The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins

Summary TheRicinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphidelinked 4 and 7 kDa polypeptides. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2 S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in theRicinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.     

Solution structure of RicC3, a 2S albumin storage protein from Ricinus communis

The three-dimensional structure in aqueous solution of recombinant (15)N labeled RicC3, a 2S albumin protein from the seeds of castor bean (Ricinus communis), has been determined by NMR methods. The computed structures were based on 1564 upper limit distance constraints derived from NOE cross-correlation intensities measured in the 2D-NOESY and 3D-HSQC-NOESY experiments, 70 phi torsion angle constraints obtained from (3)J(HNH)(alpha) couplings measured in the HNHA experiment, and 30 psi torsion angle constraints derived from (3)J(H)(alpha)(Ni+1) couplings measured in the HNHB experiment. The computed structures showed a RMSD radius of 0.64 A for the structural core. The resulting structure consists of five amphipatic helices arranged in a right-handed super helix, a folding motif first observed in nonspecific lipid transfer proteins. Different than the latter, RicC3 does have not an internal cavity, a fact that can be related to the exchange in the pairing of disulfide bridges in the segment.CXC. Previous attempts to determine high resolution structures of a 2S albumin protein by either X-ray crystallography or NMR methods failed because of the heterogeneity of the protein prepared from natural sources. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamine superfamily, some of whose members are food allergens. The solution structure for recombinant RicC3 determined here is a suitable representative structure for the broad family of seed 2S albumin proteins, which may help to establish meaningful relationships between structure and allergenicity. RicC3 is also the peptidic component of the immunomodulator Inmunoferon, a widely used pharmaceutical product, and its structure is expected to help understand its pharmaceutical activity.     

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.     

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.     

Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent

The apoprotein of yeast cytochrome c1 is made outside the mitochondria as a larger precursor which is then processed in at least two steps. In the first step, it is transported across both mitochondrial membranes and converted by a matrix-localized protease to an intermediate form whose molecular weight is between that of the precursor and the mature form. The intermediate form is bound to the outer face of the inner membrane. This first step requires an energized mitochondrial inner membrane, but no heme. In the second step, the intermediate form is converted to the mature cytochrome. This second step requires heme; it is blocked in a heme-deficient mutant or in wild type cells treated with an inhibitor of heme synthesis. Import of cytochrome c1 into mitochondria thus proceeds via two distinct heme-free precursors and at least two maturation steps, one of them dependent on heme.     

Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20 000) is processed to the mature size (relative molecular mass 14 000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr 18 000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre-incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate-treated precursor is taken up and converted to a number of polypeptides of Mr 18 000 and below.     

S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein

The IRA1 and IRA2 genes of S. cerevisiae encode closely related proteins that also share homology with mammalian GAP (ras GTPase activating protein). The RAS1 and RAS2 proteins overexpressed in ira mutants accumulated in the GTP-bound form, whereas in the wild-type strain the proteins were found mostly in the GDP-bound form, indicating that IRA1 and IRA2 negatively regulate the level of RAS-GTP. In contrast, the RAS2Val-19 or RAS2Thr-66 mutant protein was bound to GTP in high amounts irrespective of the IRA genotype. Overexpression of bovine GAP suppressed the phenotypes of ira mutants by reducing the level of RAS-GTP, suggesting that IRA proteins may be functionally analogous to mammalian GAP.     

A cotyledon regulatory region is responsible for the different spatial expression patterns of Arabidopsis 2S albumin genes

The 2S albumin genes of Arabidopsis thaliana are a model system to study gene expression during late embryogenesis. The at2S1 gene has previously been shown to be expressed essentially in the embryo axis, unlike at2S2, which is expressed throughout the embryo. Hybrid promoter constructs between at2S1 and at2S2 were introduced into Arabidopsis and used to identify a cotyledon regulatory region necessary for 2S albumin expression in palisade parenchyma and specific epidermal cells. Other promoter sequences flanking this tissue-specific promoter element were shown to control mRNA expression levels independently of the mRNA distribution throughout the embryos. Certain hybrid promoters resulted in the alteration of the time course of expression in cotyledons. Differential expression of 2S albumin genes is discussed in terms of layered cellular organization and mitotic activity throughout the embryo.     

Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl-D-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.     

RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins

The S100 protein family comprises at least 25 members which, with the exception of S100G, act as Ca2+-sensor proteins that participate in Ca2+ signal transduction by interacting with target proteins thereby modifying their activities. S100 proteins are expressed in vertebrates exclusively, display a cell-specific distribution, and regulate a large variety of intracellular activities. Some S100 proteins are released by a non-classical pathway and exert regulatory effects on several cell types. The receptor for advanced glycation end products (RAGE) has been shown to transduce extracellular effects of S100B, S100A4, S100A6, S100A11, S100A12, S100A13 and S100P. However, some S100 proteins can signal by engaging RAGE as well as non-RAGE receptors. Immune cells (i.e., monocytes/macrophages/microglia, neutrophils and lymphocytes), activated endothelial and vascular smooth muscle cells, neurons, astrocytes, chondrocytes and pancreatic tumor cells are the cell types reported to respond to certain S100 proteins via RAGE engagement. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells. S100B is unique in that it can engage RAGE in neurons at low and high concentrations with trophic and toxic effects, respectively, and S100A4 stimulates matrix metalloproteinase 13 release from chondrocytes at nanomolar doses in a RAGE-mediated manner. Oligomerization of S100 proteins under the non-reducing, high-Ca2+ conditions found extracellularly appears to play a relevant role in RAGE activation, and binding of at least S100A12 and S100B results in RAGE oligomerization. Thus, S100/RAGE interactions might have important consequences during development and in tissue homeostasis as well as in inflammatory, degenerative and tumor processes.     

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.     

Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.

The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.     

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.     

A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay

A synthesis of the N-hydroxysuccinimide ester of S-acetylthioacetic acid is described. This material is stable when stored dry and has advantages over the currently available reagents used to introduce sulfhydryl groups into a variety of proteins. Proteins modified with this reagent can be used to prepare conjugates for enzyme immunoassay. The conjugation techniques described cause little or no loss of either enzyme activity or antibody titer and function, and the conjugates contain little polymeric material.     

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (相似文献   

Electron transfer may occur in the chlorosome envelope: the CsmI and CsmJ proteins of chlorosomes are 2Fe-2S ferredoxins

Chlorosomes of the green sulfur bacterium Chlorobium tepidum have previously been shown to contain at least 10 polypeptides [Chung, S., Frank, G., Zuber, H., and Bryant, D. A. (1994) Photosynth. Res. 41, 261-275]. Based upon the N-terminal amino acid sequences determined for two of these proteins, the corresponding genes were isolated using degenerate oligonucleotide hybridization probes. The csmI and csmJ genes encode proteins of 244 and 225 amino acids, respectively. A third gene, denoted csmX, that predicts a protein of 221 amino acids with strong sequence similarity to CsmI and CsmJ, was found to be encoded immediately upstream from the csmJ gene. All three proteins have strong sequence similarity in their amino-terminal domains to [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily of ferredoxins. CsmI and CsmJ were overproduced in Escherichia coli, and both proteins were shown by EPR spectroscopy to contain iron-sulfur clusters. The g-tensor and relaxation properties are consistent with their assignment as [2Fe-2S] clusters. Isolated chlorosomes were also shown to contain [2Fe-2S] clusters whose properties were similar to those of the recombinant CsmI and CsmJ proteins. Redox titration of isolated chlorosomes showed these clusters to have potentials of about -201 and +92 mV vs SHE. The former potential is similar to that measured by redox titration of the clusters in inclusion bodies of CsmJ. Possible roles for these iron-sulfur proteins in electron transport and light harvesting are discussed.     

How different two almost identical action potentials can be: A model study on cardiac repolarization

Spatial heterogeneity in the properties of ion channels generates spatial dispersion of ventricular repolarization, which is modulated by gap junctional coupling. However, it is possible to simulate conditions in which local differences in excitation properties are electrophysiologically silent and only play a role in pathological states. We use a numerical procedure on the Luo-Rudy phase 1 model of the ventricular action potential (AP1) in order to find a modified set of model parameters which generates an action potential profile (AP2) almost identical to AP1. We show that, although the two waveforms elicited from resting conditions as a single AP are very similar and belong to membranes sharing similar passive electrical properties, the modified membrane generating AP2 is a weaker current source than the one generating AP1, has different sensitivity to up/down-regulation of ion channels and to extracellular potassium, and a different electrical restitution profile. We study electrotonic interaction of AP1- and AP2 - type membranes in cell pairs and in cable conduction, and find differences in source-sink properties which are masked in physiological conditions and become manifest during intercellular uncoupling or partial block of ion channels, leading to unidirectional block and spatial repolarization gradients. We provide contour plot representations that summarize differences and similarities. The present report characterizes an inverse problem in cardiac cells, and strengthen the recently emergent notion that a comprehensive characterization and validation of cell models and their components are necessary in order to correctly understand simulation results at higher levels of complexity.