Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the ��E Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ^E, and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onσ^E activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.The Escherichia coli σ^E-mediated envelope stress response is the major pathway to ensure homeostasis in the envelope compartment of the cell (1-3). σ^E regulon members encode periplasmic chaperones and proteases, the machinery for inserting β-barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4-6). This pathway is highly conserved among γ-proteobacteria (6).The σ^E response is initiated when periplasmic protein folding and assembly is compromised (7-9). During steady state growth, σ^E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to σ^E with picomolar affinity (10-13). Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15). This initiates a proteolytic cascade in which RseP cleaves periplasmically truncated RseA near or within the cytoplasmic membrane to release the RseA_cytoplasmic-σ^E complex, and cytoplasmic ATP-dependent proteases complete the degradation of RseA thereby releasing active σ^E (16-19).RseB, a second negative regulator of the envelope stress response (11, 20, 21), binds to the periplasmic domain of RseA with nanomolar affinity. RseB is an important regulator of the response (2, 22, 23). It prevents RseP from degrading intact RseA, thereby ensuring that proteolysis is initiated only when the DegS protease is activated by a stress signal (21). Additionally, RseB prevents activated DegS from cleaving RseA, suggesting that interaction of RseB with RseA must be altered before the signal transduction cascade is activated (23).The goal of the present studies was to explore how RseB binds to RseA. The interaction partner of RseB is the unstructured periplasmic domain of RseA (RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major binding determinant to RseB (23, 24). This peptide alone binds RseB with 6 μm affinity, and deleting this region abrogates binding to RseB (23). Additional regions of RseA-peri also contribute to RseB binding, as intact RseA-peri binds with 20 nm affinity to RseB (23). Much less is known about the regions of RseB required for interaction with RseA. RseB is homodimeric two-domain protein, whose large N-terminal domain shares structural homology with LolA, a protein that transports lipoproteins to outer membrane (24, 25). The smaller C-terminal domain is connected to the N-terminal domain by a linker, and the two domains share a large interface, which may facilitate interdomain signaling. Glutaraldehyde cross-linking studies indicate that the C-terminal domain interacts with RseA, but the regions of interaction were not identified (25).In the present report, we study the interaction of RseB and RseA. We establish that both domains of RseB interact with RseA-peri. Using a global sequence alignment, we discovered several regions in RseA and RseB that had high sequence similarity, despite the low overall sequence similarity between these two proteins, a finding that was independently confirmed by a local sequence similarity algorithm. This suggested that these regions were functionally dependent, and we performed a set of mutagenesis experiments designed to test this idea. Our studies of the binding properties of these mutants revealed that regions in both the N terminus and C terminus of RseB modulate interaction with RseA. Moreover, genetic suppression analysis and cysteine-mediated disulfide bond formation suggest that the region of RseA/B with highest similarity (RseA residues 165-191 (major binding determinant in RseA) and RseB residues 233-258) are interacting partners.     

Description of a new Brazilian Paraportanus and key to the species of the genus (Insecta,Hemiptera,?Cicadellidae,Portanini)

Paraportanus longispinus,, a new leafhopper species from Roraima and Amazonas States, North Brazil, is described and illustrated. The new species can be recognized by the male genital features, especially the distal third of ventral margin of the pygofer with a dentiform short process; plates distinctly longer than pygofer, extending posteriorly beyond pygofer by approximately 1/3 of their length and aedeagus with one pair of spiniform process long crossed and directed ventrally. A checklist and key to males of all known Paraportanus species is provided.     

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO₂, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO₂-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.     

Src-Mediated Phosphorylation of Dynamin and Cortactin Regulates the “Constitutive” Endocytosis of Transferrin

The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process.     

Rarity,Species Richness,and the Threat of Extinction—Are Plants the Same as Animals?

Assessment of conservation status is done both for areas or habitats and for species (or taxa). IUCN Red List categories have been the principal method of categorising species in terms of extinction risk, and have been shown to be robust and helpful in the groups for which they have been developed. A recent study highlights properties associated with extinction risk in flowering plants, focusing on the species-rich hot spot of the Cape region of South Africa, and concludes that merely following methods derived from studies of vertebrates may not provide the best estimates of extinction risk for plants. Biology, geography, and history all are important factors in risk, and the study poses many questions about how we categorise and assess species for conservation priorities.     

Characterization of the Differential Roles of the Twin C1a and C1b Domains of Protein Kinase C��

Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated “C1a” and “C1b” domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKCδ constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the δC1a and δC1b domains potently bound phorbol esters, but the binding of [³H]phorbol 12,13-dibutyrate ([³H]PDBu) by the δC1a domain depended much more on the presence of phosphatidylserine than did that of the δC1b domain. In intact PKCδ, the δC1b domain played the dominant role in [³H]PDBu binding, membrane translocation, and down-regulation. A contribution from the δC1a domain was nonetheless evident, as shown by retention of [³H]PDBu binding at reduced affinity, by increased [³H]PDBu affinity upon expression of a second δC1a domain substituting for the δC1b domain, and by loss of persistent plasma membrane translocation for PKCδ expressing only the δC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the δC1b domain to the normal position of the δC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C (PKC)² activation is its translocation from the cytosol to the membranes. For conventional (α, βI, βII, and γ) and novel (δ, ε, η, and θ) PKCs, this translocation is driven by interaction with the lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated from phosphatidylinositol 4,5-bisphosphate upon the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). A pair of zinc finger structures in the regulatory domain of the PKCs, the “C1” domains, are responsible for the recognition of the DAG signal. The DAG-C1 domain-membrane interaction is coupled to a conformational change in PKC, both causing the release of the pseudosubstrate domain from the catalytic site to activate the enzyme and triggering the translocation to the membrane (2). By regulating access to substrates, PKC translocation complements the intrinsic enzymatic specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino acids), which was first identified in PKC as the interaction site for DAG or phorbol esters (3). It possesses a globular structure with a hydrophilic binding cleft at one end surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1 domain caps the hydrophilic cleft and forms a continuous hydrophobic surface favoring the interaction or penetration of the C1 domain into the membrane (4). In addition to the novel and classic PKCs, six other families of proteins have also been identified, some of whose members possess DAG/phorbol ester-responsive C1 domains. These are the protein kinase D (5), the chimaerin (6), the munc-13 (7), the RasGRP (guanyl nucleotide exchange factors for Ras and Rap1) (8), the DAG kinase (9), and the recently characterized MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) families (10). Of these C1 domain-containing proteins, the PKCs have been studied most extensively and are important therapeutic targets (11). Among the drug candidates in clinical trials that target PKC, a number such as bryostatin 1 and PEP005 are directed at the C1 domains of PKC rather than at its catalytic site.Both the classic and novel PKCs contain in their N-terminal regulatory region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester (12). Multiple studies have sought to define the respective roles of these two C1 domains in PKC regulation, but the issue remains unclear. Initial in vitro binding measurements with conventional PKCs suggested that 1 mol of phorbol ester bound per mole of PKC (13-15). On the other hand, Stubbs et al., using a fluorescent phorbol ester analog, reported that PKCα bound two ligands per PKC (16). Further, site-directed mutagenesis of the C1a and C1b domains of intact PKCα indicated that the C1a and C1b domains played equivalent roles for membrane translocation in response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V (17). Likewise, deletion studies indicated that the C1a and C1b domains of PKCγ bound PDBu equally with high potency (3, 18). Using a functional assay with PKCα expression in yeast, Shieh et al. (19) deleted individual C1 domains and reported that C1a and C1b were both functional and equivalent upon stimulation by PMA, with either deletion causing a similar reduction in potency of response, whereas for mezerein the response depended essentially on the C1a domain, with much weaker response if only the C1b domain was present. Using isolated C1 domains, Irie et al. (20) suggested that the C1a domain of PKCα but not those of PKCβ or PKCγ bound [³H]PDBu preferentially; different ligands showed a generally similar pattern but with different extents of selectivity. Using synthesized dimeric bisphorbols, Newton''s group reported (21) that, although both C1 domains of PKCβII are oriented for potential membrane interaction, only one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ to study the equivalency of the twin C1 domains. The P11G point mutation of the C1a domain, which caused a 300-fold loss of binding potency in the isolated domain (22), had little effect on the phorbol ester-dependent translocation of PKCδ in NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in phorbol ester potency for inducing translocation, suggesting a major role of the C1b domain for phorbol ester binding (23). A secondary role for the C1a domain was suggested, however, because mutation in the C1a domain as well as the C1b domain caused a further 7-fold shift in potency. Using the same mutations in the C1a and C1b domains, Bögi et al. (24) found that the binding selectivity for the C1a and C1b domains of PKCδ appeared to be ligand-dependent. Whereas PMA and the indole alkaloids indolactam and octylindolactam were selectively dependent on the C1b domain, selectivity was not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and 12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1 (24). In in vitro studies using isolated C1a and C1b domains of PKCδ, Cho''s group (25) described that the two C1 domains had opposite affinities for DAG and phorbol ester; i.e. the C1a domain showed high affinity for DAG and the C1b domain showed high affinity for phorbol ester. No such difference in selectivity was observed by Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for other conditions, such as diabetic retinopathy or macular degeneration (26-30). Kinase inhibitors represent one promising approach for targeting PKC, and enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to other PKC isoforms (but still with activity on some other non-PKC kinases) is currently in multiple clinical trials. An alternative strategy for drug development has been to target the regulatory C1 domains of PKC. Strong proof of principle for this approach is provided by multiple natural products, e.g. bryostatin 1 and PEP005, which are likewise in clinical trials and which are directed at the C1 domains. A potential advantage of this approach is the lesser number of homologous targets, <30 DAG-sensitive C1 domains compared with over 500 kinases, as well as further opportunities for specificity provided by the diversity of lipid environments, which form a half-site for ligand binding to the C1 domain. Because different PKC isoforms may induce antagonistic activities, inhibition of one isoform may be functionally equivalent to activation of an antagonistic isoform (31).Along with the benzolactams (20, 32), the DAG lactones have provided a powerful synthetic platform for manipulating ligand: C1 domain interactions (31). For example, the DAG lactone derivative 130C037 displayed marked selectivity among the recombinant C1a and C1b domains of PKCα and PKCδ as well as substantial selectivity for RasGRP relative to PKCα (33). Likewise, we have shown that a modified DAG lactone (dioxolanones) can afford an additional point of contact in ligand binding to the C1b domain of PKCδ (34). Such studies provide clear examples that ligand-C1 domain interactions can be manipulated to yield novel patterns of recognition. Further selectivity might be gained with bivalent compounds, exploiting the spacing and individual characteristics of the C1a and C1b domains (35). A better understanding of the differential roles of the two C1 domains in PKC regulation is critical for the rational development of such compounds. In this study, by molecularly manipulating the C1a or C1b domains in intact PKCδ, we find that both the C1a and C1b domains play important roles in PKCδ regulation. The C1b domain is predominant for ligand binding and for membrane translocation of the whole PKCδ molecule. The C1a domain of intact PKCδ plays only a secondary role in ligand binding but stabilizes the PKCδ molecule at the plasma membrane for downstream signaling. In addition, we show that the effect of the individual C1 domains of PKCδ does not critically depend on their position within the regulatory domain.     

Coupling of the β Rhythm and Slow Electric Activity of the Cerebral Cortex during the Performance of Go/NoGo Tasks and the Identification of the Facial Expression

We investigated the interaction of β-rhythm parameters with α and θ rhythms in a paradigm of cognitive set as a response on facial expression in 35 healthy adults. Data were analyzed by means of continuous wavelet transform on the basis of “maternal” complex Morlet-wavelet in a range of 1–35 Hz. Distribution cards of the values of the wavelet-transform coefficient (WLC) module characterizing potentials amplitude were analyzed. We used indicators of the mean and maximal WLC levels. A significant interaction between β₂ and α rhythms at the mean WLC level at the stage of set formation was revealed in a group with rigid set, and a correlation coefficient was 0.57. The interaction between β₂ and θ rhythms at the mean WLC level (correlation coefficient was 0.74) and WLC maximum (correlation coefficient was 0.8) at the same experimental stage in the group with the flexible set was found.     

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the ¹⁴C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg⁻¹ protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [¹⁴C]gibberellin (GA)₉ and [¹⁴C]GA₂₀ to radiolabeled GA₄ and GA₁, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA₉ was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA₂₀. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA₁₂-aldehyde, GA₁₂, GA₁₉, GA₂₅, GA₅₃, or GA₄₄ as the open lactone (20-hydroxy-GA₅₃), whereas GA₁₅, GA₂₄, and GA₄₄ were hydroxylated to GA₃₇, GA₃₆, and GA₃₈, respectively. The open lactone of GA₁₅ (20-hydroxy-GA₁₂) was hydroxylated but less efficiently than GA₁₅. In contrast to the free acid, GA₂₅ 19,20-anhydride was 3β-hydroxylated to give GA₁₃. 2,3-Didehydro-GA₉ and GA₅ were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA₉ and GA₆.Dwarf mutants with reduced biosynthesis of the GA plant hormones have been valuable tools in studies of the function of these compounds (Ross, 1994). In Arabidopsis thaliana, mutations at six loci (GA1-GA6) that result in reduced GA biosynthesis have been identified (Koorneef and van der Veen, 1980; Sponsel et al., 1997), and three of these loci have recently been cloned. The GA1 locus was isolated by genomic subtraction (Sun et al., 1992) and shown by heterologous expression in Escherichia coli to encode the enzyme that cyclizes geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya, 1994). This enzyme was formerly referred to as ent-kaurene synthase A but has been renamed copalyl diphosphate synthase (Hedden and Kamiya, 1997; MacMillan, 1997). The GA5 locus was shown to correspond to one of the GA 20-oxidase genes (Xu et al., 1995), the products of which catalyze the conversion of GA₁₂ to GA₉ and GA₅₃ to GA₂₀ (Phillips et al., 1995; Xu et al., 1995). GA 20-oxidases are 2-oxoglutarate-dependent dioxygenases that are encoded by small multigene families, members of which are differentially expressed in plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the basis of the derived amino acid sequence, was also shown to encode a dioxygenase (Chiang et al., 1995). Several lines of evidence indicate that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a ga4 mutant, all alleles of which are semidwarf, contained reduced concentrations of the 3β-hydroxy GAs GA₁, GA₄, and GA₈ compared with the Landsberg erecta wild type, whereas levels of immediate precursors to these GAs were elevated (Talon et al., 1990). Furthermore, metabolism of [¹³C]GA₂₀ to [¹³C]GA₁ was substantially less in the mutant than in the wild type (Kobayashi et al., 1994). In the present paper we confirm by functional expression of its cDNA in E. coli that GA4 encodes a GA 3β-hydroxylase. In addition, we determine the substrate specificity of recombinant GA4 using a number of C₂₀- and C₁₉-GAs and show by kinetic analysis that the enzyme has a higher affinity for GA₉ than for GA₂₀, which is consistent with the non-13-hydroxylation pathway predominating in Arabidopsis (Talon et al., 1990).     

The N-terminal Amphipathic ��-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α₁-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.Type I interferons are the first line of defense against viral infections. The significance of the interferon pathway is illustrated by the susceptibility of interferon signaling mutants to infection and by viral mechanisms that counteract this pathway (1, 2). Although many genes are induced upon interferon stimulation, very few of these genes have been functionally characterized. Viperin is highly induced by both Type I and II interferons and has a broad range of antiviral activity, inhibiting DNA viruses, notably human cytomegalovirus (3); RNA viruses such as influenza, hepatitis C virus (HCV),² and alphaviruses (4-6); and retroviruses such as human immunodeficiency virus (7). Upon expression, viperin localizes to the endoplasmic reticulum (ER), where it interacts with farnesyl-diphosphate synthase, an enzyme involved in lipid biosynthesis. This interaction appears to result in the disruption of lipid raft microdomains and prevention of influenza virus from budding from the plasma membrane (4).Although recent studies have explored the antiviral functions of viperin, the general biochemical properties of this protein remain largely undefined. Viperin is highly conserved across both mammals and lower vertebrates and shares homology with the MoaA family of “radical S-adenosylmethionine” enzymes that bind Fe-S clusters (3, 8). In addition to a putative Fe-S cluster-binding domain, viperin has a 42-amino acid residue N-terminal amphipathic α-helix, and similar domains in other proteins have been shown to bind membranes and induce membrane curvature (9, 10).In this study, we examined the role of the viperin N-terminal α-helical domain in both cellular localization and ER membrane morphology and analyzed the biochemical properties of viperin. We discovered that viperin forms dimers and induces a tightly ordered, visually striking array of ER membranes, known as crystalloid ER(11-13), upon overexpression. In addition, viperin expression impedes the secretion of a variety of soluble proteins. Although the N-terminal amphipathic α-helix is not sufficient to induce crystalloid ER formation, it is both necessary and sufficient to mediate ER localization and to inhibit protein secretion.     

Inhibition of the Water Oxidizing Complex of Photosystem II and the Reoxidation of the Quinone Acceptor QA ? by Pb2+

The action of the environmental toxic Pb²⁺ on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb²⁺ action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb²⁺ confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S₂ state of the Mn₄Ca cluster. Electron transfer between the quinone acceptors Q_A and Q_B is also greatly retarded in the presence of Pb²⁺. This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem.     

Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for ��-Secretase

The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.Folding of proteins is a highly regulated process ensuring their correct three-dimensional structure. Under pathological circumstances, a soluble protein can be folded into highly stable cross β-sheet amyloid structures, which are believed to play pathological roles in disorders such as Alzheimer, Parkinson, and Huntington diseases. An exception to this general concept is the physiological amyloid structure of the melanosomal matrix formed by the protein Pmel17. Melanosomes are lysosome-related organelles that contain pigment granules (melanin) in melanocytes and retinal epithelial cells (reviewed in Ref. 1). Melanogenesis is believed to proceed through several sequential maturation steps, classified by melanosomes from stage I to stage IV. Maturation of stage II melanosomes requires the formation of Pmel17 intralumenal fibers (2, 3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I transmembrane glycoprotein of up to 668 amino acids in humans (reviewed in Ref. 4). The requirement of Pmel17 for the generation of functional melanin has been shown in a number of different organisms, because, for example, certain point mutations in the Pmel17/silver gene result in hypopigmentation phenotypes (5–7). The most characteristic domain within Pmel17 is a specific lumenal proline/serine/threonine rich repeat domain (see Fig. 1A), that is imperfectly repeated 13 times in the Mα fragment. Importantly, deletion of the rich repeat domain results in a complete loss of fibril formation, pointing to the requirement of Pmel17, and especially the rich repeat domain, in melanin formation (8). Pmel17 exists in different isoforms generated by alternative splicing. Pmel17-i² is the most abundant isoform, whereas the Pmel17-l isoform contains a 7-amino acid insertion close to the transmembrane domain (9, 10).Open in a separate windowFIGURE 1.Effect of the γ-secretase inhibitor DAPT on Pmel17 processing. A, schematic diagram of Pmel17 and epitopes of antibodies. Pmel17 contains five potential N-glycosylation sites indicated by branched structures. The long form of Pmel17, Pmel17-l, is characterized by a seven amino acid insertion (VPGILLT) within the lumenal domain close to the transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a potential N-glycosylation site near this insertion. The epitopes of antibodies αPep13h and HMB45 are indicated. Cleavage by a furin-like PC results in the formation of the Mα and the membrane-bound 26-kDa Mβ fragment, which are connected via disulfide bonds. Release and further processing of the Mα fragment into MαN and MαC fragments results in the formation of fibrils and marks the transition of stage I to stage II melanosomes (dashed line). B, human MNT-1 cells were incubated with increasing amounts of DAPT for 18 h, and then the lysates were separated by SDS-PAGE and analyzed by immunoblotting with αPep13h antibody. DAPT treatment resulted in the accumulation of a C-terminal fragment of Pmel17 (CTF), whereas Pmel17 P1 and Mβ fragment were unchanged. C, probing the Triton-soluble fraction with HMB45 revealed increased amounts of the highly glycosylated P2 form of Pmel17 after DAPT incubation. D, detection of Pmel17 amyloidogenic fragments (MαC) in the SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO cells treated with increasing concentrations of DAPT. Immunoblotting using antibodyαPep13h revealed the formation of CTF of similar size as in MNT-1 cells. F, time course analysis of Pmel17, Mβ, and Pmel17-CTF after DAPT treatment. The cell lysates were immunoblotted using αPep13h. Pmel17-CTF was detectable after 10 min of incubation with 1 μm DAPT. G, the size of the Pmel17-CTF was determined using an unstained low molecular range peptide standard. The marker peptides were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot using αPep13h.Pmel17 traffics through the secretory pathway as a 100-kDa protein (called P1). In the late Golgi compartment it undergoes further glycosylation, resulting in a short lived 120-kDa protein (called P2). P2 is rapidly cleaved within the post-Golgi by a furin-like proprotein convertase (PC) to generate two fragments that remain tethered to each other by disulfide bonds: a C-terminal polypeptide containing the transmembrane domain (Mβ) and a large N-terminal ectodomain (Mα) (2) (Fig. 1A). Consequently, inhibition of this furin-like activity not only prevents the generation of Mα and Mβ fragments but also inhibits the formation of melanosomal striation in HeLa cells (3). These findings suggest that Mα must first be dissociated from the Mβ for melanogenesis to proceed. It is unclear how Mα is released from the membrane. Reduction of disulfide bonds would release Mα from Mβ; alternatively, proteolytic digestion of Mβ should also free Mα from the membrane tether. It has been speculated that, given the presence of lysosomal hydrolases in melanosomes and proteolytic maturation of Pmel17, proteolysis is the more likely mechanism (4). Recently, it was shown that recombinant Mα is able to form amyloid structures in vitro in an unprecedented rapidity, and furthermore, Pmel17 amyloid also accelerated melanin formation (11). These findings demonstrate that mammalian amyloid formed by Pmel17 is functional and physiological.The insoluble pool of Pmel17 in cells consists mostly of truncated Mα C-terminal fragments (MαC) of heterogeneous sizes, indicating that further processing of Mα occurs after its release from the membrane (8, 12). MαC fragments are found in the insoluble fraction of melanocytes as well as in nonmelanotic cells, the latter after overexpression of Pmel17 (8), and are reduced or absent in amelanotic cells (8, 13, 14). Meanwhile, the C-terminal fragment derived from the Mβ fragment and recognized by a C-terminal specific epitope antibody is less stable, indicating rapid turnover (2).The presenilin (PS) family of proteins consists of two homologous integral transmembrane proteins, PS1 and PS2, which are part of the γ-secretase complex. The latter consists of presenilin 1 or 2, nicastrin, APH-1, and PEN-2 (15) and catalyzes the cleavage of the hydrophobic transmembrane domain of a burgeoning list of proteins, also called regulated intramembrane cleavage. Other substrates for the γ-secretase-mediated intramembrane cleavage include Notch, amyloid precursor protein (APP), cadherin (E-cadherin), nectin-1, the low density lipoprotein-related receptor, CD44, ErbB-4, the voltage-gated sodium channel β2-subunit, and the Notch ligands Delta and Jagged. Importantly, in Alzheimer disease, the presenilin-mediated γ-secretase cleavage of APP releases the amyloid β-protein fragment, a peptide believed to play a key role in Alzheimer disease pathogenesis. Interestingly, a recent report described the absence of melanin pigment in presenilin-deficient animals, an observation confirmed by the lack of melanin formation in cells treated with γ-secretase inhibitors (16). The mechanism responsible for this finding is unclear, leading us to ask whether Pmel17 processing is a presenilin-dependent process and, if so, whether this cleavage is involved in melanogenesis.In this study, we show the presence of an endoproteolytic activity that cleaves the extracellular domain of Pmel17-i at a juxtamembrane position between the known PC cleavage site and the transmembrane domain, which we term the S2 cleavage site, by a TAPI-sensitive ADAM (a disintegrin and metalloproteinase protein) protease. This intracellular shedding of Pmel17 after S2 cleavage results in the liberation of the Mα N-terminal ectodomain, the precursor to Pmel17 amyloid, which is able to form insoluble Pmel17 aggregates. The C-terminal transmembrane fragment generated by S2 cleavage is further processed by γ-secretase (S3 cleavage) to release the Pmel17 intracellular domain, which is then rapidly degraded.     

Isolation of the Ornithine-δ-Aminotransferase cDNA and Effect of Salt Stress on Its Expression in Arabidopsis thaliana

To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-δ-aminotransferase (δ-OAT) from Arabidopsis thaliana. The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide. Our results show that under both salt stress and normal conditions, δ-OAT activity and mRNA in young plantlets are slightly higher than in older plants. This appears to be related to the necessity to dispose of an easy recycling product, glutamate. Analysis of the expression of the gene revealed a close association with salt stress and Pro production. In young plantlets, free Pro content, Δ¹-pyrroline-5-carboxylate synthase mRNA, δ-OAT activity, and δ-OAT mRNA were all increased by salt-stress treatment. These results suggest that for A. thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress. Conversely, in 4-week-old A. thaliana plants, although free Pro level also increased under salt-stress conditions, the δ-OAT activity appeared to be unchanged and δ-OAT mRNA was not detectable. Δ¹-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level. Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.     

On newly and recently recorded species of the genus Lema Fabricius (Coleoptera,Chrysomelidae,?Criocerinae) from Taiwan

New records of four species (Lema lacertosa Lacordaire, 1845, Lema diversipes Pic, 1921, Lema cyanella (Linnaeus, 1758), Lema trivittata trivittata Say, 1824 and additional information on one recently recorded species (Lema solani Fabricius, 1798) are reported for Taiwan. Lema diversipes Pic, 1921 is removed from synonymy with Lema lacertosa Lacordaire, 1845; both species are redescribed. A lectotype is designated for Lema phungi Pic, 1924. The synonymies of Lema phungi Pic, 1924 and Lema jeanvoinei Pic, 1932 with Lema lacertosa Lacordaire, 1845 are supported. A revised key to the known species in Taiwan is provided.     

A new species of the genus Himertosoma from the Ryukyus,Japan, with a key to species from the Palaearctic and Oriental Regions (Hymenoptera,?Ichneumonidae,Banchinae)

A new species of the genus Himertosoma Schmiedeknecht, Himertosoma kuslitzkii sp. n., was discovered in Amamioshima Island, the Ryukyus. This new species resembles two Oriental species, Himertosoma philippense Chandra & Gupta and Himertosoma townesi Chandra & Gupta, in the colour pattern of the head and metasoma, number of flagellomeres, and the relatively slender first metasomal tergite, but can easily be distinguished from them by the nearly evenly punctate propodeum, different length/width ratio of the first metasomal tergite, different length of the ovipositor sheath, tricoloured mesosoma, and the whitish band along the posterior margin of the second and following metasomal tergites. A key to the Palaearctic and Oriental species of Himertosoma is also provided.