SEARCH · PubMed Health

Results for “Multiprotein Complexes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed2000

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

Animals↗

PubMed1996

Penicillin and beyond: evolution, protein fold, multimodular polypeptides, and multiprotein complexes.

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and beta-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes.

Bacterial Proteins↗

PubMed1997

Initiation of bacteriophage phi29 DNA replication in vivo: assembly of a membrane-associated multiprotein complex.

Initiation of in vitro phage phi29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase. We have analyzed membrane vesicles from phi29-infected Bacillus subtilis cells by quantitative immunoblot techniques. During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase. Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked. Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA. The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted. The above results, together with the critical role of protein p1 for in vivo phi29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo phi29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase. Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP. The ability of free TP to bind to membranes and to prime phi29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins. We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane.

Bacillus Phages↗

PubMed1992

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.

Animals↗

PubMed2005

Structural analysis of Siah1-Siah-interacting protein interactions and insights into the assembly of an E3 ligase multiprotein complex.

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.

Calcium-Binding Proteins↗

PubMed2006

Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex.

In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second beta-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.

Amino Acid Sequence↗

PubMed1997

Formation of a regulatory factor X/X2 box-binding protein/nuclear factor-Y multiprotein complex on the conserved regulatory regions of HLA class II genes.

Coordinate regulation of MHC class II genes occurs in a tissue-specific and cytokine-inducible manner. While the upstream regulatory sequences are conserved among all MHC class II genes, multiple base pair changes are found, even within the essential X box region. Analysis of all class II X boxes reveals differential binding between two transcription factors known to interact with the X box region, regulatory factor X and X2 box-binding protein (RFX and X2BP) of the HLA-DRA gene. These data presented a paradox with regard to the coordinate regulation of the class II genes if the factors though to regulate the HLA-DRA gene do not bind to the homologous sequence of all class II genes. Previous results suggested that cooperative interactions between the DNA binding proteins may be the key to understanding this paradox. Here RFX/X2BP/DNA complexes were formed on all class II isotypes regardless of the ability of the X box region to bind either factor individually. To further determine the role of the interactions between the X and Y factors, multiprotein/DNA complexes containing RFX, X2BP, NF-Y, and X-Y box DNA of the DRA and DRB genes were formed. This quaternary complex was extremely stable to competitor DNA, with a half-life > 4 h. These results suggest that the conserved X and Y boxes of class II genes function similarly and define a single multiprotein regulatory complex for class II expression in B cells.

Base Composition↗

PubMed2002

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein.

Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. Here we report the isolation of a multiprotein E(z) complex that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48. This complex, which we termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. We hypothesize that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing.

Amino Acid Sequence↗

PubMed2001

Multiprotein complex signaling and the plasticity problem.

Synaptic transmission of distinct patterns of spikes, or 'neural code', leads to plastic changes in synapses and other parts of the neuron, as well as learning in animals. Recent findings indicate that specialized multiprotein structures associated with neurotransmitter receptors and cell-adhesion proteins function as molecular devices that both read the neural code and initiate long-term changes in synaptic structure and function.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Cell-type specific multiprotein complex formation over the c-fos serum response element in vivo: ternary complex formation is not required for the induction of c-fos.

At the serum response element (SRE) of the c-fos proto-oncogene a ternary complex with two proteins, ternary complex factor (p62TCF) and serum response factor (SRF) can be formed. Its formation has previously been suggested to be necessary for efficient induction of c-fos transcription by serum in mouse NIH 3T3 fibroblasts (1) and by phorbol esters, but not by serum, in mouse BALB/c 3T3 fibroblasts (2). It is shown here by genomic dimethyl sulfate (DMS) footprinting that this ternary complex is indeed formed in NIH 3T3 cells in vivo. However, cells were found, murine F9 teratocarcinoma stem cells, in which the pattern of protection and hyperreactivity is consistent with the absence of p62TCF in the protein complex in vivo, although inducibility of the endogenous c-fos gene is not impaired. Both in NIH 3T3 cells and in F9 cells a mutated c-fos promoter that binds SRF but fails to form a ternary complex, was inducible by serum and phorbol esters to the same extent as the wild-type promoter. The data suggest that ternary complex formation is not an absolute prerequisite for the transient induction of c-fos. Ternary complex formation rather appears to enhance overall promoter efficiency. A cell type specific component determines the formation of the multicomponent transcription factor complex in vivo.

Animals↗

PubMed2002

Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins.

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.

Escherichia coli↗

PubMed1988

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus.

The genes encoding the multiprotein membrane-bound ribosomal protein (MBRP) complex (mrp genes), associated with membrane-bound ribosomes in Staphylococcus aureus, were cloned in Escherichia coli. All four components (molecular sizes 71, 60, 46, and 41 kilodaltons) of the MBRP complex were expressed from an 8.5-kilobase DNA fragment as judged by Western blot (immunoblot) analysis. The order of the individual genes within the cloned DNA fragment was determined by deletion mutagenesis and subcloning of various restriction fragments. Three RNAs, transcribed from the same DNA strand, were identified within the MBRP-coding region: one large RNA of approximately 5.9 kilobases, presumably coding for all four MBRP components, and two minor RNAs, coding for MBRP-71 and MBRP-60. The two minor RNAs seemed to be transcribed from promoters within the large transcription unit. Attempts to make insertional inactivations of the mrp genes with an internal 600-base-pair DNA fragment of the MBRP-coding region as a target were unsuccessful, presumably because such insertions are lethal.

Blotting, Northern↗

PubMed1984

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro.

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.

DNA Replication↗

PubMed2002

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors.

The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.

Animals↗

PubMed2004

Tandem mass spectrometry defines the stoichiometry and quaternary structural arrangement of tryptophan molecules in the multiprotein complex TRAP.

We have used tandem mass spectrometry to examine the stoichiometry and binding sites of trp molecules in various assemblies of the protein complex TRAP. The results show that TRAP forms oligomers containing 11 and 12 subunits. MS/MS experiments show that up to 11 trp molecules bind to the 12-mer but that during gas-phase dissociation 5 then 6 trp molecules are released reflecting the different gas-phase stabilities of the partially ligated forms. At high trp concentrations, the protein assembles to form a double ring structure. Tandem mass spectrometry reveals that it is composed of 24 subunits with up to 22 molecules of trp. Dissociation of the complex reveals the same dissociation pathway as for the single ring structure, allowing us to propose a model for the assembly of the TRAP 24-mer based on the different environments of trp molecules. More generally, these results demonstrate the power of tandem mass spectrometry for defining the stoichiometry and quaternary structural arrangement of subunits and ligands within a 46-component multiprotein multiligand complex.

Bacillus subtilis↗

PubMed2003

Mass spectrometric analysis of the editosome and other multiprotein complexes in Trypanosoma brucei.

The composition of the editosome, a multi-protein complex that catalyzes uridine insertion and deletion RNA editing to produce mature mitochondrial mRNAs in trypanosomes, was analyzed by mass spectrometry. The editosomes were isolated by column chromatography, glycerol gradient sedimentation, and monoclonal antibody affinity purifications. At least 16 proteins form the catalytic core of the editosome, and additional associated proteins were identified. Analyses of mitochondrial fractions identified several non-editosome proteins and multi-protein complexes. These studies contribute to the functional annotation of T. brucei genome.

Amino Acid Sequence↗

PubMed1993

PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer.

B-cell-specific enhancers have been identified in the immunoglobulin lambda locus 3' of each constant-region cluster. These enhancers contain two distinct domains, lambda A and lambda B, which are essential for enhancer function. lambda B contains a near-consensus binding site for the Ets family of transcription factors. In this study, we have identified a B-cell-specific protein complex which binds the lambda B motif of the lambda 2-4 enhancer in vitro and appears necessary for the activity of the enhancer in vivo, since mutations in lambda B which prevent this interaction also eliminate enhancer function. This complex contains PU.1, a member of the Ets family, and a transcriptional activator whose expression is restricted to cells of the hematopoietic system with the exception of T lymphocytes. In addition, it contains a factor which binds specifically to a region adjacent to the PU.1 binding site. This factor cannot bind lambda B autonomously but appears to require interaction with the PU.1 protein to stabilize its association with the DNA. This complex may be identical or related to the PU.1/NF-EM5 complex which interacts with a homologous DNA element in the immunoglobulin kappa 3' enhancer.

Animals↗

PubMed2004

A model for the Bacillus subtilis formylglycinamide ribonucleotide amidotransferase multiprotein complex.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity. smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood. We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively. PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices. In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer. The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core. We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex. The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ. The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer. Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site. The homology model is consistent with biochemical studies on the reconstituted complex.

Adenosine Diphosphate↗