The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

Apoplastic α-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three α-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic α-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli α-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-α-d-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside revealed that the acidic α-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.

Haemophilus influenzae pili are composite structures assembled via the HifB chaperone.

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.

A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.

Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world. These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing of AcAP reveals no homology to any previously identified anticoagulant or protease inhibitor. Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic K*i for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.

Microbial iron transport via a siderophore shuttle: A membrane ion transport paradigm

A mechanism of ion transport across membranes is reported. Microbial transport of Fe3+ generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe3+ enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

Attempted hydrogen–deuterium exchange of the protio-trimethyloxonium dication (CH3)3OH2+, study of methylating ability of (CH3)3O+ in superacids and theoretical investigations

Attempted hydrogen–deuterium exchange of trimethyloxonium ion, (CH3)3O+ with excess of 1:1 2HF/SbF5 superacid at −30°C over a period of 30 days showed no exchange. Theoretical calculations at the MP2/6–31G** level are in accord with the lack of hydrogen–deuterium exchange in the methyl group of the (CH3)3O+ cation as protonation (protosolvation) prefers the oxygen lone pair of electrons, instead of a C—H bond. Methylation of aromatics with the (CH3)3O+CF3SO3− in CF3SO3H and 2CF3SO3H:B(O3SCF3)3 was also studied. Whereas in triflic acid no alkylation was observed, in triflatoboric acid, a powerful superacid, alkylation takes place, indicating protolytic activation of the trimethyloxonium ion.

Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors

The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl− selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl−-selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1 Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2 Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

Computation of Surface Electrical Potentials of Plant Cell Membranes : Correspondence to Published Zeta Potentials from Diverse Plant Sources

A Gouy-Chapman-Stern model has been developed for the computation of surface electrical potential (ψ0) of plant cell membranes in response to ionic solutes. The present model is a modification of an earlier version developed to compute the sorption of ions by wheat (Triticum aestivum L. cv Scout 66) root plasma membranes. A single set of model parameters generates values for ψ0 that correlate highly with published ζ potentials of protoplasts and plasma membrane vesicles from diverse plant sources. The model assumes ion binding to a negatively charged site (R− = 0.3074 μmol m−2) and to a neutral site (P0 = 2.4 μmol m−2) according to the reactions R− + IΖ ⇌ RIΖ−1 and P0 + IΖ ⇌ PIΖ, where IΖ represents an ion of charge Ζ. Binding constants for the negative site are 21,500 m−1 for H+, 20,000 m−1 for Al3+, 2,200 m−1 for La3+, 30 m−1 for Ca2+ and Mg2+, and 1 m−1 for Na+ and K+. Binding constants for the neutral site are 1/180 the value for binding to the negative site. Ion activities at the membrane surface, computed on the basis of ψ0, appear to determine many aspects of plant-mineral interactions, including mineral nutrition and the induction and alleviation of mineral toxicities, according to previous and ongoing studies. A computer program with instructions for the computation of ψ0, ion binding, ion concentrations, and ion activities at membrane surfaces may be requested from the authors.

Polarity and permeation profiles in lipid membranes

The isotropic 14N-hyperfine coupling constant, a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document}, of nitroxide spin labels is dependent on the local environmental polarity. The dependence of a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document} in fluid phospholipid bilayer membranes on the C-atom position, n, of the nitroxide in the sn-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form {1 + exp [(n − no)/λ]}−1, where n = no is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with n, on a characteristic length-scale, λ. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol, no ≈ 8 and λ ≈ 0.5–1 CH2 units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35–80%, depending on the lipid. For membranes containing equimolar cholesterol, no ≈ 9–10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol no ≈ 8 and λ ≈ 1 CH2 units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e., no lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.