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At least 163 records · Page 9Linked to original sources
PubMed2005

Intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle.

Herpes virions are complex particles that consist of more than 30 different virally encoded proteins. The molecular basis of how this complicated structure is assembled is only recently beginning to emerge. After replication in the host cell nucleus viral DNA is incorporated into preformed capsids which leave the nucleus by budding at the inner nuclear membrane resulting in the formation of primary enveloped virions in the perinuclear space. The primary envelope then fuses with the outer leaflet of the nuclear membrane, thereby releasing nucleocapsids into the cytoplasm. Final envelopment including the acquisition of more than 15 tegument and more than 10 envelope (glyco)proteins occurs by budding into Golgi-derived vesicles. Mature virions are released after fusion of the vesicle membrane with the plasma membrane of the cell. Thus, herpesvirus morphogenesis requires a sequence of envelopment--de-envelopment--re-envelopment processes which are distinct not only in the subcellular compartments in which they occur but also in the viral proteins involved. This review summarizes recent advances in our understanding of the complex protein-protein interactions involved in herpesvirus assembly and egress.

Animals↗
PubMed1995

Inositol hexakisphosphate binds to clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro.

We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP6). The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180. This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles. The isolated protein-bound InsP6 with a dissociation constant of 1.2 microM and a stoichiometry of 0.9 mol of InsP6 bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP6 with a similar affinity. InsP6 inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages. We speculate that InsP6 and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

Assembly, remodeling, and histone binding capabilities of yeast nucleosome assembly protein 1.

Recombinant yeast nucleosome assembly protein (yNAP-1) facilitates the formation of uniformly spaced nucleosomes from high molecular weight DNA and core histone octamers. No additional factors or metabolites are required. The repeat length of the chromatin produced is about 146 base pairs. To obtain the most distinct nucleosomal ladders, the core histones must preexist as an octamer complex. yNAP-1 forms complexes with core histones as judged by native gel electrophoresis, chemical cross-linking, limited histone proteolysis, and affinity blotting. A discrete complex was observed with a probable ratio of yNAP-1 to histone octamer of 4:1. Chromatin produced by salt dialysis does not contain uniformly spaced nucleosomes, but subsequent incubation with yNAP-1 creates uniform spacing. Trypsin-treated core octamers that lack amino termini, although capable of forming core particles with core-length DNA by salt dialysis, are not assembled by yNAP-1 into uniformly spaced nucleosomes on high molecular weight DNA. Proteolytic removal of the amino termini of the core histones precludes complex formation between a histone octamer and yNAP-1. Affinity blotting also demonstrates that yNAP-1 binds linker histones and high mobility group (HMG)-1/HMG-2 but not HMG-14. Competition experiments with poly-L-arginine, poly-L-lysine, and protamine reveal that yNAP-1 binds to core and linker histones more tightly despite the much higher positive charge densities of the former molecules. Naturally occurring acetylated histone H4 species show no evidence for differential yNAP-1 binding. yNAP-1 is not bound tightly to the resulting chromatin after deposition and thus could act catalytically.

Cell Cycle Proteins↗
PubMed2005

Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly.

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.

Amino Acid Sequence↗
PubMed1992

Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.

A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.

Animals↗
PubMed2002

Synthesis and self-assembly of 2,9,16-tri(tert-butyl)-23-(10-mercaptodecyloxy)phthalocyanine and the application of its self-assembled monolayers in organic light-emitting diodes.

2,9,16-Tri(tert-butyl)-23-(10-mercaptodecyloxy)phthalocyanine (8) and its disulfide (9) have been synthesized and characterized, and their self-assembling behaviors on gold substrates have been studied. Characteristic Q-bands were observed at about 630 nm in the UV/visible spectra of the self-assembling monolayers (SAMs). They were broadened and blue-shifted relative to those observed in solution. Binding energies for S2p have the same values (161.70 eV) and are in accord with those for gold thiolates. The application of the SAMs in organic light-emitting diode was investigated. It shows that the SAM promotes the hole injection process from the anode.

Journal Article↗
PubMed1984

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly.

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.

Animals↗
PubMed1983

Synthesis and assembly of spectrin during avian erythropoiesis: stoichiometric assembly but unequal synthesis of alpha and beta spectrin.

The synthesis and assembly of spectrin was investigated in erythroid cells during chicken embryo development. Immunoprecipitation of Triton X-100-soluble and -insoluble cytoskeletal fractions with alpha- and beta-spectrin antisera show that, at steady state, alpha and beta spectrin are present in stoichiometric amounts, and exclusively, in the cytoskeleton. However, pulse labeling of cells and in vitro translation of total erythroid cell RNA reveal that alpha spectrin is synthesized in a two to three fold excess over beta spectrin. Pulse-chase experiments show that newly synthesized alpha and beta spectrin are present in both the cytoskeletal and soluble fractions, and that stoichiometric amounts are stably assembled in the cytoskeleton. On the other hand, there is a severalfold excess of alpha relative to beta spectrin in the soluble fraction, both of which turn over with a half-life of 50 min. In cells from 4 day old embryos, more than 80% of the newly synthesized beta spectrin, but only 10% of the alpha spectrin, are present in the cytoskeleton. Thus, early in development, the association of alpha and beta spectrin with the membrane-cytoskeleton may be rate-limited by the amount of beta spectrin synthesized. Later on in erythroid development, progressively lesser proportions of newly synthesized beta spectrin are present in the cytoskeleton, suggesting that during development, the rate of association of beta spectrin with the membrane-cytoskeleton becomes limited by some other membrane-cytoskeletal component.

Animals↗
PubMed1995

Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase.

We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms.

Amino Acid Sequence↗
PubMed2006

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly.

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.

Bacillus subtilis↗
PubMed1998

The assembly of yeast mitochondrial ATP synthase: subunit depletion in vivo suggests ordered assembly of the stalk subunits b, OSCP and d.

The abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy. Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex. A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region. Thus, subunit b is assembled first, followed by OSCP and then by subunit d.

Adenosine Triphosphatases↗
PubMed1997

A novel fluorescent marker for assembled mitochondria ATP synthase of yeast. OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo.

We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus. Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).

Adenosine Triphosphatases↗
PubMed1997

The 'assembly-promoting sequence region' of microtubule-associated protein 4 failed to promote microtubule assembly.

In order to study the function of the bovine MAP4 microtubule-binding domain (the assembly-promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system. When the fragment was mixed with purified tubulin at 37 degrees C, the fragment caused a time- and dose-dependent turbidity increase, and the fragment bound to tubulin. However, the products were cold-stable, and amorphous aggregates were observed by electron microscopy. Using axonemes as the seeds for microtubule assembly, the microtubule-elongating activity of the fragment was examined. A dose-dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose-dependently elongated from the axonemes. Consequently, the AP sequence region does not nucleate microtubules, but elongates them.

Adrenal Cortex↗
PubMed1994

Assembly of nucleosomes: do multiple assembly factors mean multiple mechanisms?

In eukaryotic cells, transcription and DNA replication occur on DNA templates associated with chromatin proteins, most notably histone octamers. Protein factors that can assemble these units have been isolated from many sources. In particular, one factor from human cells is associated with ongoing DNA synthesis; other known assembly factors are not obligately coupled to the replication process. The wide variety of histone chaperones suggests that multiple pathways for the remodeling of chromatin structure have evolved.

Animals↗
PubMed1991

Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1.

We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits.

Biological Transport↗
PubMed1998

Reversible and irreversible steps in assembly and disassembly of vesicular stomatitis virus: equilibria and kinetics of dissociation of nucleocapsid-M protein complexes assembled in vivo.

The matrix (M) protein of vesicular stomatitis virus (VSV) condenses the viral nucleoprotein core (nucleocapsid) into a tightly coiled, helical nucleocapsid-M protein (NCM) complex. Using NCM complexes assembled in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein binding to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in their M proteins were analyzed using sedimentation and light-scattering assays. At physiological ionic strength, the binding reaction had the characteristics of a dynamic reversible equilibrium. A temperature-sensitive M protein mutant lost the ability of M protein to reversibly dissociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast to the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaCl) were not able to bind M protein at low ionic strength with the same high affinity seen in NCM complexes assembled in vivo. The effect of incubation at 250 mM NaCl was shown to be due to a change in nucleocapsids rather than a change in soluble M protein. This result supports the idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.

Flow Injection Analysis↗
PubMed2006

Stepwise self-assembly of ordered supramolecular assemblies based on coordination chemistry.

Ultrathin multilayers based on transition metal complexes have been prepared by successive deposition and self-assembly. Dendrimer layers were deposited onto SiO2 wafers by alternately immersing the substrate into a solution of terpyridyl (tpy)-pendant poly(amido amine) (PAMAM) dendrimers (dend-n-tpy; n = 8, 16) dissolved in CH2Cl2, followed by the interfacial coordination reaction with cobalt (Co2+) from aqueous solution. The films derived from this simple assembly method have been characterized by electrochemical methods, synchrotron-based X-ray reflectivity (XRR), and X-ray fluorescence (XRF) recorded under grazing incidence. XRR analysis revealed a linear thickness dependence with an increase of 9.9 +/- 0.5 angstroms and 10.8 +/- 0.4 angstroms per growth cycle for both dend-8-tpy and dend-16-tpy, respectively, indicative of a layer-by-layer (LbL) growth of single dendrimer/Co2+ layers. XRF and electrochemical results showed that the amount of Co2+ increases linearly as more layers are deposited, and that the Co2+ concentration (mol/L) in dend-8-tpy/Co2+ films decays slowly as the number of growth cycles (l) increases. Moreover, a preliminary kinetics analysis indicated that the growth of a dendrimer layer in a deposition cycle is a self-limiting process.

Dendrimers↗