Proteomics of chloroplast envelope membranes

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like Arabidopsis, to the functional knowledge provided by studies of plant cell compartments, such as chloroplast envelope membranes. This review summarizes the present state of proteomic analyses of highly purified spinach and Arabidopsis envelope membranes. Methods targeted towards the hydrophobic core of the envelope allow identifying new proteins, and especially new transport systems. Common features were identified among the known and newly identified putative envelope inner membrane transporters and were used to mine the complete Arabidopsis genome to establish a virtual plastid envelope integral protein database. Arabidopsis envelope membrane proteins were extracted using different methods, that is, chloroform/methanol extraction, alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to the less hydrophobic ones. Mass spectrometry analyses lead to the identification of more than 100 proteins. More than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are (a) involved in ion and metabolite transport, (b) components of the protein import machinery and (c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism or in responses to oxidative stress, were associated with envelope membranes. Almost one third of the newly identified proteins have no known function. The present stage of the work demonstrates that a combination of different proteomics approaches together with bioinformatics and the use of different biological models indeed provide a better understanding of chloroplast envelope biochemical machinery at the molecular level.     

Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346. RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b. However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.     

Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

Evidence of Type I and Type II Transforming Growth Factor-β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia

Abstract: The peptides of the transforming growth factor-β (TGF-β) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-β receptors and their potential ligands (TGF-β1, TGF-β2, and TGF-β3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-β isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-βs and their receptors. We show that the three TGF-β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-β2. We have demonstrated that both type I and type II TGF-β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-β1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-β receptor was not altered by the ischemic insult, the pattern of the type II TGF-β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.     

Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants

For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and F_ab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and F_abfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeF_ab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or F_ab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream. ⁴Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be     

Distribution, abundance, and conservation of Vinaceous Amazons (Amazona vinacea) in Argentina and Paraguay

ABSTRACT.   Vinaceous Amazons ( Amazona vinacea ) are endemic to the Atlantic forest of southeastern Brazil, eastern Paraguay, and the province of Misiones in Argentina. We searched for Vinaceous Amazons throughout the western part of its range in Argentina and Paraguay during 1639 days of fieldwork from 1997 to 2006. These parrots have disappeared from most areas where they were historically recorded in these countries, and are now limited to a few sites in northeastern Paraguay and central Misiones (Argentina). We estimate the minimum remaining populations at 220 individuals in Paraguay and 203 individuals in Argentina. Important sites for the species are (1) the farming area from San Pedro to Tobuna (Misiones, Argentina) and (2) the Itaipú reserves complex and Reserva Natural Privada Itabó (Paraguay). In our surveys, Vinaceous Amazons were absent from the largest tracts of forest in Misiones, and were most often observed feeding, roosting, and nesting in small forest remnants and in agricultural areas that included forest fragments and isolated trees. Threats to amazons in these areas include nest poaching, forest clearing, and being shot as a crop pest. We confirmed 40 Vinaceous Amazons kept as pets in 35 homes between San Pedro and Tobuna. Environmental education and law enforcement are urgently needed to reduce threats in populated areas, and subsistence farmers need technical and logistical support to slow or stop the conversion of forest into cropland. Finally, additional study is needed to determine this amazon's habitat preferences, nest site requirements, and demography in different habitats.