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PubMed1999

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.

Binding Sites↗
PubMed1988

Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates.

We have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, we demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that our observations are not the result of slow displacement of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

Chromatin↗
PubMed2004

Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly.

The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Assembly regulatory domain of glial fibrillary acidic protein. A single phosphorylation diminishes its assembly-accelerating property.

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.

Amino Acid Sequence↗
PubMed1987

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities.

A 50-kDa polypeptide that is rapidly phosphorylated on addition of [gamma-32P]ATP to isolated clathrin-coated vesicles is shown here to be identical to the 50-kDa component (AP50) of the clathrin assembly protein (AP), a complex that promotes the assembly of clathrin coat structures under physiological conditions of pH and ionic strength. Phosphorylation of the AP50 occurred readily at 0 degrees C, almost exclusively on a threonyl residue(s). This reaction is attributable to autophosphorylation, since the AP50 was able to covalently incorporate 32P from [gamma-32P]ATP after separation by either one- or two-dimensional sodium dodecyl sulfate gel electrophoresis. Kinetic studies in solution were consistent with an intramolecular phosphorylation event; in addition, a concentration-dependent increase in AP50 phosphorylation was observed that may reflect intermolecular AP-AP activation of autophosphorylation. The phosphorylated AP50 was resistant to several inorganic phosphatases tested but was a substrate for protein phosphatases 1 and 2A, suggesting that a physiological phosphorylation-dephosphorylation cycle may exist. The phosphorylation state of the AP50 did not affect the ability of the AP to promote in vitro clathrin coat assembly. These and other data suggest that unique structural domains of the assembly protein are responsible for assembly (the 100-kDa components) and autophosphorylation (the AP50) and that the latter may be active as a protein kinase in the intact cell.

Adaptor Protein Complex 2↗
PubMed1994

In vitro analysis of microtubule assembly of isotypically pure tubulin dimers. Intrinsic differences in the assembly properties of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers in the absence of microtubule-associated proteins.

Microtubule assembly of different beta tubulin isotypes in the presence of 4 M glycerol and 6 mM magnesium ion demonstrates significantly different characteristics. alpha beta II and alpha beta IV assembled faster and to a greater extent than did unfractionated phosphocellulose-purified tubulin (PC-tubulin). Microtubule assembly from alpha beta III showed a distinctive delay in nucleation, proceeded at a slower rate than those of the other beta tubulin isotypes, and had the highest critical concentration. However, treatment of beta tubulin isotypes with subtilisin to remove the C-terminal domain of the tubulin dimer abolished these differences in microtubule assembly pattern and enhanced self-assembly. The kinetic analysis of microtubule elongation of different beta tubulin isotypes also showed significant differences. Elongation of alpha beta III from microtubule seeds had a lower apparent K alpha and a lower apparent Kd than did alpha beta II and alpha beta IV. The dynamic behaviors of different beta tubulin isotypes were qualitatively similar to each other and fit the dynamic instability model. However, microtubules formed from alpha beta III appeared to be less dynamic than microtubules formed from other beta tubulin isotypes. Our results suggest that the beta III isotype might have a different conformation than do the other beta tubulin isotypes. The distinctive nucleation and elongation behaviors of the alpha beta III dimers demonstrated in vitro may have a significant influence on microtubule functions in vivo.

Animals↗
PubMed1998

Self-assembling automata: a model of conformational self-assembly.

An abstract model of self-assembling systems is presented where assembly instructions are written as conformational switches-local rules that specify conformational changes of a component. The model, the self-assembling automaton, is defined as a sequential rule-based machine that operates on one-dimensional strings of symbols. Classes of self-assembling automata are defined based on classes of subassembly sequences in which the components self-assemble. The minimum number of conformations is provided which are necessary to encode subassembly sequences in the each class. It is shown that three conformations for each component are enough to encode any subassembly sequences of a string with arbitrary length.

Computational Biology↗
PubMed2003

Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo.

De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork. The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation. Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase. Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly. The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect. CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells. These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.

Base Sequence↗
PubMed1999

The pI and pXI assembly proteins serve separate and essential roles in filamentous phage assembly.

Three non-capsid, phage-encoded proteins, pI, pIV and pXI, are required for assembly of the filamentous bacteriophage at the envelope of Escherichia coli. pIV forms the outer membrane component of the assembly site, and pI and pXI are predicted to form the cytoplasmic membrane component. pXI is the result of an in-frame internal translational initiation event in gene I and is identical with the carboxyl-terminal third of pI in amino acid sequence, membrane localization and topology. The two proteins share a cytoplasmic domain predicted to be an amphipathic helix, a transmembrane domain, and a periplasmic domain. By mutating the initiation site for pXI, a phage was made that produced only pI and was shown to absolutely require functional plasmid-encoded pXI for growth. Further mutational analysis was done to examine the functional determinants of the amphipathic helix and periplasmic domains of the pI and pXI proteins. The results show that the amphipathic helix region is very important for pI function but not for pXI function. Mutational analysis of the periplasmic domains of pI and pXI implies that these domains also perform separate functions, and suggests that the interaction between pI and pIV in the periplasm is critical for assembly. The results are discussed with regard to the separate roles that the pI and pXI proteins play in the overall process of phage assembly.

Amino Acid Sequence↗
PubMed2000

Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses.

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.

Amino Acid Motifs↗
PubMed1995

Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3.

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Hierarchical self-assembly of a bow-shaped molecule bearing self-complementary hydrogen bonding sites into extended supramolecular assemblies.

The bow-shaped molecule 1 bearing a self-complementary DAAD-ADDA (D=donor A=acceptor) hydrogen-bonding array generates, in hydrocarbon solvents, highly ordered supramolecular sheet aggregates that subsequently give rise to gels by formation of an entangled network. The process of hierarchical self-assembly of compound 1 was investigated by the concentration and temperature dependence of UV-visible and (1)H NMR spectra, fluorescence spectra, and electron microscopy data. The temperature dependence of the UV-visible spectra indicates a highly cooperative process for the self-assembly of compound 1 in decaline. The electron micrograph of the decaline solution of compound 1 (1.0 mM) revealed supramolecular sheet aggregates forming an entangled network. The selected area electronic diffraction patterns of the supramolecular sheet aggregates were typical for single crystals, indicative of a highly ordered assembly. The results exemplify the generation, by hierarchical self-assembly, of highly organized supramolecular materials presenting novel collective properties at each level of organization.

Journal Article↗
PubMed2007

Inactivation of MAPK affects centrosome assembly, but not actin filament assembly, in mouse oocytes maturing in vitro.

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, gamma-tubulin and nuclear mitotic apparatus protein (NuMA) distribution, and actin filament assembly in mouse oocytes maturing in vitro. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 hr after the initiation of maturation cultures, was fully active at 6 hr, and remained active until metaphase II. Treatment of GV stage oocytes with 20 microM U0126 completely blocked MAPK phosphorylation, but did not affect GV breakdown (GVBD). However, the oocytes did not progress to the Metaphase I stage, which would normally occur after 9 hr in the maturation cultures. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of gamma-tubulin and NuMA, but did not affect actin filament assembly. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.

Actins↗
PubMed1993

Assembly of multimeric proteins. Effect of mutations in the alpha-subunit on membrane assembly and activity of pyridine nucleotide transhydrogenase.

The pyridine nucleotide transhydrogenase of Escherichia coli is an inner membrane protein of two different subunits (alpha and beta). It functions as a proton pump. The highly hydrophilic carboxy-terminal tail of ten amino acid residues in the alpha-subunit determines the correct folding and proper assembly of the beta-subunit leading to a functional enzyme. Premature termination of the alpha-subunit six amino acid residues from the carboxy-terminal end abolishes the activity completely. Although the two subunits are still assembled into the membrane, the conformation of the beta-subunit is perturbed. Systematic truncation and site-directed substitutions revealed that at least one positive charge in the carboxy-terminal region is required for efficient assembly of the two subunits to give a functional enzyme, while a phenylalanine residue, essential for activity, has no apparent effect on the extent of assembly of the two subunits.

Amino Acid Sequence↗
PubMed1986

Assembly analysis of ribosomes from a mutant lacking the assembly-initiator protein L24: lack of L24 induces temperature sensitivity.

Previously, we have shown that the ribosomal protein L24 is one of two assembly-initiator proteins. L24 is essential for early steps of the assembly of the 50S ribosomal subunit but it is not involved in both the late assembly and the ribosomal functions. Surprisingly, an E. coli mutant (TA109-130) exists which lacks L24. This apparent paradox is analyzed and resolved in this paper. The phenotypic is analyzed and resolved in this paper. The phenotypic features of the mutant lacking L24, are a temperature sensitivity (growth severely reduced beyond 34 degrees C), a very low growth rate already at permissive temperatures (at least six-fold slower than wild type) and an underproduction of 50S subunits (molar ratio of 30S to 50S about 1:0.5). The S value of the mutant large subunits is 47S, and they are normally active in poly(Phe) synthesis. The total protein of the mutant large subunits show negligible activity in the total reconstitution assay using the standard two-step procedure. Number analysis of the assembly-initiator proteins revealed that only one initiator protein is effective, as expected. The activity is restored upon addition of wild-type L24. However, when the temperature of the first step is lowered from 44 degrees to 36 degrees C, reconstitution of active particles occurs with a 50% efficiency in the absence of L24. The recovery of activity is accompanied by the appearance of again two initiator proteins, when the mutant TP50 lacking L24 is used in the reconstitution assay at the 'permissive' temperature of 36 degrees C during the first step.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed2005

Organization of assembly factors Cbp3p and Cbp4p and their effect on bc(1) complex assembly in Saccharomyces cerevisiae.

The bc(1) complex (complex III) of Saccharomyces cerevisae is composed of ten subunits that are assembled in the inner mitochondrial membrane. Cbp3p and Cbp4p are two mitochondrial proteins which are postulated to act as chaperones in bc(1) complex formation. Here, we show by blue native PAGE that cbp3Delta and cbp4Delta mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover, deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high-molecular-weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high-level expression of Cbp3p can partially compensate for the lack of Cbp4p. The finding that mitochondria of cbp3Delta and cbp4Delta mutants exhibit a wild-type lipid composition favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism.

Electron Transport Complex III↗
PubMed1988

The definition of mitochondrial H+ ATPase assembly defects in mit- mutants of Saccharomyces cerevisiae with a monoclonal antibody to the enzyme complex as an assembly probe.

mit- mutants with genetically defined mutations in the mitochondrial structural genes of the H+-ATPase membrane subunits 6, 8 and 9 were analysed to determine the H+-ATPase assembly defects that resulted as a consequence of the mutations. These include mutants which do not synthesize one of the membrane subunits and mutants which can synthesize these subunits, but in an altered form. Protein subunits which can still be assembled to the defective H+-ATPase in these mutants were determined by immunoprecipitation using a monoclonal antibody to the beta-subunit of the enzyme complex. The results suggest that the assembly pathway of the mitochondrially synthesized H+-ATPase subunits involves the sequential addition of subunits 9, 8 and 6 to a membrane-bound F1-sector. In addition to subunits of the F0- and F1-sectors, two other polypeptides (Mr = 18,000 and Mr = 25,000) are associated with the yeast H+-ATPase. These polypeptides were not observed in the immunoprecipitates obtained from mutants in which the F0-sector is not properly assembled.

Antibodies, Fungal↗
PubMed1984

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. III. The carboxyl termini of protein P11 are required for assembly activity.

The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y. Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer. During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm. Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases. Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge. P11 assembly activity is completely lost after removal of the first positive charge. These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex. Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids.

Amino Acid Sequence↗