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PubMed2001

A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly.

Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.

Base Sequence↗
PubMed2006

Sequence requirements for localization of human cytomegalovirus tegument protein pp28 to the virus assembly compartment and for assembly of infectious virus.

The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.

Animals↗
PubMed2004

Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis.

In eukaryotic cells, chromatin serves as the physiological template for gene transcription, DNA replication, and repair. Chromatin assembly factor 1 (CAF-1) is the prime candidate protein to mediate assembly of newly replicated DNA into chromatin. To investigate the physiological role of CAF-1 in vivo, we used RNA interference (RNAi) to silence the 60-kDa subunit of CAF-1 (p60) in human cells. Transfection of a small interfering RNA (siRNA) directed against p60 resulted in efficient silencing of p60 expression within 24 h. This silencing led to an induction of programmed cell death in proliferating but not in quiescent human cells. Concomitantly, proliferating cells lacking p60 accumulated DNA double-strand breaks and increased levels of the phosphorylated histone H2A.X. Nuclear extracts from cells lacking p60 exhibited a 10-fold reduction of nucleosome assembly activity during DNA synthesis, which was restored upon addition of recombinant p60 protein. Nascent chromatin in cell nuclei lacking p60 showed significantly increased nuclease sensitivity, indicating chromatin assembly defects during DNA synthesis in vivo. Collectively, these data identify CAF-1 as an essential factor not only for S-phase-specific chromatin assembly but also for proliferating cell viability.

Animals↗
PubMed1988

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA.

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.

Adenosine Triphosphate↗
PubMed1996

Polymorphism of F-actin assembly. 2. Effects of barbed end capping on F-actin assembly.

In the accompanying paper [Suzuki, A., Yamazaki, M., & Ito, T. (1996) Biochemistry 35, 5238-5244], we presented a quantitative phase diagram of actin filament ( (F-actin) described with the F-actin concentration and delta chi value which characterizes the affinity of F-actin with solvent. The phase diagram shows that F-actin changes its assembly structure from an isotropic disordered distribution to a dilute ordered assembly of a lyotropic crystalline with an increase in the concentration an to a concentrated ordered assembly of a crystalline-like bundle with an increase in the delta chi value (i.e., with a decrease in the affinity with the solvent), respectively, in the physiological concentration range. We report here that capping the barbed end of F-actin significantly affects the phase diagram. The F-actin capped by gelsolin (capped F-actin) decreased the delta chi value required for the formation of the concentrated ordered assembly. The time taken for the decrease in the delta chi value to reach a stationary state after the barbed end capping was proportional to the filament length (approximately 1 h/microm length). the electron microscopic morphology of the concentrated ordered assembly of the capped F-actin was a wide and loose bundle, which was distinctly different from the crystalline-like bundle of the uncapped F-actin. Fragmin from the acellular slime mould, which has similar functions to gelsolin, showed the same effects. These results suggest that the barbed end capping of F-actin gradually changes the nature of the whole filament so as to make the interaction with the solvent more unstable, and the F-actin loses the ability to make a crystalline-like bundle.

Actins↗
PubMed2004

Spontaneous assembly of organic thiocyanates on gold sufaces. Alternative precursors for gold thiolate assemblies.

Thiolate self-assembly on gold has proven to be a valuable technique for assembling monolayers on a wide variety of substrates. However, the oxidative instability of the thiols, especially aromatic thiols and alpha,omega-dithiols, presents several difficulties. Shown here is that thiocyanates, easily synthesized stable thiol derivatives, can be directly assembled on gold surfaces with no auxiliary reagents required. Assembly is complete in 24 h and leaves a similar gold thiolate structure as seen in typical thiol self-assembled monolayers.

Journal Article↗
PubMed2006

Directed assembly and separation of self-assembled monolayers via electrochemical processing.

Separated domains of 1-dodecanethiolate were fabricated via solution displacement of preformed 1-adamantanethiolate self-assembled monolayers on Au{111}. Subsequently, the 1-adamantanethiolate domains were desorbed selectively, and the substrate was exposed to a 1-octanethiol solution, creating artificially separated self-assembled monolayers of 1-dodecanethiolate and 1-octanethiolate. The molecular order of each lattice type and the apparent height differences imaged with scanning tunneling microscopy and the two distinct cathodic peaks observed with cyclic voltammetry indicated distinct separated domains of each lattice type in the separated self-assembled monolayers. By manipulating the intermolecular interaction strengths of the patterned molecules, we are able to control the structure and properties of the separated self-assembled monolayers via the exploitation of competitive adsorption and the utilization of electrochemical processing, which can be extended to other self-assembly patterning techniques such as microdisplacement printing.

Adamantane↗
PubMed1970

Structure and function of bacterial ribosomes. XI. Dependence of 50S ribosomal assembly on simultaneous assembly of 30S subunits.

Some spectinomycin-resistant mutants of Escherichia coli are cold-sensitive. They are unable to assemble both 30S and 50S ribosomal subunits at low temperatures. They accumulate two kinds of incomplete particles, related to 30S and 50S subunits respectively. A single mutation, causing an alteration in a 30S ribosomal component, is responsible for these phenotypes. These results show that assembly of 50S subunits in vivo is dependent on simultaneous assembly of 30S subunits. On the other hand, the assembly of 30S subunits appears to be independent of 50S assembly.

Bacterial Proteins↗
PubMed1998

The role of the alpha3(VI) chain in collagen VI assembly. Expression of an alpha3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an alpha3(VI)-deficient cell line.

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

Ascorbic Acid↗
PubMed2005

Assembly of cytochrome-c oxidase in the absence of assembly protein Surf1p leads to loss of the active site heme.

Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the alpha-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa(3)-type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa(3)-type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a(3)-Cu active sites. 50% of the oxidase lacked heme a(3), 10-15% contained heme a(3) but lacked Cu(BB), and 35-40% had a normal heme a(3) -Cu(B) active site with normal activity. Cu(A) assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a(3). Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a(3) into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a(3)-Cu(B) center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.

Binding Sites↗
PubMed2004

Glycoprotein hormone assembly in the endoplasmic reticulum: III. The seatbelt and its latch site determine the assembly pathway.

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their beta-subunits known as the "seatbelt" that is wrapped around loop 2 of their alpha-subunits (alpha2). The cysteine that terminates the seatbelt is "latched" by a disulfide to a cysteine in beta-subunit loop 1 (beta1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the beta-subunit NH(2) terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop alpha2 and latching it to the NH(2) terminus. This differs from hCG assembly, which occurs by threading the glycosylated end of loop alpha2 beneath the latched seatbelt through a hole in the beta-subunit. The seatbelt is the part of the beta-subunit that has the greatest influence on biological function. Changes in its sequence during the divergence of lutropins, follitropins, and thyrotropins and the speciation of teleost fish may have impeded heterodimer assembly by a threading mechanism, as observed when the hCG seatbelt was replaced with its salmon FSH counterpart. Whereas wrapping is less efficient than threading, it may have facilitated natural experimentation with the composition of the seatbelt during the co-evolution of glycoprotein hormones and their receptors. Migration of the seatbelt latch site to the NH(2)-terminal end of the beta-subunit would have facilitated teFSH assembly by a wraparound mechanism and may have contributed also to its ability to distinguish lutropin and follitropin receptors.

Amino Acid Sequence↗
PubMed2004

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly.

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of alpha-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a "zipper-like" sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the beta-subunit cystine knot, alpha-subunit loop 2, and the small seatbelt loop. The resulting intersubunit beta-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.

Amino Acid Sequence↗
PubMed1989

The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

Acanthamoeba↗
PubMed1997

Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

Actins↗
PubMed1971

Synthesis and assembly of immunoglobulins by malignant human plasmacytes and lymphocytes. II. Heterogeneity of assembly in cells producing IgM proteins.

Bone marrow or lymph node cells from 10 patients whose sera contained large amounts of monoclonal IgM proteins were incubated with radioactive amino acids in short-term tissue culture. Samples of soluble cytoplasmic extracts and secreted material were examined by immunologic precipitation with specific antisera, acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and sucrose gradient centrifugation. In all samples studied, 8S IgM was the major intracellular precursor of the fully assembled 19S protein. Cells obtained from some patients contained little or no fully assembled 19S protein intracellularly; however, cells from most patients seemed to accumulate fully assembled 19S molecules intracellularly before secretion. Secreted material from both groups contained large amounts of 19S IgM. The differentiation between accumulating and nonaccumulating cells did not correlate with heavy or light chain antigenic type. Synthesis and assembly appeared to be identical in cells obtained from different anatomic sites in the same patient Studies carried out in one patient before and after therapy revealed no qualitative differences in the pathway of assembly and reflected only a decrease in the total number of immunoglobulin-producing cells.

Blood Protein Electrophoresis↗
PubMed1996

Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position-independent functions in assembly but not in gene regulation.

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.

Base Sequence↗
PubMed2004

Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs.

We present an EST sequence assembler that specializes in reconstruction of pristine mRNA transcripts, while at the same time detecting and classifying single nucleotide polymorphisms (SNPs) occuring in different variations thereof. The assembler uses iterative multipass strategies centered on high-confidence regions within sequences and has a fallback strategy for using low-confidence regions when needed. It features special functions to assemble high numbers of highly similar sequences without prior masking, an automatic editor that edits and analyzes alignments by inspecting the underlying traces, and detection and classification of sequence properties like SNPs with a high specificity and a sensitivity down to one mutation per sequence. In addition, it includes possibilities to use incorrectly preprocessed sequences, routines to make use of additional sequencing information such as base-error probabilities, template insert sizes, strain information, etc., and functions to detect and resolve possible misassemblies. The assembler is routinely used for such various tasks as mutation detection in different cell types, similarity analysis of transcripts between organisms, and pristine assembly of sequences from various sources for oligo design in clinical microarray experiments.

Animals↗
PubMed1977

Microtubule assembly in vitro. Purification of assembly-promoting factors.

The role of microtubule-associated proteins in the assembly of tubulin to microtubules in vitro has been studied. 1. It has been confirmed that pure tubulin obtained by phosphocellulose column chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2+ concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure. 2. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations two protein fractions were purified, either by Ultrogel ACA34 chromatography or by sucrose gradient ultracentrifugation. The first one, with a high molecular weight, did not promote tubulin assembly; ageing of this material did not induce any activity. On the other hand, the second fraction, with an apparent molecular weight of 70 000 (tau protein), when almost completely purified, was active in promoting assembly. Thus a single specific protein is able to promote assembly of pure tubulin.

Animals↗