Proteomic analysis of Medicago truncatula root plastids

Despite the recognized importance of non‐photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil‐grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in‐depth analysis by nanoscale capillary LC‐MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid‐related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid‐located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.     

Desiccation tolerance in developing soybean seeds: The role of stress proteins

The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of certain "late embryogenesis abundant" (LEA) proteins suggests that these proteins reduce desiccation-induced cellular damage. The aim of the present work was to test this hypothesis. Exogenous abscisic acid (ABA) was used to elevate the level of heal-soluble LEA-like proteins in axes from immature (30 days after flowering: mid-development) seeds of soybean ( Glycine max [L.] Merrill cv. Chippewa 64). As the LEA-like proteins accumulated in response to ABA, the leakage of all elements after desiccation and subsequent rehydration markedly declined. Both LEA-like protein accumulation and the decline in desiccation-induced electrolyte leakage were apparently dependent on the presence of ABA. Both effects of ABA were inhibited by cycloheximide. Light microscopy revealed a marked effect of the ABA on cellular integrity following desiccation. Osmotic stress also caused a decrease in desiccation-induced electrolyte leakage and stimulated the accumulation of LEA-like proteins. Our data are consistent with the hypothesis that the LEA-like proteins contribute to the increase in desiccation tolerance in response to ABA, and are consistent with a general protective role for these proteins in desiccation tolerance.     

β-structure from the microfibillar proteins of Merino wool

The β-structure of S-caboxymethyl derivatives of microfibrillar proteins isolated from Merino wool was investigated by X-ray diffraction for comparison with the structur of β-keratin. The S-carboxymethylated microfibrillar proteins(SCMKA) w well-oriented β-films of SCMKA weer obtained by stretching the SCMKA cast films in steam up to about 300% extesnsion. It was found that the reflections in β-pattern of SCMKA may be indexed on a pseudo-orthorhombic unit cell with a =0.94 nm, b = 0.66 nm and c = nm, where the ab, and c axes are in the direction of the interchain hydrogen bonding, the main chain(fibre axis) and the side chain, respectively. The unit cell dimesnions evaluated for SCMKA were almost the same as those for β-keratin, suggeting that few peptide sequences containing S-carboxymethyl cystine may be involved in the formation of β-structure from SCMKA.     

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6–8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (K_i = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of ⁴⁵Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to ⁴⁵Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.     

Serpentine type receptors and heterotrimeric G-proteins in yeasts: Structural-functional organization and molecular mechanisms of action

The signal systems of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, coupled to heterotrimeric G-proteins and sensitive to pheromones and alimentary molecules, are prototypes of hormonal signal systems of the higher vertebrate animals and are widely used in studies on molecular mechanisms of their functioning. This review summarizes and analyzes data on structural-functional organization of the first two components of these systems—receptors of the serpentine type and heterotrimeric G-proteins; mechanisms of functional coupling of receptors and G-proteins both between each other and to other signal proteins are discussed. It has been shown that at the early stages of evolution of signaling systems, at the yeast level, various models of transduction of signals into the cell were tested; many of them differ essentially from the classic model of the three-component, G-protein-coupled signal system of the higher vertebrates.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons.

The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3' untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of beta-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA.     

Biogenesis of Respiratory Complex I

Proteins specifically involved in the biogenesis of respiratory complex I in eukaryotes have been characterized. The complex I intermediate associated proteins CIA30 and CIA84 are tightly bound to an assembly intermediate of the membrane arm. Like chaperones, they are involved in multiple rounds of membrane arm assembly without being part of the mature structure. Two biosynthetic subunits of eukaryotic complex I have been characterized. The acyl carrier subunit is needed for proper assembly of the peripheral arm as well as the membrane arm of complex I. It may interact with enzymes of a mitochondrial fatty acid synthetase. The 39/40-kDa subunit appears to be an isomerase with a tightly bound NADPH. It is related to a protein family of reductases/isomerases. Both subunits have been discussed to be involved in the synthesis of a postulated, novel, high-potential redox group.     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.