phosphatase was mapped to a 119-aa segment spanning residues 180 to 298. A forkhead-associated (FHA) homology region resides in this minimal KI domain. Site-directed mutagenesis of four highly conserved sites in this FHA homology region abolishes the KI domain's interaction with receptor-like protein kinases, indicating that the FHA region is essential for binding. Serial deletion analysis indicates that 30 aa on each side of the FHA region are also needed for binding; this minimal functional unit is designated as the KI domain. Kinetic studies using surface plasmon resonance indicate that the binding between the KI domain and receptor-like protein kinases has a dissociation constant (KD) of about 25-100 nM, which is similar to the binding affinity of two other well characterized phosphorylation-dependent protein-binding domains (14-3-3 and Src homology 2) and their high-affinity phosphopeptide ligands. (+info)
Evidence for functional conservation of a mammalian homologue of the light-responsive plant protein COP1.
(77/11716)
Identified in Arabidopsis as a repressor of light-regulated development, the COP1 (constitutively photomorphogenic 1) protein is characterized by a RING-finger motif and a WD40 repeat domain [1]. The subcellular localization of COP1 is light-dependent. COP1 acts within the nucleus to repress photomorphogenic development, but light inactivates COP1 and diminishes its nuclear abundance [2]. Here, we report the identification of a mammalian COP1 homologue that contains all the structural features present in Arabidopsis COP1 (AtCOP1). When expressed in plant cells, a fusion protein comprising mammalian COP1 and beta-glucuronidase (GUS) responded to light by changing its subcellular localization pattern in a manner similar to AtCOP1. Whereas the mammalian COP1 was unable to rescue the defects of Arabidopsis cop1 mutants, expression of the amino-terminal half of mammalian COP1 in Arabidopsis interfered with endogenous COP1 function, resulting in a hyperphotomorphogenic phenotype. Therefore, the regulatory modules in COP1 proteins that are responsible for the signal-dependent subcellular localization are functionally conserved between higher plants and mammals, suggesting that mammalian COP1 may share a common mode of action with its plant counterpart in regulating development and cellular signaling. (+info)
The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment.
(78/11716)
Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process. (+info)
Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana.
(79/11716)
The plant hormone auxin regulates diverse aspects of plant growth and development. We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1. The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1. Mutations in either ASK1 or TIR1 result in decreased auxin response. Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response. These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins. (+info)
Regions of the pea Lhcb1*4 promoter necessary for blue-light regulation in transgenic Arabidopsis.
(80/11716)
Pea (Pisum sativum) and Arabidopsis contain similar, if not identical, blue-light (BL)-responsive systems that alter expression of specific members of the Lhcb (light-harvesting chlorophyll-binding) gene family. In both plants a single, short pulse of low-fluence BL (threshold = 10(-1) micromol m-2) causes an increase in the rate of transcription from specific members of the Lhcb gene family in etiolated seedlings. Constructs of the BL-regulated pea Lhcb1*4 promoter (PsLhcb1*4) were created, which altered sequences previously implicated in light responses, deleted the 5'-promoter sequence, or removed the 5'-untranslated region. These constructs were tested for BL induction in transgenic Arabidopsis. The PsLhcb1*4 promoter deletions to -150 bp maintained normal fluence response, time course, and reciprocity characteristics. The 5'- untranslated region contained enhancer elements, but was not necessary for BL induction. The -95 to +2 promoter was capable of responding to BL, whereas sequences from -50 were not. Promoters that lack conserved light-regulatory elements or sequences directly implicated in phytochrome and circadian responses retained BL activity, suggesting that the low-fluence BL response utilizes regions of the promoter independent of those that modulate the phytochrome and circadian responses. (+info)