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PubMed2001

In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines.

Core assembly, a key step in the retroviral life cycle, is poorly understood. Previous studies have shown that the entire gag region is needed to form the assembled particles. In this report, we have shown that the assembly process is driven by recombinant capsid protein (p26) of feline immunodeficiency virus itself. Proteins are expressed in a bacterial system and soluble forms of wild-type and modified proteins are purified from bacterial extracts and are examined on gel-filtration chromatography fitted to an HPLC system. It has also been shown that changing residue Cys190 (one of the two conserved cysteines of feline immunodeficiency virus which are also conserved for all the immunodeficiency viruses including HIV) to serine by site-directed mutagenesis disrupts the assembly process. In addition, this modification causes considerable thermal instability of the protein while substitutions at nonconserved cysteines do not significantly affect the thermal stability and assembly of the protein. These findings indicate that conserved cysteine residues play a vital role in the capsid protein assembly and, therefore, are critical for virus infectivity.

Amino Acid Sequence↗
PubMed1995

Annexin II2-p11(2) (calpactin I) stimulates the assembly of GFAP in a calcium- and pH-dependent manner.

Annexin II2-p11(2) (calpactin I) was tested as a potential regulator of GFAP assembly into glial filaments (GF), following the observation that it interacts with GFAP and cosediments with GF in a sedimentation assay. Under conditions where GFAP assembly is reduced, e.g., at pH values > 6.8, annexin II2-p11(2) stimulates GF formation in a Ca(2+)- and dose-dependent manner. Concomitantly, an ever larger fraction of annexin II2-p11(2) can be recovered in GF pellets as the pH is raised from 6.8 to 7.35. Monomeric annexin II also stimulates GFAP assembly, although with a smaller efficacy as compared to annexin II2-p11(2), but does not cosediment with GF to a large extent, whereas p11 neither cosediments with GF nor affects GFAP assembly. On the other hand, the in vitro reconstituted annexin II2-p11(2) heterotetramer mimics native annexin II2-p11(2), and perturbation of the integrity of annexin II2-p11(2) by a mild treatment with alpha-chymotrypsin results in the nearly complete abolition of the stimulatory effect of annexin II2-p11(2) on GFAP assembly. These data suggest that annexin II2-p11(2) might be involved in the regulation of the state of assembly of GF, possibly in concert with other proteins.

Animals↗
PubMed1998

Differential assembly kinetics of alpha-tubulin isoforms in the presence of paclitaxel.

The antitumor drug paclitaxel (PTX) inhibits cell growth by binding to microtubules, the eukaryotic structures consisting of alpha- and beta-tubulin. PTX also promotes the assembly to tubulin in the absence of microtubule-associated proteins. Although recent studies have implicated beta-tubulin as the site of PTX binding, no information is available that relates alpha-tubulin to the binding site. In an effort to understand whether the alpha-tubulin is involved in the drug binding, we have studied the assembly of alpha-tubulin isoforms in the presence of PTX. The assembly results in the presence of 10 microM paclitaxel (PTX) show that the isoforms assemble at differential rates. The rate of assembly for tyrosinated M alpha 1/2 is about three-fold higher than that of the nontyrosinated M alpha 1/2 isoform. Such a strikingly different assembly behavior of the alpha-tubulin isoforms indicates that alpha-tubulin may be involved in the interaction of PTX with microtubules.

Amino Acid Sequence↗
PubMed1993

Ultrastructure of developing flight muscle in Drosophila. I. Assembly of myofibrils.

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion approximately 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from approximately 1.7 to approximately 3.2 microns, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from approximately 4-thick filaments to approximately 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.

Animals↗
PubMed1993

Network incorporation of intermediate filament molecules differs between preexisting and newly assembling filaments.

When studying the way in which intermediate filaments assemble in vivo, it is important to distinguish between the incorporation of intermediate filament proteins into an existing intermediate filament network and the ability to form a new network within cells. To distinguish between these alternatives, we have made a hybrid construct consisting of the rod and tail domains of murine glial fibrillary acidic protein (GFAP) coupled to the head domain of bovine keratin 19, called K19GFAP. The assembly characteristics of K19GFAP were analyzed in vitro and in vivo. Replacement of the head domain with the bovine K19 sequence did not prevent the incorporation of K19GFAP into the existing network of vimentin intermediate filaments in NIH 3T3 cells but it was incompatible with de novo formation of filament networks in the epithelial cell line MCF-7, which lacks an endogenous vimentin network. By in vitro assembly studies, it was confirmed that K19GFAP was unable to assemble into typical intermediate filaments. We also investigated the ability of an appropriate type II keratin partner to rescue K19GFAP from incorporation into a vimentin network and initiate de novo filament assembly, using the fibroblast cell line KF-K8(3), an NIH 3T3 fibroblast cell line expressing a single human keratin, K8. The results confirm the importance of the coiled coil interactions in determining the fate of intermediate filament proteins. The results also emphasize that filament networks can not only tolerate but also incorporate assembly-deficient intermediate filament protein subunits.

3T3 Cells↗
PubMed1995

Mechanical properties of the extracellular matrix influence fibronectin fibril assembly in vitro.

Mechanical properties of the extracellular matrix (ECM) are proposed to influence cell behavior and biological activity. The influence of the mechanical environment on fibronectin fibril assembly was evaluated. Fibroblasts were cultured in hydrated collagen gels with two distinctly different mechanical properties. Cells cultured within a stabilized collagen gel generate stress that is transmitted throughout the matrix (stressed gel). In contrast, cells that are cultured within a collagen gel that is floating freely in media do not generate stress (relaxed gel). Fibroblasts in the stressed collagen gel develop large bundles of actin microfilaments and associated fibronectin fibrils, while fibroblasts within relaxed gels do not form stress fibers or assemble fibronectin into fibrils. In addition, we have evaluated the mechanism of fibronectin fibril assembly employed by fibroblasts cultured within a stressed three-dimensional collagen matrix and the role of fibronectin fibrils in transmission of cell-generated forces to the surrounding matrix. Fibronectin fragments (70-kDa amino terminal fragment, 110-kDa cell-adhesive fragment, and GRGDS peptide) and a monoclonal antibody body blocked fibronectin fibril assembly in stressed three-dimensional collagen gels. These results suggest that the features of fibronectin required for fibronectin fibril assembly by cells in collagen gels is similar to those required by cells cultured on a planar substratum. Although fibronectin fibril assembly was blocked by these inhibiting fragments and antibody, the cells displayed prominent actin bundles and developed isometric tension, indicating that stress fiber formation and contractile force transmission is not dependent on the presence of fibronectin fibrils.

Actins↗
PubMed1994

Functional map of the alpha subunit of Escherichia coli RNA polymerase. Deletion analysis of the amino-terminal assembly domain.

The alpha subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme. The amino-terminal two-thirds of the alpha subunit, as far as position 235, are involved in this assembly. To define the site(s) within this region required for core enzyme assembly, we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene. The overexpressed alpha derivatives were purified to apparent homogeneity and examined for their abilities to assemble beta and beta' subunits into active core enzymes in vitro. Among a total of 22 alpha derivatives tested, only four mutants retained the activity form active core enzyme. These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30. The minimum fragment with full activity of the core assembly was alpha(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues. Most of the other mutants appeared to be defective in the formation of stable alpha dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates. These results, taken together, suggest that the amino-terminal region of the alpha subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation. Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacP1 promoter or by OmpR at the ompC promoter.

Chromatography, Gel↗
PubMed1995

Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain.

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.

Amino Acid Sequence↗
PubMed1997

Quasi-equivalent viruses: a paradigm for protein assemblies.

The structure and assembly of icosahedral virus capsids composed of one or more gene products and displaying quasi-equivalent subunit associations are discussed at three levels. The principles of quasi-equivalence and the related geodesic dome formation are first discussed conceptually and the geometric basis for their construction from two-dimensional assembly units is reviewed. The consequences for such an assembly when three-dimensional protein subunits are the associating components are then discussed with the coordinates of cowpea chlorotic mottle virus (CCMV) used to generate hypothetical structures in approximate agreement with the conceptual models presented in the first section. Biophysical, molecular genetic, and atomic structural data for CCMV are then reviewed, related to each other, and incorporated into an assembly model for CCMV that is discussed with respect to the modular, chemical nature of the viral subunit structure. The concepts of quasi-equivalence are then examined in some larger virus structures containing multiple subunit types and auxiliary proteins and the need for additional control points in their assembly are considered. The conclusion suggests that some viral assembly principles are limited paradigms for protein associations occurring in the broader range of cell biology including signal transduction, interaction of transcription factors and protein trafficking.

Bromovirus↗
PubMed1998

Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA.

The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one primary binding protein associated with each RNA domain and additional proteins assembled to domains I, II, V and VI. For all the combinations of two to five domains, enhanced assembly yields and/or new proteins were observed primarily to those transcripts containing either domains I+II or domains V+VI. This indicates that there are two major protein assembly centres located at the ends of the 23 S rRNA, which is consistent with an earlier view that in vitro protein assembly nucleates around proteins L24 and L3. Although similar protein assembly patterns were observed over a range of temperature and magnesium concentrations, protein L2 assembled strongly with domains II and IV at 4-8 mM Mg2+ (the first step of the two-step reconstitution procedure) and with domain IV alone at higher Mg2+ concentrations (the second step). It is proposed that this change in protein-RNA binding provides a basis for the two-step reconstitution in vitro. A chemical footprinting approach was employed on the reconstituted protein-domain complexes to localize a putative L4 binding region within domain I to a region that is partially co-structural with the site on the L4-mRNA where L4 binds and inhibits its own translation. A similar approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex.

Base Sequence↗
PubMed1999

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Amino Acid Sequence↗
PubMed1999

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.

Bacterial Proteins↗
PubMed1999

Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein.

The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells.

Capsid↗
PubMedDate not supplied

Characterization of early assembly intermediates of recombinant human keratins.

The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular, the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., "unit-length filaments" (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the "heads" are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins K8/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long, regular filaments with surprising efficiency in vitro.

Base Sequence↗
PubMed2002

Kinetic model of BiP- and PDI-mediated protein folding and assembly.

A mechanism for heavy chain binding protein (BiP)- and protein disulfide isomerase (PDI)- mediated protein folding and assembly has been proposed. It considers BiP chaperoning action and PDI catalytic activity. A kinetic model has been developed based on the proposed mechanism. The model was used for quantifying the influence of polypeptide concentration and ratio, and the effect of BiP and PDI concentration on the kinetics of folding and assembly. An optimum value for polypeptide concentration that minimizes assembly times was found, and different kinetic behaviors were identified for polypeptide concentrations higher or lower than the optimum. Pulse-chase experiments and the dependence of assembly time on unassembled polypeptides ratio predicted by the model are similar to those found during in vitro and in vivo folding and assembly of antibodies and human chorionic gonadotropin (hCG), as well as bovine pancreatic trypsin inhibitor (BPTI) folding. The model also explains the increase in folding and assembly rates during overexpression of BiP and PDI.

Animals↗
PubMed1998

Structural and functional analysis of chromatin assembled from defined histones.

In this review we describe how the extract-mediated chromatin assembly system derived from preblastoderm Drosophila embryos can be modified to assemble chromatin from defined histones. This approach combines the advantages of assembling (i) chromatin templates from homogeneous histones with (ii) an assembly system that generates chromatin with physiological nucleosome spacing and density and that contains the biological complexity of in vivo chromatin. We have used this technique to assemble nonacetylated and hyperacetylated histones into chromatin (K. P. Nightingale, R. Wellinger, J. Sogo, and P. B. Becker, 1998, EMBO J. 17, 2865-2876; W. A. Krajewski and P. B. Becker, 1998, Proc. Natl. Acad. Sci. USA 95, 1540-1545), and use this as an example to detail the structural and transcriptional assays used to compare and characterize these chromatin templates. The application of this procedure to assemble chromatin from recombinant histones should facilitate a wide variety of studies on the role(s) of histone mutants and variants.

Animals↗
PubMed1998

SR protein and snRNP requirements for assembly of the Rous sarcoma virus negative regulator of splicing complex in vitro.

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.

Animals↗
PubMed2000

Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2.

Animals↗