The yeast nuclear pore complex: composition, architecture, and transport mechanism

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.     

The SC5b-7 complex: formation, isolation, properties, and subunit composition.

Activation of C in C8-depleted serum results in the formation of a soluble complex containing C5, C6, and C7. The complex has an electrophoretic mobility of an alpha-globulin, an s-rate of 18.5S, and a m.w. of 668,000 daltons. This complex was isolated and upon SDS polyacrylamide gel electrophoresis it was found to contain, in addition to C5b, C6 and C7, an 88,000 dalton glycoprotein. The protein was identified as the band V protein of the soluble C5b-9 complex. It is referred to as SIIIs-protein, or S-protein. Since the S-protein does not bind to C5b-6, it is concluded that it is incorporated during the fusion of C5b-6 with C7. The SC5b-7 complex exhibits the same neoantigen as the SC5b-9 complex, but compared to the C5b-6 complex it appears to contain an additionally qualitatively distinct neoantigen.     

Isoform composition of antithrombin in a covalent antithrombin-heparin complex

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.     

NotI passporting to identify species composition of complex microbial systems

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Subunit composition of Rhodothermus marinus respiratory complex I

The basic structural characterization of complex I is still not trivial due to its complexity, not only in the number of its protein constituents but especially because of the different properties of the several subunits. Bacterial complex I generally contains 14 subunits: 7 hydrophilic proteins located in the peripheral arm and 7 hydrophobic proteins present in the membrane arm. It is the identification of the hydrophobic proteins that makes the characterization of complex I, and of membrane proteins in general, very difficult. In this article, we report the identification of the subunits of complex I from Rhodothermus marinus. The original approach, presented here, combined several protein and peptides separation strategies (different reversed phase materials, high-performance liquid chromatography, and gel electrophoresis) with different identification methods (electrospray ionization-tandem mass spectrometry, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and Edman degradation analysis) and represents a step forward in the characterization of membrane proteins that studies are still technically highly challenging. The combination of the different methodologies allowed the identification of complex I canonical subunits and also a possible novel subunit, namely a pterin-4α-carbinolamine dehydratase (PCD). This was the first time that a PCD was suggested to be part of complex I, and its possible regulatory role is discussed.     

A change in nuclear pore complex composition regulates cell differentiation

Nuclear pore complexes (NPCs) are built from ～30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.     

ICR heating of a plasma with a complex ion composition

Two methods for simultaneously heating ions with different masses and, accordingly, with different gyrofrequencies are considered. The first is heating in a steady uniform magnetic field by a nonmonochromatic RF field, and the second is heating in a nonuniform magnetic field by a monochromatic RF field. In the first method, the cyclotron resonance condition is ensured by the finite width of the RF field spectrum, while in the second one, by a drop in the magnetic field along the system. The study is based on the previously developed analytic model of the excitation of RF fields by current-carrying antennas. Nonlinear effects caused by variations in the longitudinal ion velocities during the ICR interaction are taken into account. It is found that ions with atomic masses in the range 85 < A < 150 (fragments of a spent nuclear fuel) can be efficiently heated in systems with moderate parameters.     

Chlorophyll-protein complex composition during chloroplast development: A species comparison

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - LHC the major light-harvesting complex associated with photosystem II - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.     

The antenna reaction center complex of heliobacteria: composition, energy conversion and electron transfer.

A survey is given of various aspects of the photosynthetic processes in heliobacteria. The review mainly refers to results obtained since 1995, which had not been covered earlier. It first discusses the antenna organization and pigmentation. The pigments of heliobacteria include some unusual species: bacteriochlorophyll (BChl) g, the main pigment, 8(1) hydroxy chlorophyll a, which acts as primary electron acceptor, and 4,4'-diaponeurosporene, a carotenoid with 30 carbon atoms. Energy conversion within the antenna is very fast: at room temperature thermal equilibrium among the approx. 35 BChls g of the antenna is largely completed within a few ps. This is then followed by primary charge separation, involving a dimer of BChl g (P798) as donor, but recent evidence indicates that excitation of the acceptor pigment 8(1) hydroxy chlorophyll a gives rise to an alternative primary reaction not involving excited P798. The final section of the review concerns secondary electron transfer, an area that is relatively poorly known in heliobacteria.     

D1-D2-cytochrome b559 complex from the aquatic plant Spirodela oligorrhiza: correlation between complex integrity, spectroscopic properties, photochemical activity, and pigment composition

A D1-D2-cyt b559 complex with about four attached chlorophylls and two pheophytins has been isolated from photosystem II of the aquatic plant Spirodela oligorrhiza and used for studying the detergent-induced changes in spectroscopic properties and photochemical activity. Spectral analyses (absorption, CD, and fluorescence) of D1-D2-cyt b559 preparations that were incubated with different concentrations of the detergent Triton X-100 indicate two forms of the D1-D2-cyt b559 complexes. One of them is photochemically active and has an absorption maximum at 676 nm, weak fluorescence at 685 nm, and a strong CD signal. The other is photochemically inactive, with an absorption maximum at 670 nm, strong fluorescence at 679 nm, and much weaker CD. The relative concentrations of the two forms determine the overall spectra of the D1-D2-cyt b559 preparation and can be deduced from the wavelength of the lowest energy absorption band: preparations having maximum absorption at 674, 672, or 670.5 nm have approximately 20, 60, or 85% inactive complexes. The active form contains two chlorophylls with maximum absorption at 679 nm and CD signals at 679 (+) and 669 nm (-). These chlorophylls make a special pair that is identified as the primary electron donor P-680. The calculated separation between the centers of these two pigments (using an extended version of the exciton theory) is about 10 A, the pigments' molecular planes are tilted by about 20 degrees, and their N1-N3 axes are rotated by 150 degrees relative to each other. The other two chlorophylls and one of the two pheophytins in the D1-D2-cyt b559 complex have their maximum absorption at 672 nm, while the maximum absorption of the photochemically active pheophytin is probably at 672-676 nm. During incubation with Triton X-100, the photochemically active complex is transformed into an inactive form with first-order kinetics. In the inactive form the maximum absorption of the 679 nm absorbing Chls is blue-shifted to 669 nm. The first-order decay of the photochemical activity suggests that the isolated D1-D2-cyt b559 complex is stable as an aggregate but becomes unstable on dissociation into individual D1-D2-cyt b559 units.     

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

(i) The method of preparing the oligomycin-insensitive F₁-ATPase by chloroform treatment of mitochondrial membranes (Beechey et al., 1975, Biochem. J.148, 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p-aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, (ii) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F₁-F₀ ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F₁ sector. The complex retains oligomycin- and uncoupler-sensitive ATP-³²P_i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. (iii) F₁-ATPase is released from the F₁-F₀ complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F₁ sector can be isolated containing either four or five subunits depending on whether the starting F₁-F₀ complex contained the 42,000 and 29,000 molecular weight polypeptides. (iv) Sensitivity of the F₁-F₀ ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.     

The Rhodospirillum rubrum cytochrome bc1 complex: peptide composition, prosthetic group content and quinone binding

A cytochrome bc1 complex, essentially free of bacteriochlorophyll, has been purified from the photosynthetic purple non-sulfur bacterium Rhodospirillum rubrum. The complex catalyzes electron flow from quinol to cytochrome c (turnover number = 75 s-1) that is inhibited by low concentrations of antimycin A and myxothiazol. The complex contains only three peptide subunits: cytochrome b (Mr = 35,000); cytochrome c1 (Mr = 31,000) and the Rieske iron-sulfur protein (Mr = 22,400). Em values (pH 7.4) were measured for cytochrome c1 (+320 mV) and the two hemes of cytochrome b (-33 and -90 mV). Electron flow from quinol to cytochrome c is inhibited when the complex is pre-illuminated in the presence of a ubiquinone photoaffinity analog (azido-Q). During illumination, the azido-Q becomes covalently attached to the cytochrome b peptide and, to a lesser extent, to cytochrome c1.     

New guinea salt fern (Asplenium acrobryum complex): Identity,distribution, and chemical composition of its salt

Fronds of a fern of theAsplenium acrobryum complex were traditionally used as a source of salt in the inland areas of Papua New Guinea. All previously published reports of the use of fern salt in the botanical and ethnobotanical literature had erroneously identified the species involved asAsplenium nidus, one of the common bird’s-nest ferns. A chemical analysis of the salts contained in the ash of the salt fern as well as those species related toAsplenium nidus revealed that the bulk of the inorganic component was potassium, calcium and chloride. These results are comparable to those published for the salt-yielding grass,Coix gigantean. There is no obvious chemical reason why onlyAsplenium acrobryum should be used for salt production in preference toAsplenium nidus and related species. In view of the low levels of consumption of such salt it is unlikely that there will be any cases of potassium toxicity attributable to the use of these plants.     

Subunit composition of the membrane glycoprotein complex of Semliki Forest virus

The quaternary structure of the membrane glycoproteins E1, E2 and E3 of Semliki Forest virus has been determined in intact virus and in the protein complexes obtained after Triton X100 solubilization. Intact and solubilized virus were treated with a cleavable cross-linking reagent and the covalently cross-linked glycoprotein complexes were isolated and characterized using antibodies specific for the E1 and E2 membrane glycoproteins. The isolation and characterization procedure was done in a low sodium dodecyl sulphate concentration which prevented non-covalent association between glycoprotein species, but did not abolish antigen-antibody binding.The major glycoprotein complex seen after cross-linking of either intact or Triton X100 solubilized virus was an approximately 100,000 molecular weight species composed of E1-E2 heterodimers only. These findings show that E1 and E2 form a complex in the virus and that this complex is retained after solubilization with Triton X100. The smallest membrane glycoprotein E3 was not cross-linked to the other proteins and was therefore lost in the isolation procedure. However, the presence of E3 together with E1 and E2 in complexes obtained after Triton X100 solubilization of intact virus suggests that an E1-E2-E3 trimer is present in the virus. It is likely that this trimer forms the spike-like structures seen on the surface of the virus.We have observed that antibody specific for one component of the virus glycoprotein complex can induce rearrangement of uncross-linked complexes in Triton X100 solubilized form. This fact should be considered when using specific antibody for characterization of protein complexes.