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Results for “Multiprotein Complexes”

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PubMed1997

Constitutive and strong association of PAF53 with RNA polymerase I.

Mouse RNA polymerase I (Pol I) is composed of 14 polypeptides, 3 of which are thought to be loosely associated with, and may be dislodged from, Pol I. To find out whether these polymerase-associated factors (PAF53, 51, and 49) serve a role in growth-dependent control of rDNA transcription, we generated polyclonal antibodies against three subunits of murine Pol I, RPA116, RPA40 and PAF53, and used different experimental approaches, e.g. immunoblot analysis, immunoprecipitation and immunofluorescence studies, to compare the stoichiometry of individual subunits both in different Pol I preparations and in extracts from cells grown under different conditions. This comparative analysis reveals that the molar ratio of the second largest subunit RPA116 to PAF53 is the same, irrespective of whether crude extracts or highly purified Pol I fractions are analyzed. Significantly, the relative level of PAF53 was comparable in exponentially growing or growth-arrested cells, indicating that growth-dependent fluctuations in Pol I activity are not accompanied by alterations in the amount of PAF53. In addition, we show by high resolution immunofluorescence analysis that, under conditions of repressed rDNA transcription, including serum starvation, actinomycin treatment und during mitosis, PAF53 remains attached to the transcriptional machinery. The finding that the Mr 53,000 protein remains in the multiprotein complex under all experimental conditions tested indicates that PAF53 is not a loosely associated regulatory factor but a bona fide subunit of Pol I.

3T3 Cells↗

PubMed1995

The yeast TATA-binding protein (TBP) core domain assembles with human TBP-associated factors into a functional TFIID complex.

In mammalian and Drosophila cells, the central RNA polymerase II general transcription factor TFIID is a multisubunit complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) bound to the conserved TBP carboxy-terminal core domain. TBP also associates with alternative TAFs in these cells to form general transcription factors required for initiation by RNA polymerases I and III. Although extracts of human HeLa cells contain little TBP that is not associated with TAFs, free TBP is readily isolated from yeast cell extracts. However, recent studies indicate that yeast TBP can also interact with other yeast polypeptides to form multiprotein complexes. We established stable human HeLa cell lines expressing yeast TBP and several yeast-human TBP hybrids to study TBP-TAF interactions. We found that the yeast TBP core domain assembles with a complete set of human TAFs into a stable TFIID complex that can support activated transcription in vitro. The fact that the yeast TBP core, which differs from human TBP core in approximately 20% of its amino acid residues, has the structural features required to form a stable complex with human TAFs implies that Saccharomyces cerevisiae probably contains TAFs that are structurally and functionally analogous to human TAFs. Surprisingly, the non-conserved amino terminus of yeast TBP inhibited association between the yeast core domain and human TAFs.

DNA-Binding Proteins↗

PubMed1993

A developmentally regulated glycoprotein complex from Dictyostelium discoideum.

The monoclonal antibody MUD50 recognizes an epitope on a family of developmentally regulated proteins in Dictyostelium discoideum (Alexander, S., Smith, E., Davis, L., Gooley, A., Por, S. B., Browne, L., and Williams, K. L. (1988) Differentiation 38, 82-90). This epitope is an O-linked oligosaccharide that requires the wild-type modB allele for expression. These glycoproteins are structurally diverse. Some are integral membrane proteins, while others are soluble and do not require detergent for solubilization. One of the soluble glycoproteins, PsB, is also recognized by the monoclonal antibody MUD102, which binds to the polypeptide backbone. The antibody recognizes a single 100-kDa band in Western analysis. In this report we demonstrate that the PsB molecule is part of a specific developmentally regulated multiprotein complex containing six different proteins. The complex is held together by both covalent and noncovalent bonds. Interestingly, only the PsB glycoprotein has the MUD50 O-linked oligosaccharide determinant. None of the proteins in the complex is N-glycosylated as assessed by sensitivity to peptide N-glycosidase F. Only one of the complex proteins is heavily phosphorylated. This work establishes the foundation for additional studies on the biosynthesis, processing, and assembly of the O-glycosylated PsB protein.

Animals↗

PubMed2003

NALPs: a novel protein family involved in inflammation.

A newly discovered family of cytoplasmic proteins--the NALPs--has been implicated in the activation of caspase-1 by the Toll-like receptors (TLRs) during the cell's response to microbial infection. Like the structurally related apoptotic protease-activating factor-1 (APAF-1), which is responsible for the activation of caspase-9, the NALP1 protein forms a large, signal-induced multiprotein complex, the inflammasome, resulting in the activation of pro-inflammatory caspases.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes.

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions.

Actins↗

PubMed1999

Maternal histone deacetylase is accumulated in the nuclei of Xenopus oocytes as protein complexes with potential enzyme activity.

Reversible acetylation of core histones plays an important regulatory role in transcription and replication of chromatin. The acetylation status of chromatin is determined by the equilibrium between activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The Xenopus protein HDACm shows sequence homology to other putative histone deacetylases, but its mRNA is expressed only during early development. Both HDACm protein and acetylated non-chromosomal histones are accumulated in developing oocytes, indicating that the key components for histone deposition into new chromatin during blastula formation are in place by the end of oogenesis. Here we show that the 57 kDa HDACm protein undergoes steady accumulation in the nucleus, where it is organized in a multiprotein complex of approx. 300 kDa. A second, major component of the nuclear complex is the retinoblastoma-associated protein p48 (RbAp48/46), which may be used as an adaptor to contact acetylated histones in newly assembled chromatin. The nuclear complex has HDAC activity that is sensitive to trichostatin A, zinc ions and phosphatase treatment. The 57 kDa protein serves as a marker for total HDAC activity throughout oogenesis and early embryogenesis. The active HDACm complex and its acetylated histone substrates appear to be kept apart until after chromatin assembly has taken place. However, recombinant HDACm, injected into the cytoplasm of oocytes, not only is translocated to the nucleus, but also is free to interact with the endogenous chromatin.

Amino Acid Sequence↗

PubMed2005

Structural organization of a Sex-comb-on-midleg/polyhomeotic copolymer.

The polycomb group proteins are required for the stable maintenance of gene repression patterns established during development. They function as part of large multiprotein complexes created via a multitude of protein-protein interaction domains. Here we examine the interaction between the SAM domains of the polycomb group proteins polyhomeotic (Ph) and Sex-comb-on-midleg (Scm). Previously we showed that Ph-SAM polymerizes as a helical structure. We find that Scm-SAM also polymerizes, and a crystal structure reveals an architecture similar to the Ph-SAM polymer. These results suggest that Ph-SAM and Scm-SAM form a copolymer. Binding affinity measurements between Scm-SAM and Ph-SAM subunits in different orientations indicate a preference for the formation of a single junction copolymer. To provide a model of the copolymer, we determined the structure of the Ph-SAM/Scm-SAM junction. Similar binding modes are observed in both homo- and heterocomplex formation with minimal change in helix axis direction at the polymer joint. The copolymer model suggests that polymeric Scm complexes could extend beyond the local domains of polymeric Ph complexes on chromatin, possibly playing a role in long range repression.

Amino Acid Sequence↗

PubMed1982

The Leeuwenhoek Lecture, 1981. The biochemical and genetic approach to the study of bioenergetics with the use of Escherichia coli: progress and prospects.

How the 'energy currency' of the cell, adenosine triphosphate (ATP), is produced consequent upon the oxidation of foodstuffs (oxidative phosphorylation) is, despite prolonged research, still a matter of debate and the molecular mechanism of the process is unknown. It appears that the problem of oxidative phosphorylation can be approached with the aid of the biochemical genetics of the bacterium Escherichia coli. The ease of manipulation of bacteria and definitive results obtained by this approach have been invaluable in solving other major biochemical problems. Mutants affected in oxidative phosphorylation have been isolated and characterized by genetic and biochemical techniques. These 'unc' mutants are affected in the adenosine triphosphatase (ATPase) multiprotein complex which is part of the cell membrane and responsible for the terminal stages of ATP synthesis. Seven distinct genes concerned with oxidative phosphorylation have been characterized in E. coli and shown to be part of an operon. The relationships between the different classes of unc genes and the various components of the ATPase have been established. Information about the assembly of the ATP synthesizing complex in the cell membrane has also been obtained and the stage set for further studies on the assembly, control and function of the ATP synthesizing system.

Adenosine Triphosphatases↗

PubMed2002

Silencing of transcription of the human luteinizing hormone receptor gene by histone deacetylase-mSin3A complex.

Modification of chromatin structure by histone acetylases and deacetylases is an important mechanism in modulation of eukaryotic gene transcription. The present study investigated regulation of the human luteinizing hormone receptor (hLHR) gene by histone deacetylases. Inhibition of histone deacetylases (HDACs) by trichostatin A (TSA) increased hLHR promoter activity by 40-fold in JAR cells and markedly elevated endogenous hLHR mRNA levels. Acetylated histones H3 and H4 accumulated in TSA-treated cells and associated predominantly with the hLHR promoter. Furthermore, TSA significantly enhanced the recruitment of RNA polymerase II to the promoter. One of the two Sp1 sites essential for basal promoter activity was identified as critical for the TSA effect, but the binding of Sp1/Sp3 to this site remained unchanged in the absence or presence of TSA. A multiprotein complex was recruited to the hLHR promoter via interaction with Sp1 and Sp3, in which HDAC1 and HDAC2 were docked directly to Sp1-bound DNA and indirectly to Sp3-bound DNA through RbAp48, while mSin3A interacted with both HDACs. HDAC1 and HDAC2 were shown to potently repress the hLHR gene transcription, and mSin3A potentiated the inhibition mediated by HDAC1. Our studies have demonstrated that the HDAC-mSin3A complex has an important role in the regulation of hLHR gene transcription by interaction with Sp1/Sp3 and by region-specific changes in histone acetylation and polymerase II recruitment within the hLHR promoter.

Blotting, Western↗

PubMed2004

Caspase-dependent, geldanamycin-enhanced cleavage of co-chaperone p23 in leukemic apoptosis.

Co-chaperone p23 is a component of the heat-shock protein (Hsp)90 multiprotein-complex and is an important modulator of Hsp90 activity. Hsp90 client proteins involved in oncogenic survival signaling are frequently mutated in leukemia, and the integrity of the Hsp90 complex could therefore be important for leukemic cell survival. We demonstrate here that p23 is cleaved to a stable 17 kDa fragment in leukemic cell lines treated with commonly used chemotherapeutic drugs. The cleavage of p23 paralleled the activation of procaspase-7 and -3 and was suppressed by the caspase-3/-7 inhibitor DEVD-FMK. In vitro translated 35S-p23 (in reticulocyte lysate) was cleaved at D142 and D145 by caspase-7 and -3. Cleavage of p23 occurred in caspase-3-deficient MCF-7 cells, suggesting a role for caspase-7 in intact cells. The Hsp90 inhibitor geldanamycin enhanced caspase-dependent p23 cleavage both in vitro and in intact cells. Geldanamycin also enhanced anthracycline-induced caspase activation and apoptosis. We conclude that p23 is a prominent target in leukemic cell apoptosis. Geldanamycin enhanced p23 cleavage both by rendering p23 more susceptible to caspases and by enhancing chemotherapy-induced caspase activation. These findings underscore the importance of the Hsp90-complex in antileukemic treatment, and suggest that p23 may have a role in survival signaling.

Apoptosis↗

PubMed2005

Development of a bi-cistronic baculovirus expression vector by the Rhopalosiphum padi virus 5' internal ribosome entry site.

A bi-cistronic baculovirus transfer vector was constructed based on the 5'UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene and green fluorescent protein gene flanking the RhPV 5'UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. Quantization by fluorescence spectrophotometry of the fluorescent proteins produced in Sf21 cells indicated that the translational efficacy of the RhPV 5'UTR IRES was about 3-fold weaker than cap-dependent translation. We also demonstrated that recombinant baculoviruses containing the human interferon-gamma gene (IFN-gamma) and green fluorescent protein gene flanking the RhPV 5'UTR IRES can produce IFN-gamma proteins as well as green fluorescent proteins. These results suggest that the RhPV IRES can be used in the development of bi-cistronic baculovirus expression vectors for production of heterologous multiprotein complexes or can be combined with selection markers to facilitate applications of baculovirus expression systems.

5' Untranslated Regions↗

PubMed2004

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation.

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.

3T3 Cells↗

PubMed2004

RoXaN, a novel cellular protein containing TPR, LD, and zinc finger motifs, forms a ternary complex with eukaryotic initiation factor 4G and rotavirus NSP3.

Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3' consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.

Amino Acid Motifs↗

PubMed2007

Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.

Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Chemical conditionality: a genetic strategy to probe organelle assembly.

The assembly of the Escherichia coli outer membrane (OM) is poorly understood. Although insight into fundamental cellular processes is often obtained from studying mutants, OM-defective mutants have not been very informative because they generally have nonspecific permeability defects. Here we show that toxic small molecules can be used in selections employing strains with permeability defects to create particular chemical conditions that demand specific suppressor mutations. Suppressor phenotypes are correlated with the physical properties of the small molecules, but the mutations are not in their target genes. Instead, mutations allow survival by partially restoring membrane impermeability. Using "chemical conditionality," we identified mutations in yfgL, and, here and in the accompanying paper by Wu et al. published in this issue of Cell (Wu et al., 2005), we show that YfgL is part of a multiprotein complex involved in the assembly of OM beta barrel proteins. We posit that panels of toxic small molecules will be useful for generating chemical conditionalities that enable identification of genes required for organelle assembly in other organisms.

Bacterial Outer Membrane Proteins↗

PubMed2006

Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs.

A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.

Adaptor Proteins, Signal Transducing↗

PubMed2006

[Regulation of cytokine production in inflammatory bowel disease].

Interleukin-1beta (IL-1beta) is a pivotal mediator in the inflammatory immune response that is characteristic of a number of chronic disorders. The etiology and pathophysiology of inflammatory bowel disease (IBD) have for many years been an enigma. The recent identification of the inflammasome, a multiprotein complex responsible for interleukin-1beta converting enzyme (ICE, caspase-1) activation, has generated new possibilities for the elucidation of the etiology and pathophysiology of IBD and as a consequence also for the identification of new treatment targets.

Caspase 1↗

PubMed2007

Exploiting network biology to improve drug discovery.

Mammalian signal transduction occurs in the context of multiprotein complexes, yet currently available drug discovery strategies do not reflect this fact. We present a strategy for screening drugs and targets in living human cells by utilizing high content protein-fragment complementation assays. Synthetic fragments of a mutant fluorescent protein ("Venus" and/or enhanced yellow fluorescent protein) are used for protein-fragment complementation assay construction, allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. Here we describe the utility of this novel strategy for high-throughput screening of known targets, and for screening previously undrugable targets and profiling drug leads for improved selectivity and safety.

Cell Survival↗